WO2009065199A1 - Enzyme composition from trichoderma reesei and aspergillus awamori - Google Patents

Abstract

The present invention refers to enzyme compositions comprising cellulose and hemicellulose containing enzymes and optionally β-glucosidase, obtained by fermentation with Trichoderma reesei as well as cellulose and hemicellulose containing enzymes, β-glucosidase and accessory enzymes, obtained by fermentation with Aspergillus awamori. The invention also includes a production process for said enzyme compositions.

Description

ENZYME COMPOSITION FROM TRICHODERMA REESEI ANDASPERGILLUS AWAMORI

Field of the Invention

The present invention refers to compositions of enhanced enzymes for degrading cellulose, hemicellulose and other polysaccharides of the biomass and efficiently- separate the lignin contained in lignocellulosic material, with said enzymes being obtained from specific lineages of the Trichoderma reesei and Aspergillus awamori microorganisms and comprise, apart from cellulases

(endoglucanases and exoglucanases) , β-glycosidase and accessory enzymes that promote degradation of hemicellulose and other polysaccharides also promoting the separation of the lignin and, by this mean, facilitating the main action of the majoritarian cellulases and increasing the yield of glucose obtainment and, consequently, the yield of the process of alcohol production from the biomass. The invention includes a production process of cellulolytic and hemicellulolytic enzymes, of β-glycosidase and of accessory enzymes that compose the composition of the invention and a production process of alcohol from biomass using, in the enzymatic hydrolysis step, the composition of the invention .

Background of the Invention

The progressive exhaustion of the world' s petroleum resources is triggering a new "gold rush" - the search for economically viable renewable fuels, all the more so because the combustion of fossil fuels is leading to an accumulation of CO2, one of the main causes of the "greenhouse effect". The use of alternative sources of energy produced from renewable raw materials, such as biomass, is becoming a priority goal at international levels so as to allow sustainable development and halt the build-up of the gases responsible for global warming; the CO₂ emitted by the combustion of biofuels is recycled into the biosphere through the process of photosynthesis (refer to Lin Y, Tanaka S (2006) . "Ethanol fermentation from biomass resources: Current state and prospects". Applied Microbiology and Biotechnology; 69: 627-642) . The production of second generation ethanol (from lignocellulosic biomass) is currently recognised worldwide as being the main technology for the production of liquid fuels to substitute gasoline produced from petroleum.

Brazil holds a privileged position in relation to second generation ethanol production (with emphasis on biomass from the strategically located sugar cane bagasse and straw) due to the huge available agricultural area, favourable climate, abundance of water and its vast experience in the production and use of ethanol derived from sugar cane (first generation). It is estimated that the sugar-ethanol sector generates approximately 16 million tons of surplus sugar cane bagasse and 76 million tons of straw (refer to MAPA, Ministerio da Agricultura , Pecuaria e Abastecimento (2005) [Ministry of Agriculture, Livestock and Supply] . Piano Nacional de Agroenergia [National Plan for Agro-energy] , Brasilia, MAPA, 118p, available at http: //www. agricultura. gov.br (accessed on 24/03/06) ) . The expansion of sugar cane cultivation triggered by the rising demand for ethanol will lead to a proportional increase in surplus bagasse, which shall also increase with the improved efficiency of boilers. Furthermore, the burning of cane straw has now been banned and shall gradually be eradicated in Sao Paulo within the next 30 years (Law 11.241 and Decree 47.700, of March, 2003).

The lignocellulosic residues, such as bagasse and straw, are basically constituted of cellulose, hemicellulose and lignin in varying proportions. Cellulose is a linear polymer formed by glucose molecules. The repetitive unit of the polymer is the disaccharide cellobiose. In natural celluloses, the chains align in a manner as to form organised fibrils of complex form that present regions with crystalline structures and regions with amorphous structures. Hemicelluloses belong to a mixed group of non-cellulosic linear and/or ramified polysaccharides that may be constituted of pentose or hexose units. Lignin is constituted of heterogeneous aromatic chains, with an extremely complex three dimensional structure.

The production of ethanol from a cellulosic component of biomass is based on the transformation of cellulose, which is the main component of bagasse, into glucose through the enzymatic hydrolysis reaction of the pretreated bagasse, with the resulting glucose being fermented by yeast of the Saccharomyces cerevisiae species (alcoholic fermentation) . This process for obtaining ethanol increases the production yield without, however, increasing the cultivated area of sugar cane.

Just as any lignocellulosic residue, the biomass needs to be pretreated before being used as a raw material for the production of ethanol. The pre-treatment has the purpose of altering the native interactions occurring between cellulose, hemicellulose and lignin so as to reduce the biomass' s recalcitrance to enzymatic hydrolysis. The pre-treatment also serves to reduce cellulose crystallinity and increase biomass porosity, (refer to Almeida JR, Modig T, Petersson A, Hahn-Hagerdal B, Liden G, Gorwa-Grauslund M-F (2007). "Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae" . J Chem Technol Biotechnol. ; 82: 340-349). The pre-treatment technology most used to destructure the lignocellulosic biomass is steam explosion, which further causes the partial depolymerisation of the hemicellulose due to exposure to high temperatures and pressure. However, this also forms inhibitors for the following stages, mainly in relation to enzymatic hydrolysis and cell growth and alcoholic fermentation, and therefore the pretreated biomass must be washed in order to remove all inhibitory material, which also removes part of the hemicelluloses and the derived sugars, (refer to Prasad S, Singh A and Joshi HC. (2007). "Ethanol as an alternative fuel from agricultural, industrial and urban residues". Resources, Conservation and Recycling; 50: 1-39).

The option for the enzymatic hydrolysis of cellulose stems from the absence of severe conditions as opposed to chemical hydrolysis that generates highly toxic hydrolysates (furfurals and aromatic substances derived from lignin) which prevent cellular metabolism and the alcoholic fermentation of the hydrolysate. Furthermore, the expense of enzymatic hydrolysis is low when compared to acid or alkaline hydrolysis since enzymatic hydrolysis is usually performed in mild conditions (pH 4.8 and temperature in a range of 45-50° C) and does not pose equipment corrosion problems (refer to Prasad et al, 2007, above) . Despite enzymatic hydrolysis of biomass being the only internationally accepted option, the cost and poor availability of the enzymes required for this process constitute the major drawback for the increased use of this technology, both in Brazil and other countries.

The cellulases - a set of enzymes that degrade cellulose - are classified in three groups: endoglucanases (EC 3.2.1.4) that cleave the internal links of the cellulosic fibres; exoglucanases (EC 3.2.1.91) that act progressively on the reducing and non-reducing ends of cellulose, releasing small cellulose oligosaccharides; and β-glucosidases (EC 3.2.1.21) that degrades cellobiose (glucose dimer) to glucose (refer to Lynd LR, Weimer PJ, van ZyI WH, Pretorius IS (2002) . "Microbial Cellulose Utilization: Fundamentals and Biotechnology". Microb. MoI. Biol. Rev.; 66: 506-577).

Enzymatic activity is generally initiated by a random action of the endogluconases that cleave the cellulose chain in the amorphous regions. This is followed by the activity of the exogluconases removing cellobiose units from the reducing and non-reducing terminals of the chains in the crystalline region. Finally, the β-glucosidases hydrolise the cellobiose into two glucose molecules. Individually, a single enzyme of the cellulolytic complex is incapable of hydrolising cellulose in an efficient manner and synergetic activity of the cellulolytic complex is necessary (refer to Beguin, P and Aubert, JP (1994). "The biological degradation of cellulose". FEMS Microbiol . Rev.; 13: 25 - 58) .

For example, document PI 8204206 describes a hydrolysis method for cellulosic substrate for the production of mono and disaccharides using cellulase enzymes. This document mentions the existence of a synergic effect when the cellulase enzyme of fungus or actinomycete is added to a culture of bacteria of genus Cellulomonas or Pseudomonas. The preferred fungus cellulose enzyme is obtained from Trichoderma reesei.

However, the high cost of the cellulolytic enzymes has rendered enzymatic hydrolysis of biomass difficult. To circumvent the difficulties in making enzymatic hydrolysis economically feasible for the production of biofuels, one of the alternatives for its large scale implementation is the local production of enzymes at the place of use instead of using commercial enzymes.

In the case of Brazil - a country combining all the necessary conditions favourable for the production of bioethanol (ethanol from biomass) from lignocellulosic material - the production of this biofuel could occur at the location of the plant producing ethanol from the cane juices and, simultaneously to processing the soluble sugars from the sugar cane, the existing facilities may be exploited to full advantage through the treatment, fermentation and distillation processes of the juices, steam and electrical power generation, residue treatment and recycling systems, collection and reuse of water, laboratories, automatic monitoring and management, amongst others. In summary, the pre-existent set of conditions associated to the in situ production of cellulases is preeminently favourable to the implementation of this technology in Brazil. Furthermore, the enzymatic hydrolysates of the biomass may be mixed in with the sugar cane juices or the molasses merely using the existing equipment and the microorganism Saccharomyces cerevisiae for the alcoholic fermentation process.

Certain fungi possess the capacity to produce complete cellulolytic complexes in large quantities. Most of the studies are directed at fungi having superior capability of producing cellulases, such as Trichoderma, Penicillium, Aspergillus, Fusarium and Humicola, with genus Trichoderma being reported as being the most efficient for degrading cellulose (refer to Lynd et al., 2002, above). Document EP 0448430 describes a composition of cellulolytic and hemicellulolytic enzymes produced from a fermentation process using as microorganism at least one strain of the fungus Trichoderma reesei, with the lineage of Trichoderma reesei RUT C30 (ATCC 56765) being one of the preferred. The composition of enzymes is used as a catalyser for the hydrolysis of a pretreated lignocellulosic substrate. However, according to the literature, the fungi of this genus may produce low levels of β-glucosidase, requiring the complementation of this enzyme produced in large quantities by other fungi in order to obtain a mixture capable of efficiently hydrolysing cellulose to glucose. Indeed, the microorganisms of genus Trichoderma produced relatively large quantities of endo-β-glucanase and exo-β-glucanase, but may present low levels of β- glucosidase, while the microorganisms of genus Aspergillus produce relatively large quantities of endo-β-glucanase and β-glucosidase, but low levels of exo-β-glucanase. (Refer to "3.2 Cellulase Production" - FAO Corporate Document Repository, available on the Web at http: //www. fao. org/docrep/w7241e/w7241e08. htm, accessed on 07/10/2007) .

