METHODS FOR PRODUCING FERMENTATION PRODUCTS

TECHNICAL FIELD

The present invention relates to methods for producing fermentation products from wood- containing materia! ustng one or more fermenting organisms.

BACKGROUND OF THE INVENTION

Due to the limited reserves of fossil fuels and worries about emission of greenhouse gasses there is an increasing focus on using renewable energy sources such as plant material and wood. Production of fermentation products from wood-containing material is known in the art and generally includes the steps of pre-treating, hydroiysing, and fermenting.

WO 2005/118828 concerns a process of producing ethanol from wood-containing materials by operating hydrolysis and fermentation at high substrate concentration and by avoiding the inhibitory effects of the hemicelluiose filtrate to reduce the amount of enzyme needed. However, even though converting wood-containing material into (bio)fυel (e.g. ethanol} is possible, it is still more expensive than making ethanoi from, e.g., starchy materials.

Consequently, there is a need for providing further methods and processes for producing fermentation products from wood-containing-materials.

SUMMARY OF THE INVENTION

The present invention relates to processes for producing fermentation products from wood- containing materia! ustng fermenting organisms. in the first aspect the invention relates to processes of producing fermentation products comprising the steps of: i) pre-treating wood-containing material; ii) hydroiysing by subjecting said pre-treated wood-containing material to one or more celiuloiytic enzymes; iii) fermenting using a fermenting organism; wherein the wood-containing material is subjected to one or more esterases before and/or during pre-treatment in step i) and/or hydrolysis in step ii) and/or fermentation in step iii).

In a preferred embodiment the wood-containing material is softwood-containing material.

DETAILED DESCRIPTION OF THE INVENTION

Wood-containing materia! that includes softwood and/or hardwood residues may, according to the invention be used for producing fermentation products, such as ethanol,

Wood-Containing Material The feedstock used in a process of the invention is wood-containing materia!. The wood- containing material may contain softwood and/or hardwood, or a combination therefore. However, the wood-containing materia! may comprise other constituents. !n a preferred embodiment a significant portion of the feedstock is wood, in preferred embodiments at least 30 wt-%, preferably at ieast 50 wt-%, more preferably at least 70 wt-%, even more preferably at Seast 90 wt-% of the wood-containing material is wood,

Especially contemplated is soft wood-containing material. The terms "soft wood" and "hard wood" are used herein as they are used in the art. The difference between softwood and hardwood has to do with plant reproduction. All trees reproduce by producing seeds, but the seed structures vary. Hardwood trees are angiosperms, which mean plants that produce seeds with some sort of covering. It might be a fruit, such as an apple, or a hard shell, such as an acorn or hickory nut Softwood trees are gymnosperms (conifers) with "naked" seed. Softwood trees are usually evergreen, bear cones, and have needles or scale-iike Sβaves.

Examples of softwoods include softwood derived from pine, spruce, fit hemlock, larch, redwood, and/or cedar tree. For instance, the fir may be Douglas fir the pine may be Ponderosa pine or Lobioily pine; the spruce may be Sitka spruce, the hemlock may be Eastern and/or Western hemlock.

Examples of hardwoods ineiudβ hardwood derived from ash, aspen, beech, basswood, birch, black cherry, black walnut/buttemut, buckeye. American chestnut , cottonwood, dogwood, elm, hackberry. hickory, holly, iocust, magnolia, mapie. oak, poplar, red aider, redbud, royal, paulownia, sassafras, sweetgum, sycamore, tupelo, wiliow, and/or yellow-pop!ar, or a combination thereof.

The generic chemicai composition of hardwood and softwood are as follows: Hardwood:

• Lignin (20-25%) ~ coniferyl & sinapyl alcohol • Cellulose (40-45%)

• Hemicellulose (25-35%) o Glucuronoxylan (15-30%) o Glucomannan (2-5%)

• Extractives (1-5%) Softwood:

• Lignin (25-35%) - conifβryl & p~coumaryl alcohol

• Cellulose (40-45%) • Hemiceliuiose (25-30%) o Galactoglucomannan (5-8%) o Giucomannan (10-15%) o Arabinogiucuronoxylan (7-10%)

• High concentration of localized esterified pectin • Extractives (1-5%)

Extractives comprise a significant fraction of raw wood (1-5%).

"Extractives" is a collective term that includes: terpenoids (e.g. resin acids), pheπolics (e.g. lignans, Tannins, flavonoicis, etc.), waxes, fats, fatty acids, steryi esters, and sterols.

Processes of the invention

The invention relates to methods for producing fermentation products such as ethanol from wood-containing materia! using one or more fermenting organisms. Before the wood-containing material can be fermented into the desired fermentation product, such as ethanol, it has to be converted into fermentable sugars. Firstly, the wood-containing materia! is pre-treated. Mechanical chemical and/or physical pre-treatments are generaiiy carried out to reduce the particle size, to disrupt fiber walls and to expose carbohydrates. This way the susceptibility of the wood material to enzymatic hydrolysis is increased. However, it also exposes, alters and redistributes extractives. This redeposition of certain extractives such as waxes, fats, steryi esters and sterols onto the newly available surface area can effectively shield the underlying carbohydrates from enzymatic hydrolysis. Therefore, these extractives make wood-containing material (e.g., wood chip) a difficult feedstock to use in fermentation product production processes. The inventors have found a way to increase the hydrolysis rate of wood-containing feedstock. Without being bound by any theory it is beiieved that esterase treatment improves the accessibility of hydroiysing enzymes to the substrate (cellulose and hemicellulose). This is more pronounced for softwood-containing material than for hardwood-containing material.

Consequently, in the first aspect the invention relates to processes for producing fermentation products, especially ethanoi, from wood-containing materials, wherein the processes comprise the steps of: i) pre-treating wood-containing material; ii) hydrolysing by subjecting satd pre-treated wood-containing material to one or more cellulolytic enzymes; iii) fermenting using a fermenting organism; wherein the wood-containing material is subjected to one or more esterases before and/or during pre-treatment in step i) and/or hydrolysis in step ii) and/or fermentation in step iii). in an embodiment the wood-containing materia! is subjected to esterase before and/or during step i) and/or ii) and/or iii). Esterase treatment may aiso take place in a separate step before step i); or in a separate step between pre-treatment step i) and hydrolysis step ii).

Hydrolysis step ii) and fermentation step iii) may be carried out separately or simultaneously. In preferred embodiments the hydrolysis and fermentation steps are carried out as HHF (hybrid hydrolysis and fermentation) or SHF (simultaneous hydrolysis and fermentation).

The pH during esterase treatment, especially when carried out as a separate step, is preferably in the range from pH 4.5-8,5. The temperature during esterase treatment, especially when carried out as a separate step, is preferably in the range from 40-90°C. in a preferred embodiment the esterase is dosed in the range from 7.5-25 LU per dry gram of substrate. In a preferred embodiment the esterase is a lipolytic enzyme, preferably lipase; a cutinase; a phospholipase, or a combination of two or more thereof. Examples of enzymes are described in the "Enzymes' -section below.

