WO2008023060A1 - Procédé de fermentation - Google Patents

Procédé de fermentation Download PDF

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Publication number
WO2008023060A1
WO2008023060A1 PCT/EP2007/058821 EP2007058821W WO2008023060A1 WO 2008023060 A1 WO2008023060 A1 WO 2008023060A1 EP 2007058821 W EP2007058821 W EP 2007058821W WO 2008023060 A1 WO2008023060 A1 WO 2008023060A1
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WIPO (PCT)
Prior art keywords
beta
fbg
glucanase
starch
mash
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PCT/EP2007/058821
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English (en)
Inventor
Soeren Trojaborg
Ole Bill Joergensen
Claudio Visigalli
Ramiro Martinez Gutierrez
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Novozymes A/S
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Publication of WO2008023060A1 publication Critical patent/WO2008023060A1/fr

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    • 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
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/244—Endo-1,3(4)-beta-glucanase (3.2.1.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01006—Endo-1,3(4)-beta-glucanase (3.2.1.6)
    • 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 invention relates to a process for producing a fermentation product wherein the viscosity of the mash is reduced by application of a thermostable beta-glucanase.
  • Fermentation processes are used for making a vast number of products of commercial interest. Fermentation is used in industry to produce simple compounds such as alcohols (in particular ethanol); acids, such as citric acid, itaconic acid, lactic acid, gluconic acid, lysine; ketones; amino acids, such as glutamic acid, but also more complex compounds such as antibiotics, such as penicillin, tetracyclin; enzymes; vitamins, such as riboflavin, B 12 , beta- carotene; hormones, such as insulin which are difficult to produce synthetically. Also in the brewing (beer and wine industry), dairy, leather, tobacco industries fermentation processes are used.
  • the object of the invention is to provide an improved method of fermentation processes for producing e.g., ethanol.
  • the present invention relates to an improved process of producing a fermentation product, in particular ethanol.
  • the invention provides in a first aspect a method of producing a fermentation product , said method comprising the steps of: a) providing a mash comprising starch and non-starch polysaccharides and water; b) preliquefying the mash of step (a), c) gelatinizing the mash of step (b), d) liquefying the mash of step (c), e) saccharifying and fermenting the mash of step (d) to produce ethanol, and f) recovering the fermentation product, wherein step b, c and/or d is performed in the presence of a thermostable beta-glucanase, said thermostable beta- glucanase having an amino acid sequence having at least 60% identity to the amino acid sequence shown as SEQ ID NO:2 or as amino acids 31-335 in SEQ ID NO:2.
  • a method of producing a fermentation product comprising the steps of: a) providing a mash comprising starch and non-starch polysaccharides and water; b) preliquefying the mash of step (a), c) gelatinizing the mash of step (b), d) liquefying the mash of step (c), e) saccharifying and fermenting the mash of step (d) to produce ethanol , and f) recovering the fermentation product, wherein step b, c and/or d is performed in the presence of a thermostable beta-glucanase, said thermostable beta- glucanase being present in an amount of at least 100 FBG/kg DS.
  • a method of producing a fermentation product comprising the steps of: a) providing a mash comprising starch and non-starch polysaccharides and water; b) preliquefying the mash of step (a), c) gelatinizing the mash of step (b), d) liquefying the mash of step (c), e) saccharifying and fermenting the mash of step (d) to produce ethanol, and f) recovering the fermentation product, wherein step b, c and/or d is performed in the presence of a thermostable beta-glucanase and step e is performed in the presence of a beta-glucosidase.
  • thermostable beta-glucanase in an amount of 500-10000 FBG/g, preferably 1000-7500 FBG/g, more preferably 500-5000 FBG/g, even more preferably 2000-4000 FBG/g and most preferably 2500-3500 FBG/g
  • a composition according to the fourth aspect in a process for production of ethanol, preferably to be used as a potable ethanol, a fuel ethanol and/or a fuel additive.
  • thermostable beta-glucanase shown in SEQ ID NO:2 and/or a thermostable beta-glucanase in an amount of at least 100 FBG/kg DS, reduces the viscosity significantly compared to using prior art compositions comprising non-starch polysaccharide degrading enzymes.