The cellulases, as with the other extracellular enzymes from hydrolysis, are only expressed when there is a need for them to be secreted by the microorganisms for them to grow into cellulose (refer to Kubicek CP, Messner R, Gruber F, Mach RL, Kubicek-Pranz EM (1993) . "The Trichoderma reesei cellulase regulatory puzzle: from the interior life of a secretory fungus". Enzyme Microb Technol; 15: 90-99). The culture conditions significantly effect the production of extracellular enzymes in general, including cellulases, hemicellulases and β-glucosidase. The carbon source plays an important role in the production of enzymes because the carbohydrates or their derivates may induce or repress the expression of the genes that encode for the cellulolytic enzymes (refer to Kubicek CP, Penttila, ME, (1998) . "Regulation of production of plant polysaccharide degrading enzymes by Trichoderma" . In: Harman, GE, Kubicek, CP, editors. Trichoderma and Gliocladium, Enzymes, biological control and commercial applications, vol. 2. Bristol: Taylor & Francis Ltd. [Chapter 3] p. 49-67) .

The production of enzymes of the hypercellulolytic fungus Trichoderma reesei has been studied in detail (refer to Persson I, Tjerneld F, Hagerdal BH (1991) . "Fungal cellulolitic enzyme production: an overview". Proc Biochem; 26: 65-74). Many substances are acknowledged as inducers of this cellulotytic complex such as cellobiose (refer to Fritscher C, Messner R, Kubicek CP (1990) . "Cellobiose metabolism and cellobiohydrolase I biosynthesis by Trichoderma reesei". Exp Mycol; 14: 405-15), lactose (refer to Morikawa Y, Ohashi T, Mantani O, Okada H (1995) . "Cellulase induction by lactose in Trichoderma reesei PC-3- 7". Appl Biochem Biotechnol ; 44: 106-11) and, especially, sophorose, (refer to Mach RL, Seiboth B, Myasnikov A, Gonzalez R, Strauss J, Harkki AM, Kubicek CP (1995) . "The bgll gene of Trichoderma reesei QM9414 encodes an extracellular, cellulose-inducible bglucosidase involved in cellulase induction by sophorose". MoI Microbiol ; 16: 687- 697). High enzymatic activity by cellulases are reported in studies concerning materials containing cellulose as a source of carbon (refer to Bhat MK, Bhat S. (1997) . "Cellulose degrading enzymes and their potential industrial applications". Biotechnol Adv; 15: 583-620; Kubicek and Penttila, 1998, above; Juhasz T, Szengyel Z, Reczey K, Siika-Aho M and Viikari L. (2005) . "Characterization of cellulases and hemicellulases produced by Trichoderma reesei on various carbon sources". Process Biochemistry; Vol. 40(11) : 3519-3525) .

More recently, research on cellulolytic enzymes has concentrated on obtaining mixtures of cellulases that enhance the hydrolysis of the glucosidic bonds of cellulose and hemicellulose, maintaining the lignin structure contained in lignocellulosic materials Rosgaard, L et al. (refer to Rosgaard L., Andric P., Dam-Johansen K., Pedersen S. and Meyer A. B. S. (2006). "Effects of Fed-Batch Loading of Substrate on Enzymatic Hydrolysis and Viscosity of Pretreated Barley Straw". The Forest Products Division, at a talk given at "The 2006 Annual Meeting" and Rosgaard L., Pedersen S, Cherry J., Harris P., and Meyer A. (2006). "Efficiency of New Fungal Cellulase Systems in Boosting Enzymatic Degradation of Barley Straw Lignocellulose" . Biotechnol Prog, 22 (2):493-498 16599567). Recent work has studied the improved efficiency of enzymatic hydrolysis of the lignocellulosic material of barley straw by the use of a mixture of enzymes composed of Celluclast® (a preparation of cellulolytic enzymes from Trichoderma reesei) and Novozym 188 (a preparation of β-glucosidase from Aspergillus niger) . These researchers concluded that additional enzymatic activity to the enzyme system of T. reesei accelerates the degradation of lignocelluloses .

Sendelius, J (Sendelius, J. (2005) "Steam Pretreatment Optimisation for Sugarcane Bagasse in Bioethanol Production"; Master's degree treatise, Departament of Chemical Engineering, Lund University, Sweden) also worked with mixture of Celluclast® and Novozym 188 enzymes for the enzymatic hydrolysis of sugar cane bagasse pretreated with steam and concluded that the glucose yield is dependant on the removal of the hemicellulose during the pretreatment stage .

It is also broadly recognised that the pretreatment of lignocellulosic material before processing by enzymatic hydrolysis has a preponderant role in the efficiency of the cellulose and hemicellulose disaggregation as well as the separation of lignin, which is polyaromatic and hydrophobic. One of the most commonly used pretreatments is the steam explosion method, which is widely known to technicians working in biofuel production from lignocellulosic material (refer, for example, to Sun, Ye (2002) "Enzymatic Hydrolysis of Rye Straw and Bermudagrass for Ethanol Production". PhD thesis treatise, North Carolina State University) .

In enzymatic hydrolysis, apart form the majoritarian cellulases (endoglucanases, exoglucanases) and β- glucosidase, other enzymes play an important role in the attack on the polysaccharide fraction of the biomass and its separation from lignin and other components of the biomass such as those of proteic nature, (refer to Saha B.C. (2003) . "Hemicellulose bioconversion" . Journal Ind. Microbiology Biotechnology 30: 279-291). These are termed accessory enzymes, and include glucuronidases, acetylesterases, xylanases, β-xylosidases, galactomannanases, glucomannanases, polygalacturonases, glucoamylases, amylogalactosidases, tannases, feruloyl esterases, proteases, amongst others. The literature reports the production of several of these accessory enzymes from the Aspergillus awamori strain, including the A. awamori B.361U2/1 lineage, amongst which the following may be mentioned:

• Xylanase (E. C. 3.2.1.8) of A. awamori B.361U2/1 (refer to Botella, C, Diaz, A., Ory, I., Webb, C, Blandino, A. (2007) "Xylanase and pectinase production by Aspergillus awamori on grape pomace in solid state fermentation". Process Biochemistry, Vol. 42 (1) : 98-101) ;

• Polygalacturonase (E. C. 3.2.1.67) of A. awamori B.361U2/1 (refer to Botella, C, Ory, I., Webb, C, Blandino, A. (2005) "Hydrolytic enzyme production by Aspergillus awamori on grape pomace". Biochemical Engineering Journal Vol. 26: 100-106);

• β-xylosidase (E. C. 3.2.1.37) of A. awamori (refer to

Kurakake, M., Fujii, T., Yata, M., Okazaki, T., Komaki, T.

(2005) "Characteristics of transxylosylation by beta- xylosidase from Aspergillus awamori K4". Biochim. Biophys.

Acta Vol. -1726:372-9) ;

• α-glucosidase (E. C. 3.2.1.20) of A. awamori (refer to Anindyawati, T., Ann, Y. G., Ito, K., Iizuka, M., Minamiura, N. (1998) "Two kinds of novel alpha-glucosidases from Aspergillus awamori K-Il: Their purification properties ans specificities". Journal of Fermentation and Bioengineering, Vol. 85:465-469);

• Glucoamylase (E. C. 3.2.1.3) of A. awamori B.361U2/1 (refer to Bon, E. & Webb, C. (1993) "Glucomylase production and Nitrogen Nutrition in Aspergillus awamori" . Applied Biochemistry and Biotecnology 39: 349-369. Bon, E. & Webb, C. (1989) "Passive immobilization of Aspergillus-awamori spores for subsequent glucoamylase production" Enzyme and Microbial Technology 11 (8): 495-499);

• β-D-Mannosidase (E. C. 3.) of A. awamori (refer to de Vries, R. P. and Visser, J. (2001) "Aspergillus Enzymes

Involved in Degradation of Plant Cell Polysaccharides". Microbiology and Molecular Biology Reviews. Vol. 65(4) :497- 522);

• Arabinoxylan arabinofuranohydrolase of A. Awamori (refer to de Vries, R. P. ad Visser, J. (2001) "Aspergillus

Enzymes Involved in Degradation of Plant Cell Polysaccharides". Microbiology and Molecular Biology Reviews. Vol. 65 (4 ): 497-522 ) ;

• Acetylxylane esterase (refer to de Vries, R. P. and Visser, J. (2001) "Aspergillus Enzymes Involved in

Degradation of Plant Cell Polysaccharides". Microbiology and Molecular Biology Reviews. Vol. 65 (4 ): 497-522 ; Koseki, T., Furuse, S., Iwano, K., Sakai,H., Matsuzawa, H. (1997) "An Aspergillus awamori acetylesterase : purification of the enzyme, and cloning and sequencing of the gene" Biochemical Journal 326: 485-490) ;

• Feruloyl esterase (refer to Topakas, E., Vafiadi, C, Christakopoulos, P. (2007) "Microbial production, characterization and applications of feruloyl esterases" Process Biochemistry Vol. 42:497-509; Koseki, T., Takahashi, K., Handa, T., Yamane, Y., Fushinobu, S., Hashizume, K. (2006) "N-linked oligosaccharides of Aspergillus awamori feruloyl esterase are important for thermostability and catalysis" Bioscience Biotechnology and Biochemistry 70 (10) : 2476-2480; Koseki, T., Takahashi, K., Fushinobu, S., Iefuji, H., Iwano, K., Hashizume, K., Matsuzawa, H. (2005) "Mutational analysis of a feruloyl esterase from Aspergillus awamori involved in substrate discrimination and pH dependence" Biochimica et Biophysica Acta-general subjects 1722 (2 ): 200-208 ; de Vries, R. P. and Visser, J. (2001) "Aspergillus Enzymes Involved in Degradation of Plant Cell Polysaccharides". Microbiology and Molecular Biology Reviews. Vol. 65 (4 ): 497-522; Koseki, T., Furuse, S., Iwano, K., Matsuzawa, H. (1998) "Purification and characterization of a feruloylesterase from Aspergillus awamori" Bioscience Biotechnology and Biochemistry 62 (10) : 2032-2034 ).

