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WO2009042622A2 - Procédés de production de produits de fermentation - Google Patents

Procédés de production de produits de fermentation Download PDF

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Publication number
WO2009042622A2
WO2009042622A2 PCT/US2008/077421 US2008077421W WO2009042622A2 WO 2009042622 A2 WO2009042622 A2 WO 2009042622A2 US 2008077421 W US2008077421 W US 2008077421W WO 2009042622 A2 WO2009042622 A2 WO 2009042622A2
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WO
WIPO (PCT)
Prior art keywords
strain
fermentation
wood
hydrolysis
containing material
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Application number
PCT/US2008/077421
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English (en)
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WO2009042622A3 (fr
Inventor
Gregory Delozier
Frank Droescher Haagensen
Mads Peter Torry Smith
Jing Luo
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Novozymes A/S
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Publication of WO2009042622A2 publication Critical patent/WO2009042622A2/fr
Publication of WO2009042622A3 publication Critical patent/WO2009042622A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/16—Butanols
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/54—Acetic acid
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to methods for producing fermentation products from wood- containing materia! ustng one or more fermenting organisms.
  • 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.
  • wood-containing material e.g. ethanol ⁇
  • it is still more expensive than making ethanoi from, e.g., starchy materials.
  • the present invention relates to processes for producing fermentation products from wood- containing materia! ustng fermenting organisms.
  • 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).
  • the wood-containing material is softwood-containing material.
  • 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.
  • the wood-containing materia! may comprise other constituents.
  • 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,
  • soft wood-containing material Especially contemplated is soft wood-containing material.
  • 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 S ⁇ aves.
  • softwoods include softwood derived from pine, spruce, fit hemlock, larch, redwood, and/or cedar tree.
  • 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.
  • the generic chemicai composition of hardwood and softwood are as follows: Hardwood:
  • Extractives comprise a significant fraction of raw wood (1-5%).
  • Extractives is a collective term that includes: terpenoids (e.g. resin acids), phe ⁇ olics (e.g. lignans, Tannins, flavonoicis, etc.), waxes, fats, fatty acids, steryi esters, and sterols.
  • terpenoids e.g. resin acids
  • phe ⁇ olics e.g. lignans, Tannins, flavonoicis, etc.
  • waxes e.g. lignans, Tannins, flavonoicis, etc.
  • the invention relates to methods for producing fermentation products such as ethanol from wood-containing materia! using one or more fermenting organisms.
  • the wood-containing material Before the wood-containing material can be fermented into the desired fermentation product, such as ethanol, it has to be converted into fermentable sugars.
  • 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.
  • 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!
  • 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.
  • 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 is preferably in the range from pH 4.5-8,5.
  • the temperature during esterase treatment is preferably in the range from 40-90°C.
  • the esterase is dosed in the range from 7.5-25 LU per dry gram of substrate.
  • 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.
  • 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.
  • the pre-treatment step may be a conventional pre-treatment step using techniques well known in the art.
  • pre-treatment takes place in aqueous slurry
  • 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-%.
  • 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.
  • chemical, mechanical and/or biological pre-treatrnent is carried out prior to the hydrolysis.
  • 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.
  • the pre-treated wood-containing material may be washed or detoxified in another way.
  • chemical treatment refers to any chemical pre-treatment which promotes the separation and/or release of cellulose, hemicellulose and/or lignin.
  • suitable chemical pre-treatment methods include treatment with; for example, dilute acid, lime, alkaline, organic solvent, ammonia, sulfur dioxide, carbon dioxide.
  • wet oxidation and pH-controlled hydrothermolysis are also considered chemical pre-treatment.
  • 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.
  • 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.
  • Alkaline chemical pre-treatment with base e.g., NaOH, Na 2 COs and/or ammonia or the like
  • base e.g., NaOH, Na 2 COs and/or ammonia or the like
  • 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.
  • oxidizing agents such as: sulphite based oxidizing agents or the like.
  • solvent pre-treatments include treatment with DMSO
  • 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.