  • the viscosity following liquefaction and cooling to 50 0 C is as low or lower than the viscosity before the gelatinization step.
  • the reduced viscosity results in increased flow rates of the liquefied mash, thereby increasing the capacity of the production plants, especially by improving heat transfer and facilitating passage of the liquefied mash through the mash coolers.
  • the process of the invention facilitates the use of higher dry matter percentage in the fermentation while still securing an efficient cooling and a correct and uniform temperature of the mash delivered to the fermentation tanks.
  • the effect on the by-products, such as the distiller's dry grain, of the prior hydrolysis of the non-starch polysaccharides is an overall improved feed conversion and better digestibility of the nutrients like minerals, protein, lipids and starch.
  • the ethanol yield may also be increased as the non-starch polysaccharides may be degraded to fermentable sugar using the thermostable beta-glucosidase in combination with a beta-glucosidase.
  • the process of the invention may be used in the production of a large number of fermentation products comprising but not limited to alcohols, such as ethanol; acids, such as citric acid, itaconic acid, lactic acid, gluconic acid, lysine; ketones; amino acids, such as glutamic acid, but also more complex compounds such as antibiotics, such as penicillin, tetracyclin; enzymes; vitamins, such as riboflavin, B12, beta-carotene; hormones, such as insulin.
  • alcohols such as ethanol
  • acids such as citric acid, itaconic acid, lactic acid, gluconic acid, lysine
  • ketones amino acids, such as glutamic acid, but also more complex compounds such as antibiotics, such as penicillin, tetracyclin
  • enzymes such as antibiotics, such as penicillin, tetracyclin
  • vitamins such as riboflavin, B12, beta-carotene
  • hormones such as insulin.
  • drinkable ethanol as
  • starch containing material may be used as raw material in the process of the present invention.
  • the starch containing material is whole grain obtained from cereals, preferably selected from the list consisting of corn (maize), wheat, barley, oat, rice, cassava, sorghum, rye, milo, and millet.
  • the starch containing material may be obtained from tubers, potato, sweet potato, cassava, tapioca, sago or banana.
  • Sugar cane or sugar beet may be utilized as described in e.g. GB 2115820 A and US4886672A1.
  • Preferred for the process of the invention are starch containing materials such as cereals, e.g.
  • Such starch containing materials also comprise amounts of non-starch polysaccharides which course increased viscosity during mashing therefore thinning is advantageous.
  • the main process steps of the present invention accoding to the first, second and third aspects be described as separated into the following main process stages: (a) mash formation; (b) preliquefaction; (c) gelatinization; (d); liquefaction; and (e) saccharification and fermentation, wherein the steps (a), (b), (c) and (d) is performed in the order (a), (b), (c), (d) and (e).
  • Step (e) may be performed as a simultaneous saccharification and fermentation (SSF) or as two separate sub steps.
  • SSF simultaneous saccharification and fermentation
  • the individual process steps of the process may be performed batch wise or as a continuous flow.
  • processes where all process steps are performed batch wise, or processes where all process steps are performed as a continuous flow, or processes where one or more process step(s) is(are) performed batch wise and one or more process step(s) is(are) performed as a continuous flow are equally preferred.
  • the cascade process is an example of a process where one or more process step(s) is(are) performed as a continuous flow and as such preferred for the invention.
  • process step(s) is(are) performed as a continuous flow and as such preferred for the invention.
  • alcohol Textbook Ethanol production by fermentation and distillation. Eds. T. P. Lyons, D. R. Kesall and J. E. Murtagh. Nottingham University Press 1995.
  • the starch containing material is milled cereals, preferably barley, and the method comprises a step of milling the cereals before step (a).
  • the invention also encompasses processes of the invention, wherein the starch containing material is obtainable by a process comprising milling of cereals, preferably dry milling, e.g. by hammer or roller mils. Grinding is also understood as milling, as is any process suitable for opening the individual grains and exposing the endosperm for further processing. Two processes of milling are normally used in ethanol production: wet and dry milling. The term "dry milling" denotes milling of the whole grain. In dry milling the whole kernel is milled and used in the remaining part of the process
  • the mash may be provided by forming a slurry comprising the milled starch containing material and water.