Pre-treatment As mentioned above the structure of wood is not directly accessible to enzymatic hydrolysis and therefore has to be pre-treated. The wood-containing materia! may according to the invention be pre-treated in any suitable way,

Pre-treatment methods including wet-oxidation and alkaline pre-treatment, target lignin, while dilute acid pre-treatment and auto-hydrolysis targets hemiceiiuiose. Steam explosion is an example of pre-treatment that targets cellulose.

According to the invention the pre-treatment step may be a conventional pre-treatment step using techniques well known in the art. in a preferred embodiment pre-treatment takes place in aqueous slurry, in preferred embodiments the wood-containing material are present during pre-

treatment in amounts between 10-80 wt-%, preferably between 20-70 wt-%, especially between 30-60 Wt.-%, such as around 50 wt-%.

Chemical, Mechanical and/or Biological Pre-treatment The wood-containing material may according to the invention be chemically, mechanically and/or biologically pre-treated before hydrolysis and/or fermentation. Mechanical pre-treatment (often referred to as "physicaP-pre-treatment) may be used alone or in combination with subsequent and/or simultaneous hydrolysis, especiaily enzymatic hydrolysis.

Preferably chemical, mechanical and/or biological pre-treatrnent is carried out prior to the hydrolysis. Alternatively, the chemical, mechanical and/or biological pre-treatment may be carried out simultaneously with hydrolysis, such as simultaneously with treatment of the wood-containing materia! to one or more cellulolytic enzymes, or other enzyme activities, to release, e.g., fermentable sugars, such as glucose and/or maltose. in an embodiment of the invention the pre-treated wood-containing material may be washed or detoxified in another way.

Chemical Pre-treatment

The term "chemical treatment" refers to any chemical pre-treatment which promotes the separation and/or release of cellulose, hemicellulose and/or lignin. Examples of suitable chemical pre-treatment methods include treatment with; for example, dilute acid, lime, alkaline, organic solvent, ammonia, sulfur dioxide, carbon dioxide. Further, wet oxidation and pH-controlled hydrothermolysis are also considered chemical pre-treatment. in a preferred embodiment the chemical pre- treatment is carried out as acid treatment, more preferably, a continuous dilute and/or mild acid treatment, such as, treatment with sulfuric acid, or another organic add, such as acetic acid, citric acid, tartaric add, succinic add, hydrogen chloride or mixtures thereof. Other acids may also be used. Mild acid treatment means that the treatment pH lies in the range from 1-5, preferably pH 1-3. In a specific embodiment the acid concentration is in the range from 0.1 to 2.0 wt % acid, preferably sulphuric acid. The acid may be contacted with the wood-containing material and the mixture may be held at a temperature in the range of 180-220°C, such as 165-195°C, for periods ranging from minutes to seconds, e.g., 1-60 minutes, such as 2-30 minutes or 3-12 minutes. Addition of strong acids, such as sulphuric acid, may be applied to remove hemicellulose, This enhances the digestibility of cellulose.

Other techniques are also contemplated. Cellulose solvent treatment has been shown to convert about 90% of cellulose to glucose. It has also been shown that enzymatic hydrolysis could be greatly enhanced when the wood structure is disrupted. Aikaiine H ₂ O ₂ , ozone, organosol v (uses Lewis adds, FeCI ₃ , (Al) ₂ SO ₄ in aqueous alcohols), glycerol, dioxane, phenol, or ethylene glycol are among solvents known to disrupt cellulose staicture and promote hydrolysis (Mosier et ai. Bioresource Technology 96 (2005), p. 673-686).

Alkaline chemical pre-treatment with base, e.g., NaOH, Na ₂ COs and/or ammonia or the like, is also contemplated according to the invention. Pre-treatment methods using ammonia are described in, e.g. , WO 2006/110891 , VWO 2006/11899, WO 2006/11900, WO 2006/110901, which are hereby incorporated by reference.

Wet oxidation techniques involve the use of oxidizing agents, such as: sulphite based oxidizing agents or the like. Examples of solvent pre-treatments include treatment with DMSO

(Dimethyl Sulfoxide) or the like. Chemical pre-treatment is generally carried out for 1 to 60 minutes, such as from 5 to 30 minutes, but may be carried out for shorter or longer periods of time depending on the materia! to be pre-treated.

Other examples of suitable pre-trβatmβnt methods are described by Schβll et ai. (2003) Appl. Biochem and Biotechn. Vol. 105-108, p. 69-85, and iVlosier et al. Biorβsource Technology 96 (2005) 673-686, and US publication no, 2002/0164730, which references are hereby ai! incorporated by reference.

Mechanical Pre-treatment

The term "mechanical pre-treatment" refers to any mechanical (or physical) pre-treatment which promotes the separation and/or release of celiuiose, hemiceSluiose and/or ϋgnin from lignoceliulose-containing materia!. For example, mechanical pre-treatment includes various types of milling, irradiation, steaming/steam explosion, and hydrothermolysis.

Mechanical pre-treatment includes comminution (mechanical reduction of the size). Comminution includes dry milling, wet milling and vibratory bal! milling. Mechanical pre-treatment may involve high pressure and/or high temperature (steam explosion), in an embodiment of the invention high pressure means pressure in the range from 300 to 600 psi, preferably 400 to 500 psi, such as around 450 psi. In an embodiment of the invention high temperature means temperatures in the range from about 100 to 300°C, preferably from about 140 to 235°C. In a preferred embodiment mechanical pre-treatment is a batch-process, steam gun hydrolyzer system which

uses high pressure and high temperature as defined above. A Sunds Hydrolyzer (available from Sunds Defibrafor AB (Sweden) may be used for this.

Combined Chemical and Mechanical Pre-treatment In a preferred embodiment the wood-containing materia! is pre-treated both chemically and mechanically. For instance, the pre-treatment step may involve dilute or mild acid treatment and high temperature and/or pressure treatment. The chemical and mechanical pre-treatments may be carried out sequentially or simultaneously, as desired.

Accordingly, in a preferred embodiment, the wood-containing material is subjected to both chemical and mechanical pre-treatment to promote the separation and/or release of cellulose, hemicellulose and/or lignin.

In a preferred embodiment pre-treatment is carried out as a dilute and/or mild add steam explosion step. In another preferred embodiment pre-treatment is carried out as an ammonia fiber explosion step (or AFEX pre-treatment step).

Biological Pre-treatment

The term "biological pre-treatment" refers to any biological pre-treatment which promotes the separation and/or release of cellulose, hemicellulose, and/or Iignin from the lignocellulose- containing material. Biological pre-treatment techniques can involve applying lignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanot: Production and Utilization, Wyman, C, E,, e&, Taylor &. Francis, Washington, DC, 179- 212; Ghosh, P., and Singh, A., 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of lignocellulosic biomass, Adv. Appi, Microbiol, 39: 295-333; McMillan, J, D., 1994, Pretreating lignoceiiulosic biomass: a review, in Enzymatic Conversion of Biomass for Fuels Production., Himmel, M. E., Baker, J. O.. and Overend, R. P., eds., ACS Symposium Series 566, American Chemical Society, Washington, DC, chapter 15; Gong, C, S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethano! production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed , Springer- Verlag Berlin Heidelberg, Germany, 65: 207-241; OSsson, L, and Hahn-Hagerdal, B., 1996, Fermentation of lignoceliuSosic hydrolysates for ethanol production, Enz, Microb, Tech. 18: 312-331; and Vallander, L, and Eriksson. K.-E. L,, 1990, Production of ethanol from lignoceiiulosic materials: State of the art, Adv. Biochetn Eng./Bioiechnoi. 42: 63-95).