Apart from the above references, document EP 1511848 is worthy of mention and describes enzyme compositions for the enzymatic hydrolysis of lignocellulosic material comprising cellulases and auxiliary enzymes such as, for example, xylanases. It should be stressed that this document mentions the fungus Trichoderma viride as the producer microorganism for xylanase (auxiliary) and the fungus Trichoderma reesei (a mutant of Trichoderma viride) as the producer microorganism for cellulase among the examples provided, both of which do not normally produce β- glucosidase in sufficient quantity for the efficient enzymatic hydrolysis of lignocellulosic material.

There have been numerous proposals for the enhancement of enzyme compositions and the methods for the enzymatic hydrolysis to degrade the cellulose and hemicellulose contained in cellulosic material, with the intent of optimising the processes for obtaining biofuels and, more especially, bioalcohol. However, limitations remain with regard availability both in terms of quantity and the types of enzymes that degrade different types of lignocellulosic materials and their efficiency in the preparation of an adequate glucose for later alcoholic fermentation. Furthermore, the diversity in the composition of biomasses from varying origins and the types of chemical interaction that the different components establish between themselves indicates the need for developing "tailor made" enzymatic mixtures not only for the different biomasses, but also for the biomass-pretreatment duo. Recent studies also indicate that the usual parameters for determining the level of the different cellulose, hemicellulase and accessory enzyme activity are not complete indicators for defining the effectiveness of the mixtures in the different biomasses and that the development of hydrolysis tests are required (refer to Zhang, Y. H. P., Himmel, M. E., Mielenz, J. R. (2006). "Outlook for cellulase improvement: Screening and selection strategies" Biotechnology Advances 24:452-481).

Furthermore, as yet uncharacterised enzymatic activity may act synergically on the efficiency of the enzymatic mixtures. The problems for the state of the art related above were the reason behind the development of the present invention for obtaining efficient mixtures for the hydrolysis of sugar cane biomass, without the use of genetically modified microorganisms (GMOs) , with all the legal and environmental advantages for the implementation of the process of the invention. The invention is described in detail below.

Summary of the Invention

The invention intends to provide enzyme compositions developed to degrade the polysaccharides contained in lignocellulosic material, and especially the cellulose and hemicellulose of sugar cane biomass, separating and maintaining the chemical characteristics of lignin with regard its hydrophobic aspect, with said enzymes being obtained from specific lineages of Trichoderma reesei and/or Aspergillus awamori microorganisms, comprising, together with the majoritarian cellulolytic enzymes (endoglucanases and exoglucanases) , β-glucosidase and accessory enzymes such as xylanase that promote the degradation of polysaccharides, except for cellulose, and also promoting the separation of the lignin and facilitating the action of the majoritarian cellulases therefore increasing the yield of glucose obtainment and, consequently, of the alcohol production process from biomass.

A primary embodiment provides enzyme compositions comprising (a) cellullolytic, hemicellullolytic and optionally β-glucosidase enzymes, obtained by fermentation with Trichoderma reesei; (b) cellullolytic, hemicellullolytic enzymes and β-glucosidase and accessory enzymes, obtained by fermentation with Aspergillus awamori;

(c) furthermore, at least one molecular species selected from the group of enzymes and/or metabolytes having CMCase activity, with said molecular species being present in the supernatant of the T. reesei and/or A. awamori culture as demonstrated in the zymogram on Figure 7; and (d) optionally, a vehicle compatible with said enzymes, whereby said enzymes (a) and (b) interact in a manner that results in a synergic effect caused by the action of the accessory enzymes that, amongst other activities, break the bonds between the lignin and the polysaccharides of the biomass, especially hemicellulose . The cellulolytic enzymes obtained from T. reesei, preferentially from the lineage of T. reesei RUT C30, substantially comprise endoglucanases and exoglucanases and, depending on culture conditions, high levels of hemicellulase and β-glucosidase . The cellulolytic and hemicellullolytic enzymes and the β-glucosidase obtained from A. awamori, preferentially from a lineage of A. awamori having a high production capacity for accessory enzymes, xylanases, pectinase and feruloyl esterases, amongst others, more preferentially from the lineage of A. awamori B.361U2/1, comprising accessory enzymes that predominantly include xylanases and feruloyl esterases.

A second embodiment of the invention refers to an enzyme composition comprising: (a) cellullolytic and hemicellullolytic enzymes and β-glucosidase, obtained by fermentation with Trichoderma reesei; (b) cellullolytic and hemicellullolytic enzymes and β-glucosidase and accessory enzymes, obtained by fermentation with Aspergillus awamori; and (c) optionally, a vehicle compatible with said enzymes, whereby said enzymes (a) and (b) interact in a manner that results in a synergic effect caused by the action of the accessory enzymes and molecular species that act on other polysaccharides of the biomass as well as on the bonds between the lignin and the hemicellulose.

A third embodiment of the invention refers to the use of the enzyme composition for the enzymatic hydrolysis of lignocellulosic material with a high glucose yield.

A fourth embodiment of the invention refers to a production process for cellulolytic and hemicellulolytic enzymes, β-glucosidase and accessory enzymes, comprising the steps of: (a) the mixture culture in an appropriate culture medium, optionally pH controlled, of a lineage of T. reesei unaffected by catabolic repression and a lineage of A. awamori having a high capability for the production of β-glucosidase and accessory enzymes, including xylanases and feruloyl esterases; (b) the separation of the supernatants of each culture of T. reesei and A. awamori containing the cellulase enzymes, the β-glucosidase and the accessory enzymes; (c) optionally, the concentration and combination of the supernatants containing said cellulase enzymes, the β-glucosidase and the accessory enzymes to obtain the enzyme and (d) optionally, drying the suspension to obtain a mixture of dried enzymes. Preferentially, the lineage of T. reesei RUT C30 and lineage of A. awamori B.361U2/1 are used for obtaining the mixture of cellulase enzymes, the β-glucosidase and the accessory enzyme, the concentration of the supernatant suspension is preferentially done using a mild concentration process, such as, for example, ultrafiltration or spray drying.

A fifth embodiment of the invention refers to a production process for alcohol from a biomass, comprising the stages of: (a) the pretreatment of the lignocellulosic material; (b) the enzymatic hydrolysis of the material treated in stage (a) with a composition of enzymes substantially comprised of cellulases obtained by fermentation with a lineage of T. reesei unaffected by catabolic repression and cellulases, β-glucosidase and accessory enzymes obtained by fermentation with a lineage of A. awamori having a high capability for the production of accessory enzymes, including pectinase, xylanases and feruloyl esterases; optionally, a composition of enzymes substantially comprised of cellulases, hemicellulases and β-glucosidase obtained by fermentation with a lineage of T. reesei unaffected by the effect of catabolic repression and cultivated in a buffer medium with a pH of 6.0 and (c) alcoholic fermentation of the glucose obtained in stage (b) to obtain alcohol. Preferentially, the pretreatment of the lignocellulosic material is done by steam explosion, optionally preceded by a compost period, with the intent of altering the lignocellulose structure overall and the native interactions occurring between the cellulose, hemicelluloses, other polysaccharides and the lignin so as to reduce the biomass's recalcitrance to enzymatic hydrolysis. This process also serves to facilitate the degradation of the hemicelluloses and the separation of the lignin. Furthermore, preferentially, the pretreatment includes at least one washing operation of the exploded lignocellulosic material. Preferentially, the alcoholic fermentation of the glucose obtained in stage (b) is performed with the microorganism Saccharomyces cerevisae. Brief Description of the Figures

Figure 1 illustrates the comparison between the effects of the mixtures of the enzymes produced from the lineages Trichoderma reesei and Aspergillus awamori used in the present invention for the enzymatic hydrolysis of untreated and steam explosion treated sugar cane bagasse.

Figure 2 illustrates the performance comparison between the commercial enzyme (a Genencor preparation) and the mixtures of enzymes produced from the lineages Trichoderma reesei and Aspergillus awamori used in the present invention, using the same concentration of FPAse

(10 FPU/g) activity for the enzymatic hydrolysis of sugar cane bagasse pretreated by steam explosion.

Figure 3 illustrates the operational stability comparison between the enzymatic mixtures produced from the lineages Trichoderma reesei and Aspergillus awamori used in the present invention and the commercial cellulases (a

Genencor preparation) during hydrolysis.

Figure 4 illustrates, graphically, the synergism of the different enzymatic activities in mixtures of different proportions of the enzymes produced from the lineages

Trichoderma reesei and Aspergillus awamori used in the present invention, through determination of CMCase

(carboxymethylcellulose activity - CMC) , FPase (paper filter activity - FPU) , β-glucosidase (cellobiose activity

- BGU) and xylanase (xylane activity) activity in the two enzymatic preparations, separately and after mixture in different proportions. Figure 5 illustrates, graphically, the enzymatic hydrolysis of sugar cane bagasse pretreated by steam explosion in the presence of a weak acid catalyst, using the enzymatic mixture produced from T. reesei RUT C30 in a medium with natural pH and in buffer medium with pH 6.0.

Figure 6 shows the zymogram (eletrophoresis of proteins in gel identifying the proteic bands with enzymatic activity) corresponding to bands with CMCase activity present in the supernatant of the T. reesei RUT C30 culture, cultivated in a medium containing wheat bran. The values indicate the quantity of enzymatic units (in IU) applied to each well.