  • the water may be heated to a suitable temperature prior to being combined with the milled starch containing material in order to achieve a mash temperature of 45 to 70 0 C, preferably of 53 to 66°C, more preferably of 55 to 60 0 C.
  • the mash is typically formed in a tank known as the slurry tank.
  • dry solids% dry solid percentage
  • dry solid percentage in the slurry tank is in the range from 1-60%, in particular 10-50%, such as 20-40%, such as 25- 35%.
  • the starch containing material (front end mash) is held in the presence of a thermostable beta-glucanase and optionally other thinning enzymes, such as a xylanase, a cellulases, and/or a hemicellulase, at a temperature of 40 to 70 0 C, more preferably to 45 to 60°C, most preferably to 48 to 55°C, such as 50 0 C.
  • the duration of the preliquefaction step is preferably 5 to 60 minutes, and more preferably 10 to 30 minutes, such as around 15 minutes.
  • An alpha-amylase, preferably a thermostable alpha-amylase may be added before or during the preliquefaction step.
  • the beta-glucanase preferred for the invention and shown in SEQ ID NO:2 is thermostable and usually need only be added once to the process, e.g. before or during the preliquefaction step (b), in order to be present and/or active during the process steps (b), (c) and/or (d).
  • the starch is gelatinized.
  • Gelatinization may be achieved by heating the starch containing slurry to a temperature above the gelatinization temperature of the particular starch used.
  • Gelatinization is preferably by jet-cooking at appropriate conditions, such as, e.g. at a temperature between 85-140 0 C, preferably 90- 125°C, such as 120 0 C to complete gelatinization of the starch.
  • gelatinization by non-pressure cooking.
  • enzymes added in the preliquefaction step will be subjected to elevated temperatures and may be partly inactivated.
  • further thinning enzymes including additional beta-glucanase may be added following the gelatinization step.
  • An alpha-amylase preferably a thermostable alpha-amylase, may be added before, during and/or following the gelatinization step.
  • the gelatinized starch (down stream mash) is broken down (hydrolyzed) into maltodextrins (dextrins).
  • a suitable enzyme preferably an alpha-amylase, is added.
  • thermostable beta-glucanase and optionally other thinning enzymes are present and/or added to the mash during liquefaction.
  • the temperature during the liquefaction step is from 60-95 0 C, preferably 80-90°C, preferably at 70-80 0 C such as 85°C, for a period of 30-240 min, preferably for 60-120 min, such as 150 min.
  • the liquefaction in step (d) is performed at a pH in the range of 3.5- 7.0, preferably pH 4.0-6.5 and more preferably pH 4.5-6.0.
  • the pH during the liquefaction is 4-5.
  • the pH of the slurry may by adjusted or not, depending on the properties of the enzymes used.
  • the pH is adjusted, e.g. about 1 unit upwards, e.g. by adding NH 3 .
  • the adjusting of pH is advantageously done at the time when the alpha-amylase is added.
  • the pH is not adjusted and the alpha-amylase has a corresponding suitable pH-activity profile, such as being active at a pH about 4.
  • An alpha-amylase preferably a thermostable alpha-amylase, may be added before or during the gelatinization step.
  • further thinning enzyme(s) is(are) added to the gelatinized mash together with the alpha-amylase.
  • the saccharification step and the fermentation step may be performed as separate process steps or as a simultaneous saccharification and fermentation (SSF) step.
  • the saccharification is carried out in the presence of a saccharifying enzyme, e.g. a glucoamylase, a beta-amylase, a beta-glucosidase or a maltogenic amylase.
  • a saccharifying enzyme e.g. a glucoamylase, a beta-amylase, a beta-glucosidase or a maltogenic amylase.
  • a phytase, and/or a protease is added.
  • the fermenting organism may be a fungal organism, such as yeast, or bacteria.
  • Suitable bacteria may e.g. be Zymomonas species, such as Zymomonas mobilis and E. coli.
  • filamentous fungi include strains of Penicillium species.
  • Preferred organisms for ethanol production are yeasts, such as e.g. Pichia or Saccharomyces.