  • mechanical pre-treatment refers to any mechanical (or physical) pre-treatment which promotes the separation and/or release of celiuiose, hemiceSluiose and/or ⁇ gnin from lignoceliulose-containing materia!.
  • 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.
  • Sunds Hydrolyzer available from Sunds Defibrafor AB (Sweden) may be used for this.
  • the wood-containing materia! is pre-treated both chemically and mechanically.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • hydrolysis is carried out enzymatically,
  • 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.
  • hydrolysis is carried out using a cellulolytic enzyme preparation comprising one or more polypeptides having celiulolytic enhancing activity.
  • 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 ⁇ olypeptides"-section below.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • the fermentation may be ongoing for between 1-48 hours, preferably 1-24 hours,
  • the fermentation is carried out at a temperature between 20 to 40°C, preferably 26 to 34°C, in particular around 32°C.
  • the pH is from pH 3-7, preferably 4-6.
  • 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 ski ⁇ ed person in the art can easily determine suitable fermentation conditions.
  • hydrolysis and fermentation is carried out as simultaneous hydrolysis and fermentation (SHF).
  • SHF simultaneous hydrolysis and fermentation
  • HHF hybrid hydrolysis and fermentation
  • 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).
  • the fermentation product may optionally be separated from the fermentation medium in any suitable way.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • fermenting organisms that can ferment C5 sugars include fungal or bacterial organism.
  • the C5 fermenting organism is yeast, preferably yeast from a strain of Pichia, preferably Pichia stipi ⁇ s, such as Pichia stipitis CBS 5773; a strain of Candida, in particular a strain of Candida utilis, Candida didd ⁇ nsiL or Candida boidinii.
  • Other fermenting organisms include strains of Zymomonas, such as Zymomonas m ⁇ bilis; Hamenula, in particular Hansenula anomaia; Klyvemmyces, in particular K. fragi ⁇ s: and Schizosaccharomyces, in particular S. pombe.
  • yeast suitable for ethanol production includes, e.g , ETHANOL RED TM yeast (available from Fermentis/Lesaffre, USA), FALJ TM ⁇ available from FSeischmann's Yeast, USA) 1 SUPERSTAR! and THERMOSACCTM 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).
  • ETHANOL RED TM yeast available from Fermentis/Lesaffre, USA
  • THERMOSACCTM 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
  • FERMIOL
  • esterases include arylesterase, triacylgiycerol lipase, acetyiesterase, acetylcholinesterase, choiinesterase, tropinesterase, pectinesterase, sterol esterase, chiorophylSase, L ⁇ arabinono!actonase, gluconoiactonase, uronolactonase, ta ⁇ nase, 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, dihydroco ⁇ marin 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, f ⁇ sarinine-C ornithinesterase, stnapine esterase, wax-ester hydrolase, phorboidiester hydrolase, phosphatidyitnositol deacylase, sialate O-ace
  • 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.
  • 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
  • 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.
  • 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 Achromobact ⁇ r, in particular Acbromobacter iopbagus, a strain of Aeromonas, a strain of Aitemaria, in particular Altemaria brassi ⁇ ola, 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 streamthermoph ⁇ lus and Bacillus subt ⁇ lis, a strain of Beauvetia
  • 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 Peni ⁇ llium, in particular Peniclilium cyclopium, Peniciliium crustosum and Penicillium expansion, a strain of Pseudomonas in particuiar Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas cepacia ⁇ syn.
  • 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 Fusa ⁇ um, 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.
  • the lipolytic enzymes is a lipase
  • Suitable lipases include upases from Humicola (synonym The ⁇ vomyces), 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),
  • 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.
  • the lipase is a variant of Therrnomyces lanuginosus (previously Humic ⁇ la lanuginosa) with one or more or a!! of the following mutations:
  • the cutinase used according to the invention may be of any origin.
  • the c ⁇ tinase 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 A ⁇ temaria, in particuiar Aiternaria brassicioia, a strain of Fusarium, in particular Fusanum solani, F ⁇ sa ⁇ um solans pisi, Fusarium rose ⁇ m 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.