Figure 7 shows the zymogram with proteic bands with with CMCase activity: channel 1; the bands present in commercial cellulase diluted 1000 times (CMCase: 4 IU/ml); channel 2; those present in the supernatant of the Aspergillus awamori culture (CMCase: 1.46 IU/ml); channel 5; those present in the supernatant of the T. reesei culture RUT C30 (CMCase: 6.59 IU/ml) and, channel 6; the same supernatant diluted 10 times; channel 3; those present in the mixture of supernatants from the T. reesei RUT C30 and Aspergillus awamori cultures (CMCase: 6.59 IU/ml) and, channel 4; the same mixture diluted 10 times.

Figure 8 presents several kinetics experiments for the enzymatic hydrolysis of pretreated sugar cane bagasse, comparing the glucose production levels produced by different enzyme preparations with the same charge of FPAse (10 UI/g of bagasse) activity, namely: (a) commercial cellulase, (b) supernatant of T. reesei RUT C30 culture, containing celullases and other enzymes, (c) supernatant of

A. awamori B.361U2/1 culture containing cellulases, β- glucosidase and accessory enzymes, (d) mixtures of the supernatants of the T. reesei RUT C30 and A. awamori B.361U2/1 cultures in proportins of: (i) 25% of the enzymatic supernatant (cellulases) of T. reesei RUT C30 +

75% of the enzymatic supernatant A. awamori B.361U2/1

(cellulases, β-glucosidase and accessory enzymes); (ii) 50% of the enzymatic supernatant of T. reesei RUT C30 + 50% of the enzymatic supernatant of A. awamori B.361U2/1; and

(iii) 75% of the enzymatic supernatant of T. reesei RUT C30

+ 25% of the enzymatic supernatant of A. awamori B.361U2/1.

Figure 9 presents photographs of the original bagasse, the ground bagasse, the bagasse treated by steam explosion, the glucose syrup with a concentration of 65 g/1 (enzymatic hydrolysate) and of the lignin residue.

Detailed Description of the Invention

The first enzyme composition of the present invention has the primary characteristic the synergistic effect resulting from the association of enzymes obtained by fermentation with a lineage of T. reesei unaffected by catabolic repression such as, for example, the lineage T. reesei RUT C30, with enzymes obtained by fermentation with a lineage of A. awamori having a high capability for the production of β-glucosidase and accessory enzymes, such as, for example, the lineage A. awamori B.361U2/1. Although the enzymes produced by this lineage must be concentrated in order to individually attack the cellulose of the pretreated biomass, they provide an important synergic action compared to the cellulases produced from T. reesei RUT C30.

Until recently, all research in relation to the enzymatic hydrolysis of lignocellulosic material was directed at microorganisms having a high cellulase production capacity, Therefore, the fungus Trichoderma reesei was, since its discovery, considered highly efficient in the production of cellulases for degrading lignocellulosic material, with the mutant lineage T. reesei RUT C30 being considered as one of the most important for the degradation of this type of material. However, due to limitations related to glucose yield, the enzymatic preparations based only on the cellulase obtained from T. reesei did not fulfil requirements due to the low levels of β-glucosidase presented by fermentation with this fungus. Thus, the next step in the search for the increased efficiency of enzymatic hydrolysis was to combine enzymes from different microorganisms so as to obtain balanced preparations for the necessary enzymatic activity instead of just exo- and endo-glucanases . Until the present invention, the combination considered optimal was of enzymes obtained by fermentation with Trichoderma reesei, with enzymes obtained by fermentation with Aspergillus niger or its mutants, such as, for example, the mixture of the Celluclast® product with the Novozym 188® product.

Surprisingly, it has now been ascertained that the performance of mixtures for the enzymatic hydrolysis of lignocellulosic materials is enhanced when the mixture of enzymes also incorporates, together with the majoritarian cellulolytic enzymes (endoglucanases, exoglucanases) , β- glucosidase, accessory enzymes selected from the group consisting of xylanases, feruloyl esterases, acetylesterases, β-xylosidases, galactomannanases, glucomannanases, polygalacturonases, glucoamylases, amylogalactosidases and tannases, amongst others. Preferentially, in accordance with the present invention, the accessory enzyme is at least one of xylanases and esterases, more preferentially, the accessory enzyme is at least one of xylanases and feruloyl esterases.

Without intending to remain restricted to a theoretical explanation, the accessory enzymes (mainly the xylanases and feruloyl esterases - E.C.3.1.1.73 and E. C.3.1.1.72, respectively), amongst other enzymes, are considered to "clean" the microfibrils of cellulose and make them vulnerable to attack by the exoglucanases and endoglucanases (majoritarian cellulases). The feruloyl esterase enzymes cleave the bond between the lignin and the hemicelluloses (-C-O-C- link) making the lignin "unstick" from the cellulose fibrils and thus making an opening for an attack by cellulases. Furthermore, the lineage of A. awamori such as, for example, A. awamori B.361U2/1, has a high production capacity for accessory enzymes such as xylanases, feruloyl esterases and pectinases and also produces high levels of β-glucosidase that hydrolises cellobiose into glucose.

The synergism observed between the enzymes produced by T. reesei and A. awamori may also be related to the new bands with cellulase activity and reduced molecular masses present in channels 3 and 4 of the zymogram of Figure 7. These new bands may be the result of the proteolythic processing of the pre-existent enzymes.

The enzyme compositions of the present invention comprise (a) cellullolytic enzymes obtained by from a lineage of T. reesei unaffected by catabolic repression;

(b) cellullolytic, hemicellullolytic and accessory enzymes obtained from a lineage of A. awamori having a high production capacity for β-glucosidase and the accessory enzymes, xylanase and feruloyl esterase; (c) furthermore, at least one molecular species selected from the group of enzymes and/or metabolites showing CMCase activity, with said molecular species being present in the supernatants of the T. reesei and/or A. awamori cultures as shown by the zymogram in Figure 7; and (d) an appropriate vehicle to maintain the stability and activity of the of said enzymes in the enzymatic hydrolysis of lignocellulosic material.

Preferentially, the lineage of T. reesei unaffected by catabolic repression used for obtaining the cellulolytic enzymes of the compositions of the present invention is the lineage T. reesei RUT C30 that is acknowledged as producing cellulases even in the presence of glucose and, therefore, remains unaffected by catabolic repression. Said cellulolytic enzymes (cellulases) are obtained by fermentation with T. reesei in conditions known in the art such as, for example, submerged fermentation, whereby the supernatant obtained is then concentrated by a mild method such as, for example, by ultrafiltration, so as to maintain the stability and activity of the cellulases. Alternatively, the supernatant is submitted to a mild drying process such as, for example, liophylisation, spray drying or the similar, so as to obtain solid cellulases, hemicellulases, β-glucosidase and accessory enzymes with preserved enzymatic activity.

Preferentially, furthermore, the lineage of A. awamori Aspergillus awamori having a high production capacity for β-glucosidase and the accessory enzymes, xylanase and feruloyl esterase is the lineage A. awamori B.361U2/1. The cellullolytic, hemicellullolytic, β-glucosidase and accessory enzymes of the composition of the present invention are obtained by fermentation such as, for example, submerged fermentation with said lineage of A. awamori . The supernatant obtained may also be concentrated or dried in the same conditions used for the treatment of the supernatant from the fermentation with T. reesei. Alternatively, the supernatants from the fermentation with T. reesei and A. awamori may be combined and then concentrated or dried by the methods described above.

)^' The second enzyme composition of the present invention has the primary characteristic of being produced from fermentation with a lineage of Trichoderma reesei unaffected by catabolic repression such as, for example, the lineage Trichoderma reesei RUT C30, in a buffer medium with pH 6.0, that provides an enzyme composition containing cellulases, hemicellulases and β-glucosidase, with an exoglucanase/β-glucosidase ratio close to 1.

The enzyme compositions of the invention may be in the form of an aqueous suspension, preferentially for immediate use after having being obtained or, alternatively, may be in a dry form. In dry form, the compositions of the invention may comprise, together with the enzymes, an appropriate vehicle that maintains their stability and activity.

Examples of such vehicles may be: neutral salts, such as ammonium sulphate, potassium sulphate or sodium chloride (for example, at 20%) that prevent microbial growth due by osmotic effect. Polyols of low molecular weight such as glycerol, sorbitol and mannitol that stabilise the enzymes and also repress microbial growth due to reduced water activity. In another embodiment, the various enzymes may be maintained in lyophilised form over long periods of time in the presence of stabilizers such as, for example, salts, carbohydrates or inert proteins, predominantly bovine serum albumin (BSA) . Other substances that may be added to the enzyme preparation of the invention as stabilisers include: substrates, thiols to maintain the reducing environment, antibiotics, benzoic acid esters as preservatives for liquid enzymatic preparations, enzyme contaminant inhibitors and chelating agents. The additives must be compatible with the final use of the enzyme in question.

The enzyme compositions of the invention are particularly useful for the enzymatic hydrolysis of lignocellulosic material, which is potentialised by the synergy between the cellulases of T. reesei and the cellulases, hemicellulases, β-glucosidase and accessory enzymes of A. awamori. This synergy is primarily the result of the activity of the accessory enzymes and more especially of the xylanases and feruloyl esterases on the hemicellulose and the bonds between hemicelluloses and lignin as well as between other polysaccharides and lignin, weakening the inter- and intramolecular bonds of these components and, consequently, making the cellulose available for enzymatic hydrolysis. It is then the activity of the cellulases and hemicellulase that breaks the glucosidic bonds and degrades these substances into glucose and xylose.

The present invention also contemplates a production process for cellulolytic and hemicellulolytic enzymes comprising the stages of: (a) culturing, separately, in an appropriate medium, a lineage of T. reesei unaffected by catabolic repression, optionally in a buffer medium of pH 6.0 and a lineage of A. awamori having a high capacity for the production of β-glucosidase and accessory enzymes, including xylanases and feruloyl esterases; (b) separating the supernatants of each culture of T. reesei and A. awamori containing the cellulase enzymes, the β-glucosidase and the accessory enzymes; (c) optionally, concentrating and combining the supernatants containing said cellulase enzymes, the β-glucosidase and the accessory enzymes to obtain the enzyme mixture and (d) optionally, drying the suspension to obtain a mixture of dried enzymes.