  • Preferred yeast according to the invention is Saccharomyces species, in particular Saccharomyces cerevisiae or bakers yeast.
  • the yeast cells may be added in amounts of 10 5 to 10 12 , preferably from 10 7 to 10 10 , especially 5x10 7 viable yeast count per ml of fermentation broth.
  • yeast cell count should preferably be in the range from 10 7 to 10 10 , especially around 2 x 10 8 .
  • the alcohol Textbook Editors K. Jacques, T. P. Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom 1999, which is hereby incorporated by reference
  • the microorganism used for the fermentation is added to the mash and the fermentation is ongoing until the desired amount of fermentation product is produced; in a preferred embodiment wherein the fermentation product is ethanol to be recovered this may, e.g., be for 24-96 hours, such as 35-72 hours, or such as 40-60 hours.
  • the temperature and pH during fermentation is at a temperature and pH suitable for the microorganism in question and with regard to the intended use of the fermentation product, such as, e.g., in an embodiment wherein the fermenting organism is yeast and the product is ethanol for recovery the preferred temperature is in the range about 26-34°C, e.g. about 32°C, and at a pH e.g. in the range about pH 3-6, e.g. about pH 4-5.
  • the temperature of the mash is around 12-16°C, such around 14°C.
  • a simultaneous saccharification and fermentation (SSF) process is employed where there is no holding stage for the saccharification, meaning that yeast and saccharification enzyme is added essentially together.
  • SSF simultaneous saccharification and fermentation
  • a pre-saccharification step at a temperature above 50 0 C is introduced just prior to the fermentation.
  • the method of the invention may further comprise recovering of the fermentation product, e.g. ethanol; hence the ethanol may be separated from the fermented material and purified.
  • the method of the invention comprises distillation to obtain the ethanol.
  • the aqueous by-product (Whole Stillage) from the distillation process is separated into two fractions, for instance by centrifugation: 1 ) Wet Grain (solid phase), and 2) Thin Stillage (supernatant).
  • the Wet Grain fraction is dried, typically in a drum dryer.
  • the dried product is referred to as "Distillers Dried Grain", and can be used as animal feed.
  • the Thin Stillage fraction may be evaporated to produce a syrup fraction, mainly consisting of limit dextrins and non fermentable sugars.
  • the syrup fraction may be introduced into a dryer together with the Wet Grain (from the Whole Stillage separation step) to provide a product referred to as "Distillers Dried Grain", which can be used as animal feed.
  • the composition provided by the invention comprises a thermostable beta-glucanase in an amount of 500-10000 FBG/g, preferably 1000-7500 FBG/g, more preferably 500-5000 FBG/g, even more preferably 2000-4000 FBG/g and most preferably 2500-3500 FBG/g.
  • the composition may further comprise an enzyme selected from the group consisting of xylanase, cellulase, hemicellulase, alpha-amylase, glucoamylase, beta-glucosidase. protease and phytase.
  • composition may be applied in any process comprising hydrolysis of a starch, e.g, in the process of the invention, i.e. in a fermentation process, such as an ethanol process,
  • the ethanol obtained by the process of the invention may be used as, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits, or industrial ethanol, including fuel additive.
  • Beta-qlucanase (E.C. 3.2.1.4)
  • the beta-glucanase to be applied in the process and/or composition of the present invention is a thermostable beta-glucanase.
  • the beta-glucanase may be of microbial origin, such as derivable from a strain of a bacterium (e.g. Bacillus) or from a filamentous fungus (e.g., Aspergillus, Trichoderma, Humicola, Fusa ⁇ um).
  • a bacterium e.g. Bacillus
  • a filamentous fungus e.g., Aspergillus, Trichoderma, Humicola, Fusa ⁇ um
  • the beta-glucanase is a thermostable beta-glucanase.
  • the beta-glucanase is derived from Thermoascus aurantiacus.
  • beta-glucanase is derived from Thermoascus aurantiacus and has the sequence shown as amino acids 31-335 SEQ ID NO:2 which is encoded by the DNA sequence shown as nucleotides 91 to 1005 in SEQ ID NO:1.