  • the cufinase is derived from a strain of Humicoia insolens, in particular the strain Humicofa insolens
  • 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 NOVOZYMTM 51032 (available from Novozymes AZS 1 Denmark). Phospholipases
  • 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.
  • 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 release diacyl glycerol or phosphatide acid respectively.
  • 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.
  • phospho ⁇ pase 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.
  • the phospho ⁇ pase 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.
  • animal origin such as, e.g., mammalian ⁇ , e.g. from pancreas (e.g., bovine or porcine pancreas), or snake venom or bee venom.
  • 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 N ⁇ ufospora, 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.
  • the phosphatase may be fungal, e.g., from the class Pyrenomycetes, such as the genus Fusarium, such as a strain of F, culmorum, F, heterosp ⁇ rum, F. solani, or a strain of F. oxyspo ⁇ m.
  • the phosphoiipase may also be from a filamentous fungus strain within the genus Aspergillus, such as a strain of Aspergillus awamo ⁇ , 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.
  • 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 LECITASETM and LECfTASETM ULTRA, YlELSMAX, or UPOPAN F (available from Novozymes A/S, Denmark ⁇ .
  • Celtulolytic 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-gl ⁇ canase activity (EC 3,2.1.4) and beta-glucosidase activity (EC 3.2.121 ).
  • At least three categories of enzymes are important for converting DCiulose 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.
  • endc-glucanases EC 3.2.1.4 ⁇ that cut the cellulose chains at random
  • cellobiohydrolases EC 3.2.1.91
  • faeta-glucossdases EC 3.2.1.21 ⁇ that convert celiobiose and soluble cellodextrins into glucose.
  • 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 Chrys ⁇ sporium lucknowense.
  • 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 Chrys ⁇ sporium lucknowense.
  • 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.
  • cellulolytic enzyme preparation is a composition concerned in co-pending application US application # 60/941,251 , which is hereby incorporated by reference.
  • 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 T ⁇ choderma, Aspergillus or PerticiiHum, including the fusion protein having beta-glucosidase activity disclosed in co-pending application US 60/832.511 ⁇ Novozymes).
  • the cellulolytic enzyme preparation may also comprises a CBH II enzyme, preferably Thielavia termstns cellobiohydrolase SI CEL6A.
  • the cellulolytic enzyme preparation may also comprises cellulolytic enzymes, preferably one derived from T ⁇ choderma 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 DCiuiolytic enzymes derived from Trichoderma reesei. in an embodiment the cellulolytic enzyme composition is the commercially available product
  • 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.
  • 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. Cft ⁇ tv. 59: 257-268, in a preferred embodiment enclogl ⁇ canases may be derived from a strain of the genus Trichoderma, preferably a strain of Trichoderma reese ⁇ , a strain of the genus Humicola, such as a strain of Humicola insolens; or a strain of Chrysosponum, preferably a strain of Chrysosporium lucknowense.
  • CMC carboxymethyl cellulose
  • 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.
  • cellobiohydroloses examples include CBH I and CBH Il from Trichoderma reseei; H ⁇ rnicoSa insoiens and CBH ! I from Thielavia tetvestris c ⁇ lSobiohydrolase (CELL6A)
  • Cellobiohydrolase activity may be determined according to the procedures described by Lever ⁇ t ai. 1972. Anal Biochem, 47: 273-279 and by van Tiibeurgh ⁇ i aL, 1982, FEBS L ⁇ tters 149: 152-156; van Tiibeurgh and Claeyssens, 1985 r FEBS L ⁇ tters 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-glucosidases may be present during hydrolysis.
  • 'beta-gl ⁇ cosidase means a beta-D-gl ⁇ coside 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.
  • beta-glucosidase activity is determined according to the basic procedure described by Venturi et at., 2002, J. Basic Microbiol.
  • beta-glucosidase activity is defined as 1.0 ⁇ mole 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.
  • beta-glucosidase is of fungal origin, such as a strain of the genus Trichoderma.. Aspergillus or Penicillium.
  • 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).
  • 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),
  • 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.
  • hemicellulase suitable for use in hydrolyzing hemicellulose, preferably into xylose may be used.