The appropriate culture medium for the fermentation of T. reesei and A. awamori, may be those known in the art such as, for example, Mandels' medium (refer to MANDELS, M. & WEBER, J. (1969) "Production of cellulases" Adv. Chem. Ser. 95: 391-414), the modified Mandels' medium or Breccia medium (Breccia et al., 1995). It is also possible to use media containing potentially inductor substances or that encourage the production of the enzymes of interest, such as lactose and wheat bran, that are the media used in the present invention. Preferentially, the lineage T. reesei RUT C30 and the lineage A. awamori B.361U2/1 are used to obtain the mixture of celullases, β-glucosidase and the accessory enzymes. The concentration and drying of the supernatant suspension is preferentially done using mild processes such as, for example, ultrafiltration, lyophilisation or spray drying, amongst others.

The present invention also includes a production process of alcohol from biomass, comprising the steps of:

(a) the pretreatment of the lignocellulosic material; (b) the enzymatic hydrolysis of the material treated in step (a) with compositions of enzymes substantially comprised of cellulases, hemicellulases and, depending on culture conditions, β-glucosidase at high levels, obtained by fermentation with a lineage of T. reesei unaffected by catabolic repression and cellulases, β-glucosidase and accessory enzymes obtained by fermentation with a lineage of A. awamori having a high production capacity for accessory enzymes, including xylanases and feruloyl esterases; and (c) the alcoholic fermentation of the glucose obtained in step (b) to obtain alcohol.

The methods for pretreating lignocellulosic material for subsequent enzymatic hydrolyis are broadly known in the art. These include: steam explosion (or autohydrolysis) , steam explosion in the presence of weak acid, grinding, explosion of the fibres by ammonia, explosion with CO2, amongst others (refer to Sun Ye, (2002) above, for more details). Preferentially, in accordance with the present invention, the steam explosion method is used and this may be preceded by a "composting" step that encourages the growth of microorganisms within the bagasse stored in the open, with this growth being further encouraged by the residual saccharose and producing CO₂ which is transformed into carbonic acid and thus constitutes a natural pretreatment by CO2. More preferentially, the exploded lignocellulosic material is washed following the pretreatment by steam explosion. This procedure not only facilitates hydrolysis of the hemicellulose but also helps separate the lignin in the subsequent enzymatic hydrolysis step.

In accordance with the present invention, the enzymatic hydrolysis is performed by addition of the enzyme compositions of the present invention to pretreated lignocellulosic material to obtain glucose which is then submitted to alcoholic fermentation with Saccharomyces cerevisae. The conditions used for the enzymatic hydrolysis and alcoholic fermentation are known in the art (refer, for example to US 5628830; EP 0448430; Sun Ye (2002), above and Sendelius J (2005), above).

It must be understood that the examples and embodiments described herein are merely for illustrative purposes and, in the light of these, various modifications or alterations may be considered by those skilled in the art whereby these should, therefore, be included within the spirit and range of this description and the scope of the accompanying claims.

EXAMPLES

Example 1: Fermentation for the Production of Cellulases

The propagation of microorganisms for obtaining spores was performed by culture in solid medium using PDA (Potato Dextrose Agar) culture. Suspensions of spores were obtained through the addition of saline solution (0.85% w/v) and scraping the sporulated plates. The suspensions obtained were centrifuged and preserved in solutions of glycerol at 10% w/v at -20° C.

Submerged fermentation - fermentation conditions and culture media:

For the preparation of the pre-inoculant , 3 ml of the spore suspension was inoculated in 300 ml of Mandels' medium or in Breccia medium (refer to Tables Ia and Ib) , contained in 1000 ml Erlenmeyer flasks. Variations of the

Mandels' medium, principally in relation to sources of nitrogen are included in the scope of the present invention. The culture was maintained at 30° C and 200 RPM for four days.

The cultures intended for the production of cellulases were done in 1000 ml Erlenmeyer flasks, containing 300 ml of modified Mandels' or Breccia medium (Table Ia and Ib) , (refer to MANDELS & WEBER, (1969), above) or its variations. After sterilisation, the culture- media were inoculated with 30 ml of pre-inoculant. The initial pH of all the media were adjusted to 4.8 and monitored during the entire culture. The flasks were incubated at 30° C and 200 RPM, for a maximum period of nine days. The equipment used was of the New Brunswick Scientific brand, Innova 4340 model .

Table Ia: Mandels' Medium (1); Modified Mandels' Medium (2)

Reagent (1) Concentration (2) Concentration

Urea 0.3 g/1 0.3 g/i (NH₄) ₂SO₄ 1.4 g/1 1.4 g/i KH₂PO₄ 2.0 g/1 2.0 g/i CaCl₂ 0.3 g/1 0.3 g/i MgSO₄ 7 H₂O 0.3 g/1 0.3 g/i FeSO₄ 7 H₂O 5 mg/1 5 mg/1 CoCl₂ 6 H₂O 20 mg/1 20 mg/1 MnSO₄ 4 H₂O 1.6 mg/1 1.6 mg/1 ZnSO₄ 7 H₂O 1.4 mg/1 1.4 mg/1 Peptone 0.75 g/1

Cellulose (Avicel^' 7.5 g/1 Yeast Extract 0.25 g/1 6.0 g/i Corn Steep Liquor 0.6^ \ w/v Carbon source 30 g/i

The cellulose was substituted for the desired carbon source in the modified Mandels' medium, at a concentration of 30 g/1 (3% w/v) . The carbon sources selected for assessment were wheat bran and lactose. A vitamin an nitrogen source, corn steep liquor and/or yeast extract, were also added to the medium. Table Ib: Breccia Medium (Breccia et al. , 1995]

Reagent Concentration

Sodium nitrate 1.2 g/1

KH₂PO₄ 3.0 g/1

K₂HPO₄ 6.0 g/1

CaCl₂.2H₂O 0.05 g/1

MgSO₄ ^• 7 H₂O 0.2 g/1

MnSO₄.7H₂O 10 mg/1

ZnSO₄.7H₂O 1 mg/1

FeSO₄.7H₂O 5 mg/1

CoCl₂.6H₂O 20 mg/1

Yeast extract 12 g/1

Carbon source 30 g/1

Follow-up of the fermentation:

Samples of the supernatant from the submerged fermentation were obtained daily (not exceeding a total of 20% of the initial volume) and centrifuged at 3000 RPM during 15 minutes in a Beckman Coulter Allegra 6R centrifuge.

The supernatants were used to determine the concentration of reducing sugars, the concentration of the enzymes of the cellulolytic complex (CMCase, FPase and β- glucosidase) , of accessory enzymes and of pH. pH was determined in Beckman φ390 apparatus.

Example 2: Enzymatic tests:

The supernatants from the fermentation were used to determine CMCase, FPase and β-glucosidase activity and, optionally, also xylanase activity. All tests were performed in duplicate. One unit of emzymatic activity is defined by the production of one μmol of product/minute.

CMCase Activity:

This was determined in accordance with the standard methodology described by IUPAC (refer to Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem; 59: 257-268). The method is based on dosing the concentration of reducing sugar released during the degradation of carboxymethylcellulose (CMC) .

The reaction medium is formed of 3.0 ml of a CMC solution at 4% w/v in a buffer of sodium citrate 50 mM at pH 4.8 and 3.0 ml of supernatant from the fermentation, containing the enzymes of the invention (diluted in a buffer of sodium citrate 50 mM at pH 4.8, when necessary). The reaction mixture is then incubated at 50° C, during 10 minutes, under agitation. Aliquots of 0.5 ml were removed at 2 minute intervals to determine reducing sugar concentration. The enzymatic reaction of the aliquots was interrupted by the immediate addition of these samples to a test tube containing 0.5 ml of DNS (3.5-dinitrosalicylic acid) . The DNS reagent ends enzymatic reaction and allows dosing the concentration of the reducing sugar produced by the enzymatic activity (refer to Miller GL (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Biochem; 31: 426-428) .

FPase Activity:

This was determined in accordance with the standard methodology described by IUPAC (refer to Ghose TK (1987), above) . The method is based on dosing the concentration of reducing sugar released during the degradation of a strip of filter paper (FP) .

The reaction medium is formed of 0.5 ml of supernatant from the fermentation (diluted in a buffer of sodium citrate 50 mM at pH 4.8, when necessary) containing the enzymes of the invention, 1.0 ml of a buffer of sodium citrate 50 mM at pH 4.8 and a strip of Whatman n. 1 filter paper measuring 1.0 cm X 6.0 cm (approximately 50 mg) . The reaction mixture is then incubated at 50° C, during 60 minutes, under agitation. The enzymatic reaction was interrupted by the immediate addition of 0.5 ml of the reaction mixture to a test tube containing 0.5 ml of DNS. The concentration of the reducing sugar was the determined.

β-glucosidase Activity:

This was determined in accordance with the standard methodology described by IUPAC (Ghose, 1987). The essay is based on dosing the concentration of the glucose released by the enzymatic hydrolysis of cellobiose.

The reaction medium is formed of 1.0 ml of supernatant from the fermentation, containing the enzymes of the invention (diluted in a buffer of sodium citrate 50 mM at pH 4.8, when necessary) and 1.0 ml of substrate solution

(cellobiose 15 mM in a buffer solution of sodium citrate 50 mM at pH 4.8). The mixture is then incubated at 50° C, during 30 minutes, under agitation. The enzymatic reaction was interrupted by immersing the tubes in boiling water for 5 minutes. Xylanase Activity:

Xylanase activity is based on the determination of the concentration of reducing sugars released during the degradation of xylane.

The reaction medium is formed of 0.5 ml of the supernatant from fermentation (diluted in a buffer of sodium citrate 50 mM at pH 4.8, when necessary) containing the enzymes of the invention and 1.0 ml of xylane solution l%^(w/v) (of the oat spelt type, Sigma code X-0627) . The reaction mixture is then incubated at 50° C, during 5 minutes. The reaction is interrupted by the immediate addition of 0.5 ml of the reaction mixture to a test tube containing 0.5 ml of DNS. The concentration of the reducing sugar is then determined.