  • the beta-glucanase preferably has at least 70% identity to the amino acid sequence shown in SEQ ID NO:2, and preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to the amino acid sequence shown in SEQ ID NO:2.
  • the beta- glucanase to be applied in the process and/or composition of the present invention is a beta- glucanase having at least 70% identity to the sequence shown as amino acids 31-335 in SEQ ID NO:2, and preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to the sequence shown in as amino acids 31-335 SEQ ID NO:2.
  • thermostable beta- glucanase is added and/or present in the preliquefaction step, gelatinizion step, liquefaction step, saccharification step and/or fermentation step in an amount of 150-2000 FBG/kg DS, preferably 200-1500 FBG/kg DS, more preferably 250-1200 FBG/kg DS, even more preferably 300-1000 FBG/kg DS, yet more preferably 350-800 FBG/kg DS, and most preferably 400-600 FBG/kg DS.
  • the process of the invention is carried out in the presence of an effective amount of a suitable xylanase which may be derived from a variety of organisms, including fungal and bacterial organisms, such as Aspergillus, Disporotrichum, Penicillium, Neurospora, Fusarium and Trichoderma.
  • a suitable xylanase which may be derived from a variety of organisms, including fungal and bacterial organisms, such as Aspergillus, Disporotrichum, Penicillium, Neurospora, Fusarium and Trichoderma.
  • xylanases examples include xylanases derived from H. insolens (WO 92/17573; Aspergillus tubigensis (WO 92/01793); A. niger (S hei et al., 1985, Biotech, and Bioeng. Vol. XXVII, pp. 533-538, and Fournier et al., 1985, Bio-tech. Bioeng. Vol. XXVII, pp. 539-546; WO 91/19782 and EP 463 706); A. aculeatus (WO 94/21785).
  • the xylanase may also be a 1 ,3-beta-D-xylan xylanohydrolase (EC. 3.2.1.32).
  • the xylanase having is Xylanase Il disclosed in WO 94/21785.
  • compositions comprising xylanase include SHEARZYME® 200L, SHEARZYME® 500L, BIOFEED WHEAT®, and PULPZYMETM HC (from Novozymes) and GC 880, SPEZYME® CP (from Genencor Int).
  • Xylanases may be added in the amounts of 1.0-1000 FXU/kg dry solids, preferably from 5-500 FXU/kg dry solids, preferably from 5-100 FXU/kg dry solids and most preferably from 10-100 FXU/kg dry solids.
  • the process of the invention is carried out in the presence of an effective amount of a suitable alpha-amylases.
  • Preferred alpha-amylases are of bacterial or fungal origin.
  • a Bacillus alpha-amylases (often referred to as "Termamyl-like alpha-amylases"), variant and hybrids thereof, are preferred according to the invention.
  • Well-known Termamyl- like alpha-amylases include alpha-amylase derived from a strain of B. licheniformis (commercially available as TermamylTM), B. amyloliquefaciens, and B. stearothermophilus alpha-amylase.
  • a suitable bacterial alpha-amylase may be the alpha-amylase derived from B. stearothermophilus and having the amino acid sequence disclosed as SEQ.NO:4 in WO99/19467.
  • a suitable fungal alpha-amylases may be derived from Aspergillus, such as an acid fungal alpha-amylase derived from Aspergillus niger.
  • the acid fungal alpha-amylase may preferably comprise a starch binding domain (SBD).
  • Fungal alpha-amylases may be added in the liquefaction step (d) in an amount of 0.001-1.0 AFAU/g dry solids, preferably from 0.002-0.5 AFAU/g dry solids, preferably 0.02- 0.1 AFAU/g dry solids.
  • Bacillus alpha-amylases may be added in effective amounts well known to the person skilled in the art.
  • alpha-amylase products and products containing alpha-amylases include TERMAMYLTM SC, FUNGAMYLTM, LIQUOZYMETM SC, SANTM SUPER, SPIRIZYMETM FUEL and SPIRIZYMETM PLUS (Novozymes A/S, Denmark) and DEX-LOTM, SPEZYMETM AA, SPEZYMETM DELTA AA and SPEZYMETM ETHYL (from Genencor Int.).