Hydrolysis

Before the pre-treated wood-containing material is fermented it may be hydrolyzed to break down cellulose and hemicellulose. As mentioned above esterase treatment may be carried out as a separate step between the pre-treaiment step i) and the hydrolysis step ii). but may also be carried out during hydrolysis with cellulolytic enzymes.

The dry solids content during hydrolysis may be in the range from 5-50 wt.-%, preferably 10- 40 wt-%, preferably 20-30 wt.-%. Hydrolysis may in a preferred embodiment be carried out as a fed batch process where ihe pre-treated wood-containing material (substrate) is fed gradually to, e.g., an enzyme containing hydrolysis solution. In a preferred embodiment hydrolysis is carried out enzymatically, According to the invention the pre-treated wood-containing material may be hydroiyzed by one or more cellulolytic enzymes, such as one or more cellullases. One or more hemicellulolytic enzymes, such as hemicellulases may also be present during hydrolysis. in a preferred embodiment hydrolysis is carried out using a cellulolytic enzyme preparation comprising one or more polypeptides having celiulolytic enhancing activity. In a preferred embodiment the polypeptide(s) having cellulolytic enhancing activity is(are) of family GH61A origin. Examples of suitable and preferred celiulolytic enzyme preparations and polypeptides having celiulolytic enhancing activity are described in the "Cellulolytic Enzymes'- section and "Cellulolytic Enhancing ρolypeptides"-section below. As the wood-containing material contain other constituents than lignin cellulose, hemicellulose and extractives hydrolysis and/or fermentation in steps ii) and iii) may be carried out in the presence of additional enzyme activities such as: protease activity, amyiase activity, carbohydrate-generating enzyme activity; and pectinase activity.

Suitable enzyme activities are described in the "Enzymes"-section below. Enzymatic hydrolysis is preferably carried out in a suitable aqueous environment under conditions which can readily be determined by one skilled in the art. in a preferred embodiment hydrolysis is carried out at suitable, preferably optimal, conditions for the enzyme(s) in question.

Suitable process time, temperature and pH conditions can readily be determined by one skilled in the art. Preferably, hydrolysis is carried out at a temperature between 25 and 70°C, preferably between 40 and 60°C, especially around 50°C. The step is preferably carried out at a pH in the range from 3-8, preferably pH 4-6, especialiy around pH 5. Hydroiysis is typically carried out for between 12 and 96 hours, preferable 16 to 72 hours, more preferabiy between 24 and 48 hours.

Fermentation

According to the invention fermentable sugars from pre-treated and hydrolyzed wood- containing material may be fermented by one or more fermenting organisms capable of fermenting sugars, such as glucose, xylose, mannose. and galactose directly or indirectly into a desired fermentation product. The fermentation conditions depend on the desired fermentation product and fermenting organism and can easily be determined by one of ordinary skill in the art.

Especially in the case of ethanol fermentation the fermentation may be ongoing for between 1-48 hours, preferably 1-24 hours, In an embodiment the fermentation is carried out at a temperature between 20 to 40°C, preferably 26 to 34°C, in particular around 32°C. In an embodiment the pH is from pH 3-7, preferably 4-6. However, some, e.g., bacterial fermenting organisms have higher fermentation temperature optima. Therefore, in an embodiment the fermentation is carried out at temperature between 40-80°C, such as 50-60°C. The skiϋed person in the art can easily determine suitable fermentation conditions.

SHF and HHF

In a preferred embodiment hydrolysis and fermentation is carried out as simultaneous hydrolysis and fermentation (SHF). In general this means that combined hydrolysis and fermentation is carried out at conditions (e.g. temperature and/or pH) suitable for the fermenting organism in question. in another preferred embodiment hydrolysis steps ii) and fermentation step isi) is carried out as hybrid hydrolysis and fermentation (HHF). HHF begins with a separate hydrolysis step and ends with a simultaneous hydrolysis and fermentation step. The separate hydrolysis step is an enzymatic cellulose saccharification step typically carried out at conditions (e.g., at higher temperatures) suitable, preferably optimal, for the hydrolysing enzyme(s) in question. The following simultaneous hydrolysis and fermentation step is typically carried out at conditions suitable for the fermenting organism (often at lower temperatures that the separate hydrolysis step).

Recovery Subsequent to fermentation, the fermentation product may optionally be separated from the fermentation medium in any suitable way. For instance, the medium may be distilled to extract the fermentation product or the fermentation product may be extracted from the fermentation medium

by micro or membrane filtration techniques. Alternatively the fermentation product may be recovered by stripping. Recovery methods are well known in the art.

Fermentation Products The present invention may be used for producing any fermentation product Preferred fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids {e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid, succinic acid, fumaric add); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H ₂ and CO ₂ ); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B12, beta-carotene); and hormones. Other products include consumable alcohol industry products, e.g., beer and wine; dairy industry products, e.g., fermented dairy products; leather industry products and tobacco industry products. In a preferred embodiment the fermentation product is an alcohol, especially ethanol. The fermentation product, such as ethanol, obtained according to the invention, may preferably be used as fuel alcohol/ethanol However, in the case of ethanol it may also be used as potable ethanol.

Fermenting Organism

The term 'termenting organism" refers to any organism, including bacteria! and fungal organisms, suitable for producing a desired fermentation product. The fermenting organism may be C ₆ and/or C ₅ fermenting organisms, or a combination thereof. Both C ₆ and C ₅ fermenting organisms are well known in the art.

Suitable fermenting organisms are able to ferment, i.e.. convert, fermentable sugars, such as giucose, xylulose, and maltose, directly or indirectly into the desired fermentation product.

Examples of bacterial and fungai fermenting organisms producing ethanol can be found in Un et ai (2006). AppS. Microbiol, Biotechnoi 69:627-642, see especially pages 630-632, Tabie 1 and 2 (which is hereby incorporated by reference).

Examples of fermenting organisms that can ferment C6 sugars include fungal and bacteria! organisms, especially yeast Preferred yeast includes strains of Saccharomyces, in particuSar strains of Sacchammyces cerevisiae or Sacchammyces uvarutn

Examples of fermenting organisms that can ferment C5 sugars include fungal or bacterial organism. In a preferably embodiment the C5 fermenting organism is yeast, preferably yeast from a strain of Pichia, preferably Pichia stipiϋs, such as Pichia stipitis CBS 5773; a strain of Candida, in particular a strain of Candida utilis, Candida diddθnsiL or Candida boidinii.

Other fermenting organisms include strains of Zymomonas, such as Zymomonas mαbilis; Hamenula, in particular Hansenula anomaia; Klyvemmyces, in particular K. fragiϋs: and Schizosaccharomyces, in particular S. pombe.