Example 3: Analytic tests:

Dosage of the Reducing Sugar Concentration

The concentration of reducing sugars was determined according to Miller (1959) . In this method, test tubes containing 0.5 ml of the reaction mixture and 0.5 ml of DNS are placed in a hot water bath at 100° C during 5 minutes. Immediately afterwards, the mixture is diluted with 6.5 ml of distilled water. The reading is performed by spectrophotometer at 540 nm. The absorbancies obtained are transformed into the concentrations of reducing sugar by means of a standard glucose curve.

Dosage of the Glucose Concentration

The concentration of glucose was determined using a glucose concentration analysis kit based on the reaction of glucose oxidase and peroxidase enzymes. In this process, the glucose is transformed into gluconic acid and hydrogen peroxide through the action of the glucose oxidase. The hydrogem peroxide formed together with 4-aminoantipyrine and phenol is transformed through the action of peroxidise into quinoneimine, a colouring compound. At the end of the reaction, each mol of glucose is transformed into a mol of quinoneimine. For such, 10 μL of the reaction mixture are pipetted to test tubes containing 1 ml of the glucose oxydase and peroxidise reagent (BioSystems®) . The tubes are incubated for 10 minutes at ambient temperature (25° C) , during which time the enzymatic reaction, occurs. The resulting colour is read by spectrophotometer at 500 nm.

Dosage of Total Sugars and Ethanol

The concentration of total sugar was determined by initially performing chemical hydrolysis with HCl 2N at 100° C/10 min, which was then neutralised with NaOH. In this manner the non-reducing sugars were converted into reducing sugars. The dosage of the sugar was then performed by the dinitrosalicylate (DNS) method (Miller, 1959) . The ethanol concentration in the fermentation must was determined by the alcohol dehydrogenase enzymatic method (refer to Bernt, E., Gutmann, I., 1974. Determination with alcohol Dehydrogenase and NAD, in: Hans, U. B. (Ed.), Methods of Enzymatic Analysis. Academic Press Inc., New York, pp. 1499-1502) . This test is based on the formation of NADH measured by the increase of the absorbancy at 340 nm, which is proportional to the concentration of ethanol. The reaction medium contains: 3.5 ml of glycine-NaOH buffer 100 mM at pH 9.0, semicarbazide 75 mM; 0.2 ml NAD⁺ (16 mM) ; 0.1 ml alcohol dehydrogenase from S. cerevisiae (120 U) in a phosphate buffer 20 mM at pH 9.0 containing BSA 0.1%. This mixture is used for zeroing the spectrophotometer. After the addition of 0.2 ml of fermented must, the mixture was homogenised and absorbancy at 340 nm was monitored until there was no increase in absorbancy. The fermented must is previously diluted to an approximate ethanol content of 0.1%.

Assessment of Saccharomyces cerevisiae cell growth in the enzymatic hydrolysates of sugar cane bagasse

The assessment of the cellular concentration was determined by measuring absorbancy at 570 nm of a cell suspension converted into a cell concentration (by mg of dry weight/ml) . The conversion factor for the dry weight was calculated by filtering an adequate volume of the cell suspension in a Millipore (0.45Dm) that was then placed in an oven at 80° C until attaining constant weight.

Example 4 : Production of Cellulases by Trichoderma reesei RUT C30

The lineage of T. reesei RUT C30 (ATCC 56765) is one of the most used in scientific research development due to its availability in culture collections and because its use is not restricted by property rights, which are common for other lineages of T. reesei. However, the fact that the lineage of T. reesei RUT C30 is capable of producing cellulases even in the presence of glucose and is unaffected by catabolic repression is of paramount importance to the embodiment of the present invention. Compositions of the media tested, in relation to the main sources of carbon or nitrogen sources, for production in an agitated flask

- 3% Wheat bran + 0.6% corn steep liquor + 0.6% yeast extract

- 3% Wheat bran + 0.6% corn steep liquor

- 3% Wheat bran + 1.2% corn steep liquor

- 3% DDG + 0.6% corn steep liquor + 0.6% yeast extract

- 3% Sugar cane bagasse + 0.6% corn steep liquor + 0.6% yeast extract

- 3% Lactose + 0.6% corn steep liquor + 0.6% yeast extract

- 3% Lactose + 1.2% corn steep liquor.

- 3% Lactose + 0.6% corn steep liquor + 0.6% yeast extract in a buffer medium at pH 6.0 with a sodium phosphate buffer 100 mM.

Production in an agitated flask

The maximum levels of CMCase, FPase and beta- glucosidase activity obtained for T. reesei RUT C30 in agitated flasks are shown in Table 2. It must be stressed that not all fermentation were monitored up to the ninth day. As such, the true production peaks may occur on other days.

Table 2: Data of maximum activity of the cellulases and Beta-glucosidase in the supernatant of the T. reesei RUT C30 culture.

Key to the abbreviations on Table 2 : WB = Wheat bran; CSL = corn steep liquor; YE = yeast extract; DDG = dried distilled grains; SCB=sugar cane bagasse; LAC = lactose.

The production of β-glucosidase is expressively greater when the pH is maintained around 6.0, while the production of exoglucanases and endoglucanases is not particularly effected by broader variations of pH. Therefore, a more balanced enzymatic mixture in relation to the cellulolytic activity (exoglucanase FPase/β- glucosidase) may be obtained through controlling the pH of the medium by the addition of sodium phosphate buffer 100 mM. Furthermore, xylanase activity levels of 20 IU/ml were also obtained in this medium. Example 5: Production of Cellulases by Aspergillus awamori B.361U2/1 (Commonwealth Mycological Institute - UK)

This lineage is a is a sequential mutant of NRRL 3112, which is a lineage classified as Aspergillus niger. The fungus Aspergillus niger are known for their greater capacity of producing β-glucosidase, a necessary enzyme generally produced in smaller quantity by the fungus of genus Trichoderma.

Compositions of the media tested - 3% Wheat bran

- 3% Wheat bran + 0.7% yeast extract

- 3% Wheat bran + 0.3% corn steep liquor

- 3% Wheat bran + 1.2% corn steep liquor

- 3% DDG - 3% DDG + 0.7% yeast extract

- 3% DDG + 1.2% corn steep liquor

- 3% Sugar cane bagasse + 1.2% corn steep liquor

- 3% Wheat bran + 0.7% yeast extract

The maximum levels of CMCase, FPase and beta- glucosidase activity obtained by fermentation with A. awamori in agitated flasks are shown in Table 3. The highest CMCase activity attained was 10.0 IU/ml in a medium containing 3% wheat bran and 0.7% yeast extract. The greatest production of β-glucosidase attained was in a medium containing 3% DDG and 0.7% yeast extract, with the rate being of 2.9 IU/ml. The greatest FPase activity attained was of only 0.18 IU/ml. Table 3. Concentration peaks of cellulases in the supernatants of A. awamori B.361U2/1 cultures

Medium CMCase Day FPase Day β-glucosidase Day Xylanase

(IU/ml) (IU/ml) (IU/ml) (IU/ml)

3% WB 7.8 4th * ND - 2.0 4th* ND

3% WB + 0.7% 10.0 4th * ND - 1.0 4th * ND YE

3% WB + 0.3% 5.9 4th * ND - 0.9 4th * ND CSL

3% WB + 1.2% 4.7 7th ND - 0.8 7th ND CSL

3% DDG 5.8 4th * ND — 1.6 4th * ND

3% DDG + 6.8 4th * ND - 2.9 4th * ND 0.7% YE

3% DDG + 4.0 7th ND - 1.1 7th ND 1.2% CSL

3% SCB + 0.4 7th 0.18 7th 0.3 7th ND 1.2% CSL

3% WB + 1.2% 2.5 7th 0.2 7th 15 7th 25 YE

*only dosed on the fourth day; ND= Not determined.

Key to the abbreviations on Table 3 : WB = wheat bran; CST = corn steep liquor; YE = yeast extract; DDG = dried distilled grains; SCB = sugar cane bagasse.

Example 6: Separation and Concentration of the Cellulolytic Enzymes

After production of the enzymes, the supernatants containing the cellulolytic enzymes, the β-glucosidase and the accessory enzymes were separated from the microorganisms by vacuum filtration using a glass-fibre micro filter. The enzymatic mixtures were prepared from these supernatants and enzyme activity was measured.

The enzymatic hydrolysis experiments used 10 FPU/g of biomass and the enzymatic mixture had to be initially concentrated by ultrafiltration using a 30.000 cut-off membrane (Millipore®) retaining proteins having a molecular weight of over 30.000 Daltons, with these proteins remaining in the concentrate. The ultrafiltration experiments used the AMICON system, conventional filtration model 8400 (circular module with a membrane area of

41.8 cm²). Filtration was performed in batches under mild agitation. All experiments used a pressure of 2 bar

(compressed air line) . This procedure enables the enzymatic mixture to be concentrated up to 25 times.

Example 7: Stability of the FPase, β-glucosidase , CMCase and xylanase enzymes present in the supernatants of fermentation with T. reesei RUT C30 and the CMCase xylanase enzymes resulting from fermentation with A. awamori B.361U2/1

The thermal stability of the FPase and β-glucosidase enzymes present in the supernatant of T. reesei was assessed at 50° C, 60° C and 70° C for a period of up to 24 hrs. The results are shown in Table 4, below.

Table 4: Thermostability of the FPase and β-glucosidase activity of the raw extracts of T. reesei RUT C30 incubated at 50° C, 60° C and 70° C.