  • Preferred hemiceiiulases include xylanases, arabinofuranosidases, acetyi xylan esterase, fer ⁇ loyl esterase, glucuronidases, endo-galactanase. mannases, endo or exo arabinases, exo- galactanses, and mixtures of two or more thereof.
  • 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 ViSCOZYIvlETM (available from Novozymes A/S, Denmark). in an embodiment the hemicellulase is a xylanase.
  • the xylanase may preferably be of microbial origin, such as of fungal origin (e.g., Trichoderma, Me ⁇ pilus, 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.
  • fungal origin e.g., Trichoderma, Me ⁇ pilus, Humicoia, Aspergillus, Fusarium
  • a bacterium e.g., Bacillus ⁇
  • the xylanase is derived from a filamentous fungus, preferably derived from a strain of Aspergillus, such as Aspergillus aculeaius;
  • 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 SHEARZYfviETM and BiOFEED WHEATTM 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.-% ⁇ f 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.
  • ce ⁇ uiolyfjc enhancing activity is defined herein as a biological activity that enhances the hydrolysis of a lignocellulose derived materia! by proteins having cellulolytic activity.
  • c ⁇ lluiolytic 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
  • 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.
  • 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 T ⁇ choderma 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • proteases within E.C. 3.4.22.* 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).
  • the protease is a protease preparation derived from a strain of
  • the protease is derived from a strain of Rhizomucor, preferabiy Rhizom ⁇ cor mehei.
  • 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 Rhizom ⁇ cor 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.
  • 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.
  • 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 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
  • FPU assay Cellulase Activitv Usino Filter Paper 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, P ⁇ re & Appi. Chem. 59, pp. 257-268, 1987.
  • 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.
  • 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.
  • a reagent blank is prepared by adding 1 ,5 mL of citrate buffer to a test tube.
  • 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.
  • Glucose Sisndards 24.1 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.
  • Glucose standard tubes are prepared by adding 0,5 mL of each dilution to 1.0 mL of citrate buffer.
  • glucose standard tubes are assayed in the same manner as the enzyme assay tubes, and done along with them
  • 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.
  • 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 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

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Abstract

La présente invention concerne des procédés de production d'un produit de fermentation à partir d'un matériau contenant du bois, le procédé comprenant les étapes de i) pré-traitement du matériau contenant du bois ; ii) hydrolyse en soumettant ledit matériau pré-traité contenant du bois à une ou des enzymes cellulolytiques ; iii) fermentation à l'aide d'un organisme de fermentation ; le matériau contenant du bois étant soumis à une ou des estérases avant et/ou pendant le pré-traitement de l'étape i) et/ou l'hydrolyse de l'étape ii) et/ou la fermentation de l'étape iii).
PCT/US2008/077421 2007-09-25 2008-09-24 Procédés de production de produits de fermentation WO2009042622A2 (fr)

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WO2013016115A1 (fr) 2011-07-22 2013-01-31 Novozymes North America, Inc. Procédés pour prétraiter un matériau cellulosique et améliorer l'hydrolyse de celui-ci
US20130109071A1 (en) * 2010-07-07 2013-05-02 Novozymes North America, Inc. Fermentation Process
US20130288296A1 (en) * 2010-11-02 2013-10-31 Novozymes, Inc. Methods of Pretreating Cellulosic Material with a Family 61 Polypeptide
US9752168B2 (en) 2010-08-12 2017-09-05 Novozymes, Inc. Compositions comprising a polypeptide having cellulolytic enhancing activity and a quinone compound and uses thereof
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US9783793B2 (en) 2011-04-06 2017-10-10 Teknologian Tutkimuskeskus Vtt Polypeptides and active fragments of polypeptides having at least one esterase activity
CN109022518A (zh) * 2011-07-22 2018-12-18 诺维信北美公司 用于预处理纤维素材料和改进其水解的方法
CN103703139A (zh) * 2011-07-22 2014-04-02 诺维信北美公司 用于预处理纤维素材料和改进其水解的方法
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CN107858393B (zh) * 2017-12-13 2021-06-08 云南省林业科学院 一种从核桃粕中提取蛋白多肽的方法
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