  • the alpha-amylase may be a maltogenic alpha-amylase.
  • Maltogenic amylases (glucan 1 ,4-alpha-maltohydrolase, E. C. 3.2.1.133) are able to hydrolyse amylose and amylopectin to maltose in the alpha-configuration.
  • a maltogenic amylase is able to hydrolyse maltotriose as well as cyclodextrin.
  • a specifically contemplated maltogenic amylase includes the one disclosed in EP patent no. 120,693 derived from Bacillus stearothermophilus C599.
  • a commercially available maltogenic amylase is MALTOGENASETM from Novozymes A/S.
  • the saccharification step or the simultaneous saccharification and fermentation step may be carried out in the presence of a glucoamylase.
  • the glucoamylase may be of any origin, e.g. derived from a microorganism or a plant.
  • Preferred is glucoamylase of fungal or bacterial origin selected from the group consisting of Aspergillus niger glucoamylase, in particular A. niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1 102), or variants thereof, such as disclosed in WO 92/00381 and WO 00/04136; the A. awamori glucoamylase (WO 84/02921 ), A. oryzae (Agric. Biol. Chem. (1991 ), 55 (4), p. 941-949), or variants or fragments thereof.
  • Glucoamylases may in an embodiment be added in the saccharification and/or fermentation step (e) in an amount of 0.02-2 AGU/g dry solids, preferably 0.1-1 AGU/g dry solids, such as 0.2 AGU/g dry solids.
  • Addition of protease(s) in the saccharification step, the SSF step and/or the fermentation step increase(s) the FAN (Free amino nitrogen) level and increase the rate of metabolism of the yeast and may increase the fermentation efficiency.
  • FAN Free amino nitrogen
  • Suitable proteases include microbial proteases, such as fungal and bacterial proteases.
  • Preferred proteases are acidic proteases, i.e., proteases characterized by the ability to hydrolyze proteins under acidic conditions below pH 7.
  • Suitable acid fungal proteases include fungal proteases derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra, Irpex, Penicillium, Sclerotiumand Torulopsis.
  • 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. Chem. Soc.
  • ALCALASETM is a Bacillus licheniformis protease (subtilisin Carlsberg). ALCALASETM may according to the invention preferably be added is amounts of 10 "7 to 10 "3 gram active protease protein/g dry solids, in particular 10 "6 to 10 "4 gram active protease protein/g dry solids, or in amounts of 0.1-0.0001 AU/g dry solids, preferably 0.00025-0.001 AU/g dry solids.
  • F LAVO U RZYM E TM (available from Novozymes A/S) is a protease preparation derived from Aspergillus oryzae.
  • F LAVO U RZYM E TM may according to the invention preferably be added in amounts of 0.01-1.0 LAPU/g dry solids, preferably 0.05-0.5 LAPU/g dry solids.
  • a suitable dosage of the protease is in the range in an amount of 10 "7 to 10 "3 gram active protease protein/g dry solids, in particular 10 "6 to 10 "4 gram active protease protein/g dry solids.
  • Acid protease is often present as significant side effects in commercial preparations of fungal alpha-amylases and/or fungal glucoamylases.
  • the phytase used according to the invention may be any enzyme capable of effecting the liberation of inorganic phosphate from phytic acid (myo-inositol hexakisphosphate) or from any salt thereof (phytates).
  • a suitable dosage of the phytase is in the range from 0.005-25 FYT/g dry solids, preferably 0.01-10 FYT/g, such as 0.1-1 FYT/g dry solids.
  • beta-glucosidase is defined herein as a beta-D-glucoside glucohydrolase
  • Suitable beta-glucosidases include microbial beta-glucosidases, such as fungal and bacterial beta-glucosidases.
  • beta-glucosidases include, but are not limited to, an Aspergillus oryzae beta-glucosidase (WO 02/095014; WO 04/099228); Aspergillus aculeatus beta-glucosidase (Kawaguchi et ai, 1996, Gene 173: 287-288); Aspergillus avenaceus (GenBankTM accession no. AY943971 ); Aspergillus fumigatus (GenBankTM accession no.