Activity Activity retained (%) after incubation period

FPase - 50° C 85 ^ξs after 24 hours

FPase - 60° C 65% after 24 hours

FPase - 70° C Inactive after 30 minutes β-glucosidase - 50° C 85% after 24 hours β-glucosidase - 60° C 22% after 24 hours β-glucosidase - 70° C 20% after 30 minutes and inactive after 1 hour The FPase e β-glucosidase activity proved to be stable when incubated at a temperature of 50° C and maintained over 80% of initial activity. When the temperature was increased to 60° C, FPase maintained 25% of initial activity but β-glucosidase activity was reduced to only 22%. The enzymes proved to be unstable at 70° C, with reduced or no activity after 30 minutes of incubation. The thermal stability of CMCase and xylanase enzymes was assessed at 50° C and 60° C, both with and without cysteine and tryptophane. The results are shown in Tables 5, 6 and 7, below.

Table 5: Thermostability of the xylanase and CMCase activity of the raw extracts of T. reesei RUT C30 and A. awamori incubated at 50° C and 60° C.

Enzyme Activity Half-life (h)

T. reesei RUT C30 Xylase - 50° C Over 288 h*

A. awamori Xylanase - 50° C Over 288 h*

T. reesei RUT C30 Xylanase - 60° C 46

A. awamori Xylanase - 60° C 40 min

T. reesei RUT C30 CMCase - 50° C 96

A. awamori CMCase - 50° C 96

T. reesei RUT C30 CMCase - 60° C 96

A. awamori CMCase - 6O⁰C 24

* the enzyme maintained 100% activity after 288 hrs of incubation Table 6: Thermostability of the xylanase and CMCase activity of the raw extracts of T. reesei RUT C30 and A. awamori incubated at 50° C and 60° C in the presence of L- cysteine .

Enzyme Activity Half-life (h)

T. reesei RUT C30 Xylanase - 50° C 7

A. awamori Xylanase - 50° C 24

T. reesei RUT C30 Xylanase - 60° C 7

A. awamori Xylanase - 60° C 5

T. reesei RUT C30 CMCase - 50° C Over 144 h*

A. awamori CMCase - 50° C 18

T. reesei RUT C30 CMCase - 60° C 96

A. awamori CMCase - 60° C 7

* the enzyme maintained 100% activity after 144 hrs of incubation

Table 7: Thermostability of the xylanase and CMCase activity of the raw extracts of T. reesei RUT C30 and A. awamori incubated at 50° C and 60° C in the presence of L- tryptophane .

Enzyme Activity Half-life (h)

T. reesei RUT C30 Xylanase - 50° C 216

A. awamori Xylanase - 50° C 192

T. reesei RUT C30 Xylanase - 60° C 4

A. awamori Xylanase - 60° C 4

T. reesei RUT C30 CMCase - 50° C 216

A. awamori CMCase - 50° C 216

T. reesei RUT C30 CMCase - 60° C 16

A. awamori CMCase - 60° C 24

The activity of xylanase and CMCase was stable at 50° C and 60° C, with the exception of the xylanase of A. awamori at 60° C, and the need to add substances for the stabilisation of the activity was not verified. The use of cysteine and tryptophane did, however, present an advantage for the half-life of xylanase and CMCase of A. awamori .

Example 8: Enzymatic Hydrolysis of Sugar Cane Bagasse

The sugar cane bagasse used for enzymatic hydrolysis was: (i) provided by the Centro de Tecnologia Canavieira

(CTC) [Cane Cultivation Technology Centre] and pretreated by sieving by Tyler 20 which resulted in a bagasse with a particle size of under 0.84 mm, known as ground bagasse or

(ii) pretreated by steam explosion (origin: Vale do Rosario mill) and washed with water.

The steam exploded bagasse was washed with warm water aiming to remove possible inhibitors for the enzymatic hydrolysis and fermentation stages. The washed bagasse was then submitted to enzymatic hydrolysis using the enzyme composition of the present invention.

The purpose of these two pretreatments was to compare the performance of the subsequent enzymatic hydrolysis using the two methods.

The enzymatic hydrolysis tests were performed using the ground bagasse and the exploded bagasse under the following conditions: the enzyme preparations of the following invention, optionally concentrated, were added to the dry bagasse in a specified concentration. The temperature used for the hydrolysis tests was 50° C under agitation at 200 RPM with 1 ml samples being collected at various times over 72 hrs for the analysis of sugar concentration. Figure 1 shows the advantage of pretreatment by steam explosion for the optimisation of the enzymatic hydrolysis following the pretreament.

Example 9: Enzymatic Hydrolysis with Sugar Cane Bagasse

Enzymatic hydrolysis experiments were performed with bagasse pretreated by steam explosion, washing and drying, using the following enzyme compositions: (a) a composition of the present invention corresponding to a mixture of supernatants from fermentation with T. reesei RUT C30 and fermentation with A. awamori B.361U2/1; (c) product acquired from the GENENCOR corporation (Spezyme CP, containing CMCase, FPase and B-glucosidase activity) .

Table 8 shows the characteristics of the samples tested for CMCase, FPase and B-glucosidase activity

Table 8: Characterisation of the enzymatic preparations used for the hydrolysis of sugar cane bagasse

Experiments were performed to provide concentrated glucose syrups, using both the product acquired from the GENENCOR corporation and the enzyme compositions of the present invention, with both enzyme mixtures having the same activity of 10 FPU/g of sugar cane bagasse. The concentration of the steam pretreated bagasse used was of 130 g/1. Figure 2 shows that the enzymatic hydrolysis of the bagasse using the composition of the present invention demonstrated better performance (obtaining 65 g/1 of glucose) . This result seems to indicate that the composition of the present invention contains enzymes with a greater specificity in relation to the steam pretreated bagasse when compared to the commercially available product .

Example 10: Assessment of the Operational Stability of the Cellulolytic enzymes during Enzymatic Hydrolysis

During the experiments performed in accordance with Example 8 also served to monitor the operational stability of the cellulolytic enzymes during the enzymatic hydrolysis of the sugar cane bagasse. The results presented in Figure 3 show that the cellulases (FPase, CMCase and β- glucosidase) are also quite stable under the operational conditions tested. Both the commercial product acquired from the GENENCOR corporation and enzyme composition of the present invention proved to be stable over the period of 72 hours in which the experiment took place.

Example 11: Synergism between the Enzymes present in the Supernatant from Fermentation with T. reesei RUT C30 and A. awamori B.361U2/1

The synergic effect of mixtures containing different proportions of supernatant from the cultures of T. reesei Rut C30 and A. awamori B.361U2/1 was evaluated for CMCase, FPase and B-glucosidase and xylanase activity. Figure 4 shows that, with exception of the xylanase activity, synergy was observed in CMCase, β-glucosidase and FPase activity, with preponderance in the two former. The synergy for CMCase activity was greatest at a ratio of 30% T. reesei and 70% A. awamori and for β-glucosidase activity at a ratio of 70% T. reesei and 30% A. awamori , with this result being representative considering the importance in obtaining glucose syrups for alcoholic fermentation and the degradation of cellobiose to prevent the inhibition of the cellulases .

Example 12: Assessment of the Performance of the Enzymatic Mixture from T. reesei RUT C30 cultivated in a Natural

Medium and in Buffer Medium on the Enzymatic Hydrolysis of Pretreated Sugar Cane Bagasse

The enzymatic hydrolysis of bagasse pretreated by steam explosion was assessed in the presence of an acid catalyser using different mixtures of supernatant from the culture of T. reesei Rut C30 in natural medium and buffer medium at pH 6.0.

As shown by Figure 5, the glucose concentration in the mixture of enzymes produced by T. reesei Rut C30 without pH control resulted in a maximum production of 3.7 g/1 of glucose and attained 25.8% of cellulose degradation. This probably indicated that the cellobiose and oligosaccharides generated by the activity of endo and exoglucanases are not being completely processed due to an insufficiency of β- glucosidase in this enzymatic preparation. When the mixture used was of enzymes produced in buffer medium, it was seen that the yield from the hydrolysis of cellulose to glucose rose to 80% and provided 11.4 g/1 of glucose. It is therefore possible to conclude that the pH control of T. reesei cultures are essential for obtaining balanced enzymatic mixtures for the hydrolysis of sugar cane bagasse .

Example 13: Zymogram of the Electrophoretic Pattern of the CMCase present in the Supernatant of the T. reesei RUT C30 Culture

The zymogram was performed with the supernatant on the eighth day of culture with T. reesei RUT C30 in a culture medium containing wheat bran. This experiment was intended to identify the protein bands corresponding to CMCase activity. The supernatant was diluted so as to be applied to the gel in preparations containing a total CMCase activity of 0.13, 0.026, 0.013 and 0.006 IU, per well. The best visualisation occurred with application of the 0.026 IU dilution. Figure 6 shows two strong bands corresponding to CMCase activity.

Example 14: Zymogram of the Electrophoretic Pattern of the CMCase present in the Supernatant of the T. reesei RUT C30 culture, in the Supernatant of the A. awamori B.361U2/1 culture and the CMCase Present in the Mixture of the Supernatants of T. reesei RUT C30 and A. awamori B.361U2/1

The zymogram, shown in Figure 7, was performed in denaturing conditions, using SDS and B-mercaptoethanol . The run was followed by a renaturation step (30 minutes) before incubating the gel at 50° C for the enzymatic hydrolysis of the CMC present in the gel. The dilutions of the GENENCOR corporation product, of the supernatant of A. awamori and the mixture of supernatants of T. reesei RUT C30 and A. awamori B.361U2/1 resulted in the separation of CMCase bands of different molecular weights. However, the dilution of the sample of the enzymes present in the supernatant of the fermentation with Trichoderma was not sufficient for separation of the proteins and did not allow visualisation of well defined bands. It was not possible to infer the approximate molecular weight of these enzymes due to the absence of the molecular weight marker. Therefore, in the case of this gel, the qualitative identification of the molecular weights was given as "low" and "high" molecular weight, using the gel centreline as a divider.