Commercially available yeast suitable for ethanol production includes, e.g , ETHANOL RED ™ yeast (available from Fermentis/Lesaffre, USA), FALJ ™ {available from FSeischmann's Yeast, USA) ₁ SUPERSTAR! and THERMOSACC™ fresh yeast {available from Ethanol Technology, Wi, USA), BIOFERM AFT and XR (available from NABC - North American Bioproducts Corporation, GA, USA), GERT STRAND {available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).

Enzymes

Even if not specifically mentioned in context of a method or process of the invention, it is to be understood that the enzyme(s) (as we!! as other compounds) are used in an "effective amount".

Esterases

An "esterase", also referred to as a carboxySic ester hydrolases, refers to enzymes acting on ester bonds, and includes enzymes classified in EC 3.1.1 Carboxylic Ester Hydrolases ac-cording to Enzyme Nomenclature {available at http://www,chem,qmw.ac.υK/iubmb/enzyme or from Enzyme Nomenclature 1992, Academic Press, San Diego, California, with Supplement 1 (1993), Supplement 2 {1994}, Supplement 3 {1995), Supplement 4 (1997) and Supplement 5, in Eur. J. Biochem. 1994, 223, 1-5; Eur. J, Biochem. 1995, 232, 1-6; Eur. j. Bio-chem. 1996, 237, 1-5; Eur. J, Biochem. 1997, 250; 1-6, and Eur. J, Biochem, 1999, 284, 610-650: respectively). Non-limiting examples of esterases include arylesterase, triacylgiycerol lipase, acetyiesterase, acetylcholinesterase, choiinesterase, tropinesterase, pectinesterase, sterol esterase, chiorophylSase, L~arabinono!actonase, gluconoiactonase, uronolactonase, taπnase, retinyl-palmitate esterase, hydroxybutyrate-dimer hydrolase, acyigiycero! lipase, 3-oxoadipaie enol-lactonase, 1,4- lactonase, gaiactolipase, 4-pyridoxoiactonase, acyicarnitine hydrolase, aminoacyl-tRNA hydrolase, D-arabinonolactonase, 6-phosphogiuconoiactonase, phospholipase A1. 6-acetylglucose deacetylase, lipoprotein lipase, dihydrocoυmarin lipase, iimonin-D-ring-lactonase, steroid-iactonase, triacetate-iactonase, actinomycin lactonase, orseliinate-depside hydrolase, cephaiosporin-C deacetyiase, chlorogenate hydrolase, alpha-amino-acid esterase, 4-methy!oxaloacetate esterase, carboxymethylenebutenolidase, deoxylimonate A-ring-lactonase, 2-acetyi-1- alkylglycerophosphocholine esterase, fυsarinine-C ornithinesterase, stnapine esterase, wax-ester

hydrolase, phorboidiester hydrolase, phosphatidyitnositol deacylase, sialate O-acetalesterase, acetoxybufynyibithiαphene deacetylase, acetylsaiicylate deacetylase, methylumbelliferyl-acetate deacetylase, 2-pyrone-4,6-dicarboxylate lactonase, N-acetylgalactosaminoglycan deacetylase, juvenile-hormone esterase, bis{2-ethylhexyl)phthalate esterase, protein-glutamate methylesterase, 11~cis-retinyl~palmitate hy-drolase, all-trans-retinyl-palmitate hydrolase, L~rhamnono-1,4-!actonase, 5-(3,4-diacetoxybut- 1 -ynyl)-2,2'-bithiophene deacetyiase, fatty-acylethyi-ester synthase, xylono-1,4- lactonase, N-acetyiglucosaminyiphosphatidyiinositol deacetylase. cetraxate benzyles-terase, acetylalkylgiycero! acetyl hydrolase, and acetyixylan esterase.

Preferred esterases for use in the present invention are lipolytic enzymes, such as, lipases (as classified by EC 3.1.1.3, EC 3.1.1.23 and/or EC 3.1.1.26), phospholipases (as classified by EC 3.1.1.4 and/or EC 3.1.1.32, including lysophosphoiipases as classified by EC 3.1.1.5), and cutinases classified as EC 3,11.74.

An esterase enzyme may in an embodiment of the invention be dosed in the range from 7.5-25 LU per dry gram of substrate.

Lipolytic Enzymes

The lipolytic enzyme is preferably of microbial origin, in particular of bacterial, fungal or yeast origin. The iipolytic enzyme used may be derived from any source, including, for exampie, a strain of Absidia, in particular Absidia blakesleena and Absidia coryrnbifera, a strain of Achromobactθr, in particular Acbromobacter iopbagus, a strain of Aeromonas, a strain of Aitemaria, in particular Altemaria brassiάola, a strain of Aspergillus, in particular Aspergillus niger and Aspergillus flavus, a strain of Achromobacter, in particular Achromobacter iophagus, a strain of Aureobasidium, in particular Aureobasidium puliutans, a strain of Bacillus, in particular Bacillus pumilus, Bacillus streamthermophϊlus and Bacillus subtϊlis, a strain of Beauvetia, a strain of Brochothήx, in particular Brochothήx thermosohata, a strain of Candida, in particular Candida cylindracea {Candida tvgosa), Candida paraiipolytϊca, and Candida antartica, a strain of Chmmobacter, in particular Chromobacter viscosum, a strain of Coprinus, in particular Copπnus cineήus, a strain of Fusarium, in particular Fusarium oxysporum, Fusaήum solani, Fusarium solani pisi, and Fusarium ωseum culmorum, a strain of Geotricυm, in particular Geotricum penicfflatum, a strain of Hansenula, in particular Hansenula anomala, a strain of Hutncola, in particular Humicola bmvispom. Humicola bmvis var. thermoidea, Humicola insolens, and Humicola lanuginosa (also know as Thennomyces lanuginosus), a strain of Hyphozyma, a strain of Lactobacillus, in particular Lactobacillus cutvatus, a strain of Meterhizium, a strain of Mucor, a strain of Paecilomyces, a strain

of Peniάllium, in particular Peniclilium cyclopium, Peniciliium crustosum and Penicillium expansion, a strain of Pseudomonas in particuiar Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas cepacia {syn. Burkholdena cepacia), Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas maitophilia, Pseudomonas mendocina, Pseudomonas mephiiica lipolytics, Pseudomonas alcaligenes, Pseudomonas planiati, Pseudomonas pseudoalcaljgenes, Pseudomonas puiida, Pseudomonas stutzeti, and Pseudomonas wisconsinensis, a strain of Rhizoctonia, in particular Rhizoctonia solani, a strain of Rhizomucor, in particuiar Rhizomucor miehei, a strain of Rhizopus, in particular Rhizopus japonicus, Rhizopus micrαspαrus and Rhizopus nodosus, a strain of Rhodospoπdium, in particular Rhodospoήdiυm toruloides, a strain of Rhodotorula, in particular Rhodotoruia glutinis, a strain of Sporobolomyces, in particuiar Sporobolomyces shibatanus, a strain of Thermomyces, in particular Thermomyces lanuginosus (also know as Hυmicola lanuginosa), a strain of Thiarosporella, in particular Thiarosporella phasøolina, a strain of Tricboderma, in particular Trichodθfma harzianum, and Trichoderma reesei, and/or a strain of Verticiliium. In a preferred embodiment, the lipolytic enzyme used according to the invention is derived from a strain of Aspergillus, a strain of Achromobacter, a strain of Bacillus, a strain of Candida, a strain of Cbromobacter, a strain of Fusaήum, a strain of Humicola, a strain of Hyphozyma, a strain of Pseudomonas, a strain of Rhizomucor, a strain of Rhizopus, or a strain of Thermomyces.