  • Preferred for the present invention is a beta-glucosidase derived from Aspergillus, preferably A. niger.
  • a beta-glucosidase is available as NOVOZYMTM 188 from Novozymes A/S, Denmark and may be present in the saccharification and/or fermentation step in an amount of preferably 0.001-1.00 kg/t dry solids, more preferably 0.05-0.50 kg/t dry solids and most preferably 0.01-0.10 kg/t dry solids.
  • Another protein which may be applied in the process of the present invention is a protein having cellulytic enhancement activity, such as a protein belonging to glycosyl hydrolase family 61 (GH61 ).
  • GH61 glycosyl hydrolase family 61
  • the protein is derived from Trichoderma reesei or Aspergillus oryzae. More preferably the protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even at least 100% homology to the amino acid sequences disclosed as SEQ ID NO: 2 in WO2005074656 or as SEQ ID NO: 2 in PCT/US2006/038556.
  • polypeptide identity is understood as the degree of identity between two sequences indicating a derivation of the first sequence from the second.
  • the identity may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 5371 1 ) (Needleman, S. B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453.
  • GAP creation penalty of 3.0
  • GAP extension penalty of 0.1.
  • the relevant part of the amino acid sequence for the identity determination is the mature polypeptide, i.e., without the signal peptide.
  • Experimental conditions for determining hybridization at low, medium, medium/high, high or very high stringency between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989) for 10 min, and prehybridization of the filter in a solution of 5 x SSC, 5 x Denhardt's solution (Sambrook et al. 1989), 0.5% SDS and 100 micrograms/ml of denatured sonicated salmon sperm DNA (Sambrook et al.
  • 5 x SSC Sodium chloride/Sodium citrate, Sambrook et al. 1989
  • 5 x Denhardt's solution Standardbrook et al. 1989
  • 0.5% SDS 100 micrograms/ml of denatured sonicated salmon sperm DNA
  • Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using an x-ray film.
  • the endoxylanase activity is determined by an assay, in which the xylanase sample is incubated with a remazol-xylan substrate (4-O-methyl-D-glucurono-D-xylan dyed with Remazol Brilliant Blue R, Fluka), pH 6.0. The incubation is performed at 5O 0 C for 30 min. The background of non-degraded dyed substrate is precipitated by ethanol. The remaining blue colour in the supernatant is determined spectrophotometrically at 585 nm and is proportional to the endoxylanase activity. The endoxylanase activity of the sample is determined relatively to an enzyme standard.
  • the assay is further described in the publication AF 293.6/1 -GB, available upon request from Novo Nordisk A/S, Denmark.
  • the cellulytic activity may be measured in fungal beta glucanase units (FBG).
  • FBG fungal beta glucanase units
  • One FBG is the amount of enzyme which according to the below outlined standard conditions, releases glucose or reducing carbohydrate with a reduction capacity equivalent to 1 mol glucose per minute.
  • Fungal beta glucanase reacts with beta glucan during the formation process to glucose or reducing carbohydrate which is determined as reducing sugar according to the Somogyi Nelson method.
  • the sample should be diluted to give an activity between 0.02-0.10 FBG/ml.
  • Substrate 0.5% beta glucan
  • the fungal beta glucanase activity is preferably determined using the method described in Novozymes Standard Method NZCN-RD-AD-SM-030. A folder describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.
  • the cellulytic activity may be measured in beta-glucanase units (BGU).
  • Beta-glucanase reacts with beta-glucan to form glucose or reducing carbohydrate which is determined as reducing sugar using the Somogyi-Nelson method.
  • 1 beta-glucanase unit (BGU) is the amount of enzyme which, under standard conditions, releases glucose or reducing carbohydrate with a reduction capacity equivalent to 1 ⁇ mol glucose per minute. Standard conditions are 0.5% beta-glucan as substrate at pH 7.5 and 30 0 C for a reaction time of 30 minutes.
  • a detailed description of the analytical method (EB-SM-0070.02/01 ) is available on request from Novozymes A/S.
  • the cellulytic activity may be measured in endo-glucanase units (EGU), determined at pH 6.0 with carboxymethyl cellulose (CMC) as substrate.