As may be seen from Figure 9, the commercial enzyme product (ENGENCOR corporation) analysed (channel 1) presented a well defined band in the upper part of the gel, with "high" molecular weight close to the weight ascertained for the enzymes of Trichoderma (channels 5 and 6) . The CMCase activity enzymes of Aspergillus awamori presented a total of four well defined bands, with both "high" and "low" molecular weights. Three new bands of "low" molecular weight were observed in the channels where the enzyme mixture of the present invention was applied. There are two possible explanations: Firstly, there was interaction between the enzymes of T. reesei RUT C30 and A. awamori , possibly either due to the cleaving of some enzymes by proteases but, nevertheless, retaining the biological activity of the enzymes or due to deglycosylation, and secondly, the supernatant of Aspergilus awamori (channel 2 - concentration of 1.46 U/ml) was more dilute than the enzyme mixture (3 - 6.59 U/ml) and it was not possible to detect bands with lower molecular weights. However, channel 4 with a charge of 0.65 U/ml allows the visualisation of seven bands.

Example 15 Assessment of the Performance of Different

Proprtions of Supernatant of T. reesei RUT C30 and A. awamori B.361U2/1 Cultures in the Enzymatic Hydrolysis od Sugar Cane Bagasse

The synergic effect of mixtures containing different proportions of supernatant from the cultures of T. reesei Rut C30 and A. awamori B.361U2/1 was evaluated in relation to their performance in the enzymatic hydrolysis of sugar cane bagasse. The hydrolyses were performed in parallel, with: (a) 100% A. awamori; (b) 100% T. reesei RUT C30; (c) 75% T. reesei RUT C30 + 25% A. awamori; (d) 50% T. reesei RUT C30 + 50% A. awamori; (e) 25% T. reesei RUT C30 + 75% A. awamori; (f) commercial product (acquired from the GENENCOR corporation) .

As can be seen from Figure 8, the glucose concentrations in the mixture containing 50% supernatant of T. reesei + 50% supernatant of A. awamori and in the mixture containing 75% supernatant of T. reesei + 25% supernatant of A. awamori presented equivalent values. This demonstrates that this mixture is ideal for obtaining the best yield.

Example 16: Alcoholic Fermentation of the Syrup obtained by the Enzymatic Hydrolysis of Steam Exploded Sugar Cane Bagasse

The hydrolysates obtained from the bagasse were first verified for possible inhibitory effect on the growth of Saccharomyces cerevisiae. The experiments used a strain isolated from baker's yeast. Results showed that the cells grew in the hydrolysates undisturbed, thus demonstrating that the syrup obtained contained no growth inhibitors. The cellular yield after 24 hrs growth in molasses and in the hydrolysate was similar (approximately 3 mg cells/ml) , using the same inoculant and total sugar concentration.

An industrial strain was then used to assess ethanol production from the hydrolysate produced by the bagasse. For purposes of comparison, diluted molasses were used, with the total sugar concentration being approximately equal to that of the hydrolysate (around 400 mM) . The results are shown in Table 9.

Table 9: Results of the ethanol production from the hydrolysate and from molasses

By the results obtained from hydrolysate, it was observed that of the 274 mM of total sugar consumed, 233 mM were of consumed glucose. This glucose came from hydrolysis of bagasse cellulose. The remaining 41 mM were hexoses, probably from hemicellulose. The conversion of the sugars consumed in ethanol during the fermentative process may be calculated from the following equation:

Conversion (%) = [Product formed/Substrate consumed] * 100

Molasses practically only contain saccharose (non reducing sugar), glucose and fructose (reducing sugars). It is acknowledged that all these sugars may be fermented to ethanol. However, it is not known what sugars are present in the hydrolysate nor the proportion of fermentable sugars. Therefore, it was decided to calculate the conversion in terms of total consumed sugar (rather than glucose) , which is appropriate since the ethanol produced in the case of molasses is also result of the saccharose and fructose presence.

According to the results, the hydrolysed must produced the largest conversion to ethanol after fermenting 24 hours (approximately 40%). The conversion to ethanol from the hydrolysate of the bagasse was approximately 2 times greater than that obtained from the cane molasses (24%), a common source for the production of ethanol in Brazilian distilleries .

Example 17: Visualisation of the sugar cane bagasse "in natura" , of the pretreated bagasse, of the lignin residue after enzymatic hydrolysis of the cellulose_^ and other polysaccharides using the mixture of supernatants from T. reesei RUT C30 and A. awamori B.361U2/1 cultures and also of the glucose syrup obtained. The photographs shown in Figure 9 serve to illustrate the visual aspect of the bagasse and other products of the enzymatic hydrolysis, sugar syrup and lignin.

All the publications and patent requests mentioned in the above description are indicative of the level of those skilled in the art relating to the present invention. All the publications and patent requests are included herein as references in the extent that each individual publication and patent request is specifically and individually appropriate to be included as a reference.

Despite the above invention having been described in certain aspects by means of illustrations and examples in the interests of clarity and comprehension, it is obvious that certain alterations and modifications may be made within the scope of the claims attached to this description.

Claims

1. Enzyme compositions comprising: (a) cellullolytic and hemicellullolytic enzymes and optionally β-glucosidase, obtained by fermentation with Trichoderma reesei; (b) cellullolytic and hemicellullolytic enzymes, ^' β-glucosidase and accessory enzymes, obtained by fermentation with Aspergillus awamori; (c) furthermore, at least one molecular species selected from the group of enzymes and/or metabolites having CMCase activity, with said molecular species being present in the supernatant of the T. reesei and/or A. awamori culture as demonstrated by the zymogram of Figure 7; and (d) optionally, a vehicle compatible with said enzymes, whereby said enzymes (a) and (b) and the molecular species (c) interact in a manner that results in a synergic effect caused by the activity of the accessory enzymes on other polysaccharides of the biomass as well as on the bonds between the lignin and the hemicellulose .

2. Enzyme compositions comprising: (a) cellullolytic and hemicellullolytic enzymes and optionally β-glucosidase, obtained by fermentation with Trichoderma reesei; (b) cellullolytic and hemicellullolytic enzymes, β-glucosidase and accessory enzymes, obtained by fermentation with Aspergillus awamori; (c) optionally, a vehicle compatible with said enzymes, whereby said enzymes (a) and (b) interact in a manner that results in a synergic effect caused by the activity of the accessory enzymes and molecular species on other polysaccharides of the biomass as well as on the bonds between the lignin and the hemicellulose .

3. Composition according to claims 1 and 2 wherein the cellullolytic enzymes obtained from fermentation with

Trichoderma reesei are present in the fermentation supernatant .

4. Composition according to claims 1 and 2 wherein the cellullolytic enzymes, β-glucosidase and accessory enzymes obtained from fermentation with Aspergillus awamori are present in the fermentation supernatant.

5. Composition according to claims 1 and 2 wherein said Trichoderma reesei is a lineage of T. reesei unaffected by catabolic repression.

6. Composition according to claim 5 wherein said Trichoderma reesei is the lineage of Trichoderma reesei RUT C30.

7. Composition according to claims 1 and 2 wherein said Aspergillus awamori is a lineage of A. awamori with a high capacity for producing accessory enzymes such as xylanases and feruloyl esterases, amongst others.

8. Composition according to claim 7 wherein said Aspergillus awamori is the lineage A. awamori B.361U2/1.

9. Composition according to claim 1 wherein said vehicle compatible with said enzymes is selected from the group consisting of neutral salts, such as ammonium sulphate, potassium phosphate or sodium chloride, polyols of low molecular weight such as glycerol, sorbitol and mannitol, amongst other compounds.

10. Composition according to claim 1 wherein said composition is in liquid form.

11. Composition according to claim 1 wherein said composition is in solid form.

12. Production process for enzymes that degrade the polysaccharide fraction of the biomass, wherein said process comprises the steps of: (a) separately culturing, in an appropriate medium, optionally buffered, a lineage of T. reesei unaffected by catabolic repression and a lineage of A. awamori having a high capacity for the production of accessory enzymes such as xylanases and esterases, amongst others; (b) separating the supernatants of each culture of T. reesei and A. awamori containing the cellulase enzymes and the accessory enzymes; (c) optionally, concentrating and combining the supernatants containing said cellulase enzymes and accessory enzymes to obtain the enzyme mixture; and (d) optionally, drying the suspension to obtain a mixture of dried enzymes.

13. Process according to claim 12 wherein said lineage of Trichoderma reesei unaffected by catabolic repression is the lineage T. reesei RUT C30.

14. Process according to claim 12 wherein said lineage of Aspergillus awamori with a high capacity for producing accessory enzymes such as xylanases and feruloyl esterases, amongst others, is the lineage A. awamori B.361U2/1.

15. Process according to claim 12 wherein said separation of the supernatants performed in step (b) is done by microfiltration.

16. Process according to claim 12 wherein said concentration of the supernatants performed in step (c) is done by microfiltration.

17. Process according to claim 11 wherein said drying performed in step (d) is done by a process selected from the group consisting of lyophilisation and spray drying.

18. Process for the production of alcohol from a biomass comprising the steps of: (a) pretreatment of the lignocellulosic material; (b) enzymatic hydrolysis of the material pretreated in step (a) with a composition as defined in claim 1 or 2; and (c) the alcoholic fermentation of the glucose obtained in step (b) to obtain alcohol.

19. Process according to claim 18 wherein the pretreatment of the lignocellulosic material is done by steam explosion.

20. Process according to claim 19 wherein said pretreatment is optionally preceded by a composting step followed by steam explosion.

21. Process according to claim 18 wherein said pretreatment includes at least one washing operation of the exploded lignocellulosic material before enzymatic hydrolysis .

22. Process according to claim 18 wherein said alcoholic fermentation of the glucose obtained in step (b) is performed using the microorganism Saccharomyces cerevisae.

23. Process according to claim 18 wherein said lineage of Trichoderma reesei unaffected by catabolic repression is the lineage T. reesei RUT C30.

24. Process according to claim 18 wherein said lineage of Aspergillus awamori with a high capacity for producing accessory enzymes such as xylanases and feruloyl esterases, amongst others, is the lineage A. awamori B.361U2/1.

25. Use of the enzyme compositions as defined in claim 1 or 2 wherein said compositions are used for the enzymatic hydrolysis of lignocellulosic material.