Upases in a preferred embodiment the lipolytic enzymes is a lipase,

Examples of suitable lipases include upases from Humicola (synonym Theπvomyces), e.g. from H. lanuginosa (T. ianuginosus) as described in EP 258 068 and EP 305216 or from H, insolens as described in WO 96/135S0, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoaicaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1372,034), P fluorescein Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from S, subtilis (Dartois et ai. (1993),

Biochβmica et Biophysica Acta, 1131 253-360), 8. stearothennophiius (JP 64/744992) or B. pumilus (WO 91/16422), Candida antarcitca lipases, such as Candida antarcitca lipase A, Candida antarcitca lipase (lipase B), Candida cytindracea lipase, and PeniciHium camemhettsi lipase, in an embodiment the lipase is a variant of Humicola lanuginosa. Examples of specifically contemplated lipase variant are described in WD 1992/GG5249; WO 1992/019726; WO

1995/022615; WO 1997/007202; WO 2000/060063; and WO 2002/055679 (which references are all incorporated by reference). in a preferred embodiment the lipase is a variant of Therrnomyces lanuginosus (previously Humicαla lanuginosa) with one or more or a!! of the following mutations:

A2x, RESINASE™ HT (available from Novozymes A/S) and G AMANO 50 (avaiiabls from Arnano),

Cutinase

The cutinase used according to the invention may be of any origin. Preferably the cυtinase is of microbial origin, in particuiar of bacteria!, of fungal or of yeast origin, in a preferred embodiment, the cutinase is derived from a strain of Aspergillus, in particuiar Aspergillus oryzae, a strain of Aϊtemaria, in particuiar Aiternaria brassicioia, a strain of Fusarium, in particular Fusanum solani, Fυsaήum solans pisi, Fusarium roseυm culmorum, or

Fusaiium mseum sambucium, a strain of Hetminthosporum, in particular Helminthosporum sativum, a strain of Humi ^' cola, in particular Huivicola Insolens, a strain of Pseudomonas, in particuiar Pseudomonas mendocina, or Pseudomonas putida, a strain of Rhizoctonia, in particular Rhizoctonia solani, a strain of Streptomyces, in particular Streptomyces scabies, or a strain of Uiocladium, in particular Ufocladium consortiale. In a most preferred embodiment the cufinase is derived from a strain of Humicoia insolens, in particular the strain Humicofa insolens

DSM 1800. Humicoia snsoiens cutinase is described in WO 96/13580 which is herby incorporated by reference. The cutinase may be a variant, such as one of the variants disclosed in

WO 00/34450 and WO 01/92502, which are hereby incorporated by reference. Preferred cutinase variants include variants listed in Example 2 of WO 01/92502, which is hereby specifically incorporated by reference.

Preferred commercial cutinases include NOVOZYM™ 51032 (available from Novozymes AZS ₁ Denmark).

Phospholipases

As used herein, the term phospholipase is an enzyme which has activity towards phospholipids Phospholipids, such as lecithin or phosphatidylcholine, consist of glycerol esterified wtth two fatty acids in an outer (sn-1) and the middle (sn-2) positions and esterified with phosphoric acid in the third position: the phosphoric acid, in turn, may be esterified to an amino-aicohoS. Phospholipases are enzymes which participate in the hydrolysis of phospholipids. Several types of phospholipase activity can be distinguished, including phospho- lipases A ₁ and A ₂ which hydroiyze one fatty acyi group (in the sn-1 and sn-2 position, respectively) to form lysophospholipid, and lysophospholipase (or phospholipase B) which can hydroiyze the remaining fatty acyl group in lysophospholipid. Phospholipase C and phospholipase D (phosphodiesterases) release diacyl glycerol or phosphatide acid respectively.

The term phosphoiipase includes enzymes with phospholipase activity, e.g., phospholipase A (A ₁ or A ₂ ), phospholtpase B activity, phospholipase C activity or phospholipase D activity. The term "phosphoϋpase A" used herein in connection with an enzyme of the invention is intended to cover an enzyme with Phospholipase A ₁ and/or Phospholipase A ₂ activity The phospholipase activity may be provided by enzymes having other activities as well, such as, e.g., a lipase with phospholipase activity. The phospholipase activity may, e.g., be from a lipase with phospholipase side activity. In other embodiments of the invention the phosphoϋpase enzyme activity is provided by an enzyme having essentiaiiy oniy phospholipase activity and wherein the phospholipase enzyme activity is not a side activity.

The phospholipase may be of any origin, e.g., of animal origin (such as, e.g., mammalian}, e.g. from pancreas (e.g., bovine or porcine pancreas), or snake venom or bee venom. Preferably the phospholipase may be of microbiai origin, e.g., from filamentous fungi, yeast or bacteria, such as the genus or species Aspergillus, e.g , A. niger Dictyosteiium, e g., D. discoideum: Mucor, e.g. Af. javanlcus, M mucedo M. subtiltssimus: Nθufospora, e.g N. crassa: Rhizomucor, e.g., R pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus, R. stoionifer. Sclerotica, e.g., S. libesiiana: Trichophyton _^ e.g. 7. rubrum; Whetzeiinia, e.g., W. sclerotiorum; BaciHus, e.g , S. megateriurn, B subiilis; Citmbacter, e.g., C- freundii; Enterobacter, e.g., E. aerogenes, E, cloacae Edwardsieila, E. tarύa; Erwϊnia, e.g., E. herϋϊcola; Escherichia, e.g., E. coll; Klebsiella, e.g., K. pneumoniae; Proteus, e.g.. R vulgaris: Providencia. e.g.. P. stuartii: Salmonella, e.g. S. typhimunum, Serratia, e g.. S. liquefasciens. S. matcescens: Shigella, e g., S. flexneπ; Strepiomyces, e.g., S. vioieceαruber, Yersinia, e.g., V. enterocoMica, Thus, the

phosphatase may be fungal, e.g., from the class Pyrenomycetes, such as the genus Fusarium, such as a strain of F, culmorum, F, heterospαrum, F. solani, or a strain of F. oxyspoωm. The phosphoiipase may also be from a filamentous fungus strain within the genus Aspergillus, such as a strain of Aspergillus awamoή, Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger or Aspergillus oryzae.

Preferred phosphoii pases are derived from a strain of Hutnicola, especially Humicola lanuginosa. The phosphoiipase may be a variant, such as one of the variants disclosed in WO 00/32758, which are hereby incorporated by reference. Preferred phosphoiipase variants include variants listed in Example 5 of WO 00/32758, which is hereby specifically incorporated by reference, in another preferred embodiment the phosphoiipase is one described in WO 04/111218, especially the variants listed in the table in Example 1. in another preferred embodiment the phosphoiipase is derived from a strain of Fusarium, especially Fusarium oxysporum. The phospholipase may be the one concerned in WO 98/026057 displayed in SEQ ID NO: 2 derived from Fusarium oxysporum DSM 2672, or variants thereof.

Examples of commercial phospholipases include LECITASE™ and LECfTASE™ ULTRA, YlELSMAX, or UPOPAN F (available from Novozymes A/S, Denmark}.

Celtulolytic Activity The term "cellulolytic activity" as used herein are understood as comprising enzymes having cellobiohydrolase activity (EC 3,2.1.91), e.g., cellobiohydrolase I and cellobiohydrolase II, as well as endo-glυcanase activity (EC 3,2.1.4) and beta-glucosidase activity (EC 3.2.121 ).

At least three categories of enzymes are important for converting ceiiulose into fermentable sugars: endc-glucanases (EC 3.2.1.4} that cut the cellulose chains at random; cellobiohydrolases (EC 3.2.1.91) which cleave celiobiosyl units from the cellulose chain ends and faeta-glucossdases (EC 3.2.1.21} that convert celiobiose and soluble cellodextrins into glucose. Among these three categories of enzymes involved in the biodegradation of ceiiulose. cellobiohydrolases seems to be the key enzymes for degrading native crystalline cellulose.

The cellulolytic activity may, in a preferred embodiment, be in the form of a preparation of enzymes of fungai origin, such as from a strain of the genus Trichoderma, preferably a strain of Trichoderma reesei; a strain of the genus Humicola, such as a strain of Humicola insolens; or a strain of Chrysosporium, preferably a strain of Chrysσsporium lucknowense.

in preferred embodiment the cellulolytic enzyme preparation contains one or more of the following activities: cellulase, hemicellulase, cellulolytic enzyme enhancing activity, beta- glucosidase activity, endoglucanase, celiubiohydroiase, or xylose-isormerase. in a preferred embodiment cellulolytic enzyme preparation is a composition concerned in co-pending application US application # 60/941,251 , which is hereby incorporated by reference.

In a preferred embodiment the celiulolytic enzyme preparation comprising a polypeptide having cellulolytic enhancing activity, preferably a family GH61A polypeptide, preferably the one disclosed in WO 2005/074656 (Novozymes). The cellulolytic enzyme preparation may further comprise a beta-glucosidase, such as a beta-glucosidase derived from a strain of the genus Tήchoderma, Aspergillus or PerticiiHum, including the fusion protein having beta-glucosidase activity disclosed in co-pending application US 60/832.511 {Novozymes). In a preferred embodiment the cellulolytic enzyme preparation may also comprises a CBH II enzyme, preferably Thielavia termstns cellobiohydrolase SI CEL6A. In another preferred embodiment the cellulolytic enzyme preparation may also comprises cellulolytic enzymes, preferably one derived from Tήchoderma reesei or Humicola insolens.

The cellulolytic enzyme preparation may also comprising a polypeptide having cellulolytic enhancing activity (GH61A) disclosed in WO 2005/074656; a beta-glucosidase (fusion protein disclosed in US 60/832,511} and ceiiuiolytic enzymes derived from Trichoderma reesei. in an embodiment the cellulolytic enzyme composition is the commercially available product

CELLUCLAST™ ISL or CELLUZYME™ (Novozymes A/S, Denmark).

The cellulolytic activity may be dosed sn the range from 0.1-100 FPU per gram total solids (TS) ₁ preferably 0.5-50 FPU per gram TS, especially 1-20 FPU per gram TS.

Endoglucanase (EG)

The term "endoglucanase" means an endo-1,4-{1 ,3,1 ,4)-beta-D-g!ucan 4- glucanohydrolase (E.G. No. 3.2.1.4), which catalyses endo-hydrolysis of 1 ,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta- 1 ,3 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components. Endoglucanase activity may be determined using carboxymethyl cellulose (CMC) hydrolysis according to the procedure of Ghose, 1987, Pure andAppt. Cftθtv. 59: 257-268,

in a preferred embodiment encloglυcanases may be derived from a strain of the genus Trichoderma, preferably a strain of Trichoderma reeseϊ, a strain of the genus Humicola, such as a strain of Humicola insolens; or a strain of Chrysosponum, preferably a strain of Chrysosporium lucknowense.

Cellobiohydrolase (CBH)

The term "cellobiohydrolase" means a 1 ,4-beta-D-glucan cellobiohydrolase (E.G. 3.2.1.91), which catalyzes the hydrolysis of 1,4-beia-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1,4-Sinked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain.

Examples of cellobiohydroloses are mentioned above including CBH I and CBH Il from Trichoderma reseei; HυrnicoSa insoiens and CBH ! I from Thielavia tetvestris cβlSobiohydrolase (CELL6A)

Cellobiohydrolase activity may be determined according to the procedures described by Lever θt ai. 1972. Anal Biochem, 47: 273-279 and by van Tiibeurgh θi aL, 1982, FEBS Lθtters 149: 152-156; van Tiibeurgh and Claeyssens, 1985 _r FEBS Lθtters 187. 283-288. The Lever et a/, method is suitable for assessing hydrolysis of cellulose in corn stover and the method of van Tiibeurgh et a/, is suitable for determining the celiobiohydralase activity on a fluorescent disaccharide derivative.

Beta-glucosidase

One or more beta-glucosidases may be present during hydrolysis. The term "'beta-glυcosidase" means a beta-D-glυcoside glucohydrolase (E.G. 3.2,1.21 }, which catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D-glucose. For purposes of the present invention, beta-glucosidase activity is determined according to the basic procedure described by Venturi et at., 2002, J. Basic Microbiol. 42: 55-66, except different conditions were employed as described herein One unit of beta-glucosidase activity is defined as 1.0 μmole of p-nitrophenol produced per minute at 5(TC, pH 5 from 4 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodium citrate, 0.01% TWEEN® 2Q.

In a preferred embodiment the beta-glucosidase is of fungal origin, such as a strain of the genus Trichoderma.. Aspergillus or Penicillium. In a preferred embodiment the beta- glucosidase is a derived from Trichoderma reesei, such as the beta-giucosidase encoded by the

bgl1 gene (see Fig. 1 of EP 562003). In anotlw preferred embodiment the befa-glucosidase is derived from Aspergillus oryzae (recombinantly produced in Aspergillus oryzae according to WO 02/095014), Aspergillus fumigaius (recombinants produced in Aspergillus oryzae according to Example 22 of WO 02/095014) or Aspergillus niger (1981 j. Appi. VoI 3, pp 157-163),

Hemicellulolytic enzymes

According to the invention the pre-treated lignocellulose-containing material may further be subjected to one or more hemicellulolytic enzymes, e.g., one or more hemicellulases.

Hemicellulose can be broken down by hemicellulases and/or acid hydrolysis to release its five and six carbon sugar components, in an embodiment of the invention the lignocellulose derived material may be treated with one or more hemicellulases.

Any hemicellulase suitable for use in hydrolyzing hemicellulose, preferably into xylose, may be used. Preferred hemiceiiulases include xylanases, arabinofuranosidases, acetyi xylan esterase, ferυloyl esterase, glucuronidases, endo-galactanase. mannases, endo or exo arabinases, exo- galactanses, and mixtures of two or more thereof. Preferably, the hemicellulase for use in the present invention is an exo-acting hemicellulase, and more preferably, the hemicellulase is an exo~ acting hemicellulase which has the ability to hydrolyze hemicellulose under acidic conditions of below pH 7, preferably pH 3-7. An example of hemicellulase suitable for use in the present invention includes ViSCOZYIvlE™ (available from Novozymes A/S, Denmark). in an embodiment the hemicellulase is a xylanase. In an embodiment the xylanase may preferably be of microbial origin, such as of fungal origin (e.g., Trichoderma, Meήpilus, Humicoia, Aspergillus, Fusarium) or from a bacterium (e.g., Bacillus}, in a preferred embodiment the xylanase is derived from a filamentous fungus, preferably derived from a strain of Aspergillus, such as Aspergillus aculeaius; or a strain of Humicoia, preferably Humicoia lanuginosa. The xylanase may preferably be an endo- 1 ,4-beta- xylanase, more preferably an endo-1,4-beta-xy!anase of GH 10 or GH11. Examples of commercial xylanases include SHEARZYfviE™ and BiOFEED WHEAT™ from Novozymes A/S, Denmark,

The hemicellulase may be added in an amount effective to hydrolyze hemicellulose, such as, in amounts from about 0,001 to 0.5 wt-% of total solids (TS), more preferably from about 0.05 to 0.5 wt.-% αf TS.

Xylanases may be added in amounts of 0,001-1.0 g/kg DM (dry matter) substrate, preferably in the amounts of 0.005-0.5 g/kg DiVl substrate, and most preferably from 0.05-0.10 g/kg DiVi substrate.

€ellulolytic Enhancing Activity

The term "ceϋuiolyfjc enhancing activity" is defined herein as a biological activity that enhances the hydrolysis of a lignocellulose derived materia! by proteins having cellulolytic activity. For purposes of the present invention, cβlluiolytic enhancing activity is determined by measuring the increase in reducing sugars or in the increase of the total of cellobiose and glucose from the hydrolysis of a Signocellulose derived material, e.g., pre-treateci Signocellulose- containing material by cellulolytic protein under the following conditions: 1-50 mg of total protein/g of cellulose in PCS (pre-treated corn stover), wherein total protein is comprised of 80- 99.5% w/w celiulolytic protein/g of cellulose in PCS and 0,5-20% w/w protein of cellulolytic enhancing activity for 1-7 day at 50"C compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1-50 mg of ceilulolytic protein/g of cellulose in PCS).

The polypeptides having cellulolytic enhancing activity enhance the hydrolysis of a lignoceSSuiose derived materia! catalyzed by proteins having celluloiytic activity by reducing the amount of celluloiytic enzyme required to reach the same degree of hydrolysis preferably at least 0.1-fold, more at least 0.2-fold, more preferably at least 0.3-fold, more preferably at least 0.4-fo!d, more preferably at least 0.5-foid, more preferably at least 1-fold, more preferably at least 3-fold, more preferably at least 4-fold, more preferably at least 5-fold, more preferably at least 10-fold, more preferably at least 20-fold. even more preferably at feast 30-fold, most preferably at ieast 50-fold, and even most preferably at least 100-fold, Sn a preferred embodiment the hydrolysis and/or fermentation is carried out in the presence of a cellulolytic enzyme in combination with a polypeptide having enhancing activity, in a preferred embodiment the polypeptide having enhancing activity is a family GH61A polypeptide. VVO 2005/074647 discloses isolated polypeptides having celluloiytic enhancing activity and polynucleotides thereof from Thietavia terrestris. WO 2005/074658 discloses an isolated polypeptide having ceSluioSytic enhancing activity and a polynucleotide thereof from Themjoascus aurantiacus. U.S. Published Application Serial No. 2007/0077630 discloses an isolated polypeptide having cellulolytic enhancing activity and a polynucleotide thereof from Tήchoderma reesei.

Proteases

A protease may be added during hydrolysis in step ii), fermentation in step iii) or simultaneous hydrolysis and fermentation. The protease may be any protease. In a preferred embodiment the protease is an acid protease of microbial origin, preferably of fungal or bacterial origin, An acid fungal protease is preferred, but also other proteases can be used.

Suitable proteases include microbial proteases, such as fungal and bacterial proteases. Preferred proteases are acidic proteases, i.e., proteases charactered by the ability to hydrolyze proteins under acidic conditions below pH 7. Contemplated acid fungal proteases include funga! proteases derived from Aspergillus,

Mucor, Rhizopus, Candida, Coήolus, Endothia, Enthomophtra, //pex, Penicillium, Sderotiumand Tomiopsis. Especially contemplated are proteases derived from Aspergillus niger {see, e.g., Koaze et al., (1964), Agr. Biol, Chem, Japan, 28, 216), Aspergillus saitoi (see, e.g., Yoshida, (1954) J. Agr, Cherrs. Soc. Japan, 28, 66), Aspergillus awamori (Hayashida et a!., {1977} Agric. Biol. Chem., 42(5), 927-933, Aspergillus acuieatus {WO 95/02044), or Aspergillus oryzae, such as the pepA protease, and acidic proteases from Mucor pusillus or Mucor nniehei.

Contemplated are also neutral or alkaline proteases, such as a protease derived from a strain of Bacillus. A particular protease contemplated for the invention is derived from Bacillus amyloliquefadens and has the sequence obtainable at Swissprot as Accession No. P06832. Also contemplated are the proteases having at least 90% identity to amino acid sequence obtainable at Swissprot as Accession No. P06832 such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% identity.

Further contemplated are the proteases having at least 90% identity to amino acid sequence disclosed as SEQJD.NO:1 in the WO 2003/048353 such as at 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% identity.

Also contemplated are papain-like proteases such as proteases within E.C. 3.4.22.* (cysteine protease), such as EC 3.4.22.2 (papain), EC 3.4.22.6 {chymopapain), EC 3.4.22.7 (asclepain), EC 3.4.22.14 (actinidain), EC 34.22.15 (cathepsin L), EC 3.4.22 25 (giycyl enclopeptidase) and EC 3.4,22.30 (caricain). in an embodiment the protease is a protease preparation derived from a strain of

Aspergillus, such as Aspergillus oryzae. in another embodiment the protease is derived from a strain of Rhizomucor, preferabiy Rhizomυcor mehei. In another contemplated embodiment the protease is a protease preparation, preferably a mixture of a proteolytic preparation derived

from a strain of Aspergillus, such as Aspergillus oryzae, and a protease derived from a strain of Rhizomucor, preferably Rhizomυcor mehei.

Aspartic acid proteases are described in, for example, Hand-book of Proteolytic Enzymes, Edited by A.J. Barrett, N. D. Rawiings and J. F. Woessner, Aca-demic Press, San Diego, 1998, Chapter 270). Suitable examples of aspartic acid protease include, e.g., those disclosed in R.M, Berka et ai. Gene. 96, 313 (1990)}; (R, M. Berka et a!. Gene, 125, 195-198 (1993)}; and Gomi et al. Biosci. Biotech. Biochem, 57, 1095-1100 (1993), which are hereby incorporated by reference.

Commercially available products include ALCALASE®, ESPERASE™, FLAVOU RZYME ™\ PROMIX™, NEUϊRASE®, RENNILASE®, NOVOZYM™ FM 2.0L, and NOVOZYM™ 50006 (available from Novozymes A/S, Denmark) and GC106™ and SPE2YME™ FAN from Genencor Int., Inc., USA.

The protease may be present in an amount of 0.0001-1 mg enzyme protein per g DS, preferably 0.001 to 0,1 mg enzyme protein per g DS. Alternatively, the protease may be present in an anount of 0.0001 to 1 LAPU/g DS ₁ preferably 0,001 to 0.1 LAPU/g DS and/or 0.0001 to 1 mAU-RH/g DS, preferably 0.001 to 0.1 mAU-RH/g DS,

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention, indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fa!! within the scope of the appended claims, in the case of conflict, the present disclosure including definitions will control. Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

MATERIALS & METHODS Methods

Determination of identity

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".

The degree of identity between two amino acid sequences may be determined by the

Clustal method (Higgins, 1989, CABIOS 5: 151-153} using the LASERGENE™ MEGAUGN ™ software (DNASTAR, Inc., Madison, WI) with an identity table and the following multiple alignment parameters; Gap penalty of 10 and gap length penalty of 10 Pairwise alignment parameters are Ktuple=1, gap penaity=3, windows=5, and diagonals=5.

The degree of identity between two nucleotide sequences may be determined by the Wilbur-Upman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA 80: 726-730) using the LASERGENE™ MEGAUGN™ software (DNASTAR, Inc., Madison, Wl) with an identity table and the following multiple aiignment parameters: Gap penalty of 10 and gap length penalty of 10. Pasrwise alignment parameters are Ktupie=3, gap ρenalty=3, and windows=20.

Measurement of Cellulase Activitv Usino Filter Paper Assay (FPU assay) 1 , Source of Method 1.1 The method is disclosed in a document entitled "Measurement of Cellulase Activities" by Adney, B. and Baker, J. 1996. Laboratory Analytical Procedure, LAP-006, National Renewable Energy Laboratory (NREL). it is based on the IUPAC method for measuring ceilulase activity (Ghose, T.K., Measurement of Ceiluise Activities, Pυre & Appi. Chem. 59, pp. 257-268, 1987.

2. Procedure

2.1 The method is carried out as described by Adney and Baker, 1996, supra., except for the use of a 96 well plates to read the absorbance values after color development, as described below.

2.2 Enzyme Assay Tubes: 2.2.1 A rolled filter paper strip (#1 Whatman; 1 X 6 cm; 50 mg) is added to the bottom of a test tube (13 X 100 mm).

2.2.2 To the tube is added 1.0 mL of 0.05 M Na-citrate buffer (pH 4.80).

2.2.3 The tubes containing filter paper and buffer are incubated 5 min. at 50* C (± 0.1° C) in a circulating water bath. 2,2.4 Following incubation, 0.5 mL of enzyme dilution in citrate buffer is added to the tube. Enzyme dilutions are designed to produce values slightly above and below the target value of 2.0 mg glucose. 2.2.5 The tube contents are mixed by gently vortexing for 3 seconds.

2.2.6 After vortexing, the tubes are incubated for 60 mins, at 50'- C (± 0.1° C) in a circulating water bath.

2.2.7 immediately following the 60 min. incubation, the tubes are removed from the water bath, and 3.0 mL of DNS reagent is added to each tube to stop the reaction. The tubes are vortexed 3 seconds to mix.

2.3 Blank and Controls

2.3.1 A reagent blank is prepared by adding 1 ,5 mL of citrate buffer to a test tube.

2.3.2 A substrate control is prepared by placing a rolled filter paper strip into the bottom of a test tube, and adding 1.5 mL of citrate buffer 2.3.3 Enzyme controls are prepared for each enzyme dilution by mixing 1.0 mL of citrate buffer with 0.5 mL of the appropriate enzyme dilution.

2.3.4 The reagent blank, substrate control, and enzyme controls are assayed in the same manner as the enzyme assay tubes, and done along with them.

2.4 Glucose Sisndards 2,4.1 A 100 mL stock solution of glucose (10.0 mg/mL} is prepared, and 5 mL aiiquots are frozen. Prior to use, aiiquots are thawed and vortexed to mix.

2.4.2 Dilutions of the stock solution are made in citrate buffer as follows. G1 = 1.0 ml stock + 0.5 mL buffer = 6.7 mg/mL = 3.3 mg/0.5 mL

G2 = 0.75 mL stock + 0.75 ml buffer = 5.0 mg/mL = 2.5 mg/0.5 mL G3 = 0.5 mL stock + 1O mL buffer = 3.3 mg/rnl = 1.7 mg/0.5 mL G4 = 0.2 mL stock + 0.8 mL buffer = 2.0 mg/mL = 1.0 mg/0.5 mL

2.4.3 Glucose standard tubes are prepared by adding 0,5 mL of each dilution to 1.0 mL of citrate buffer.

2.4.4 The glucose standard tubes are assayed in the same manner as the enzyme assay tubes, and done along with them

2.5 Color Development

2.5.1 Following the 80 min, incubation and addition of DNS, the tubes are all boiled together for 5 mins. in a water bath,

2.5.2 After boiling, they are immediately cooled in an ice/water bath. 2.5,3 When cool, the tubes are briefly vortexed, and the puip is allowed to settie. Then each tube is diluted by adding 50 mtcroL from the tube to 200 mieroL of ddH2O in a 96-well plate, Each well is mixed, and the absorbance is read at 540 nm.

2.6 Calculations (examples am given in the NREL document)

2.6,1 A glucose standard curve is prepared by graphing glucose concentration (mg/0.5 mL) for the four standards {G1-G4} vs. A ₅₄₀ . This is fitted using a linear regression (Prism Software), and the equation for the line is used to determine the glucose produced for each of the enzyme assay tubes. 2.6.2 A plot of glucose produced (mg/0.5 mL) vs. total enzyme dilution is prepared, with the Y- axis (enzyme dilution) being on a log scale.

2.6.3 A line is drawn between the enzyme dilution that produced just above 2.0 mg glucose and the dilution that produced just below that. From this line, it is determined the enzyme dilution that would have produced exactly 2.0 mg of glucose. 2.6.4 The Filter Paper Units/ml (FPU/mL) are calculated as follows;

FPU/mL = 0.37/ enzyme dilution producing 2.0 mg glucose

Lipase activity (LU)

The esterase activity is determined using thbutynne as substrate. This method is based on the hydrolysis of tributyrin by the enzyme, and the alkali consumption is registered as a function of time.

One Lipase Unit (LU) is defined as the amount of enzyme which, under standard conditions (i.e., at 30° C; pH 7.0; with Gum Arabic as emuisifier and tributyrine as substrate) liberates 1 micrO-moi titrable butyric acid per minute. Folders AF 95/5 or EB-SM-0095-02-D which describes this analytical method in more detail is available upon request to Novozymes

A/S, Denmark. The folders are hereby included by reference.