  • EGU endo-glucanase units
  • CMC carboxymethyl cellulose
  • a substrate solution is prepared, containing 34.0 g/l CMC (Hercules 7 LFD) in 0.1 M phosphate buffer at pH 6.0.
  • the enzyme sample to be analyzed is dissolved in the same buffer.
  • 5 ml substrate solution and 0.15 ml enzyme solution are mixed and transferred to a vibration viscosimeter (e.g. MIVI 3000 from Sofraser, France), thermostated at 4O 0 C for 30 minutes.
  • One EGU is defined as the amount of enzyme that reduces the viscosity to one half under these conditions.
  • the amount of enzyme sample should be adjusted to provide 0.01-0.02 EGU/ml in the reaction mixture.
  • the Novo Glucoamylase Unit is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute at 37°C and pH 4.3.
  • the activity is determined as AGU/ml by a method modified after (AEL-SM-0131 , available on request from Novozymes) using the Glucose GOD-Perid kit from Boehringer Mannheim, 124036. Standard: AMG-standard, batch 7-1195, 195 AGU/ml. 375 microL substrate (1 % maltose in 50 mM Sodium acetate, pH 4.3) is incubated 5 minutes at 37°C. 25 microL enzyme diluted in sodium acetate is added. The reaction is stopped after 10 minutes by adding 100 microL 0.25 M NaOH. 20 microL is transferred to a 96 well microtitre plate and 200 microL GOD-Perid solution (124036, Boehringer Mannheim) is added. After 30 minutes at room temperature, the absorbance is measured at 650 nm and the activity calculated in AGU/ml from the AMG-standard. A detailed description of the analytical method (AEL-SM- 0131 ) is available on request from Novozymes.
  • Acid alpha-amylase activity may be measured in AFAU (Acid Fungal Alpha-amylase Units), which are determined relative to an enzyme standard. 1 FAU is defined as the amount of enzyme which degrades 5.260 mg starch dry matter per hour under the below mentioned standard conditions.
  • Acid alpha-amylase an endo-alpha-amylase (1 ,4-alpha-D-glucan-glucanohydrolase, E. C. 3.2.1.1 ) hydrolyzes alpha-1 ,4-glucosidic bonds in the inner regions of the starch molecule to form dextrins and oligosaccharides with different chain lengths.
  • the intensity of color formed with iodine is directly proportional to the concentration of starch.
  • Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under the specified analytical conditions.
  • Buffer Citrate, approx. 0.03 M Iodine (12): 0.03 g/L
  • the amylolytic activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue color is formed, but during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which is compared to a colored glass standard.
  • KNU Kilo Novo alpha amylase Unit
  • the proteolytic activity may be determined with denatured hemoglobin as substrate.
  • Anson-Hemoglobin method for the determination of proteolytic activity denatured hemoglobin is digested, and the undigested hemoglobin is precipitated with trichloroacetic acid (TCA).
  • TCA trichloroacetic acid
  • the amount of TCA soluble product is determined with phenol reagent, which gives a blue color with tyrosine and tryptophan.
  • One Anson Unit is defined as the amount of enzyme which under standard conditions (i.e. 25°C, pH 7.5 and 10 min. reaction time) digests hemoglobin at an initial rate such that there is liberated per minute an amount of TCA soluble product which gives the same color with phenol reagent as one milliequivalent of tyrosine.
  • LAPU Leucine Amino Peptidase Unit
  • CBU Beta-glucosidase Activity
  • Beta-glucosidase (cellobiase EC 3.2.1.21 ) hydrolyzes beta-1 ,4 bonds in cellobiose to release two glucose molecules. The amount of glucose released is determined specifically and quantitatively using the hexokinase method as follows:
  • the increase in absorbance is then measured at 340 nm as the absorbance value for NADPH is high at this wavelength.
  • CBU beta-glucosidase unit
  • Composition 1 comprising 3,210 FBG/g of thermostable beta-glucanase derived from Thermoascus aurenticus and having the amino acid sequence shown in SEQ ID NO:1.
  • Composition 2 commercially available from Genencor Int. as "Laminex Super” comprising 267 FBG/g, 91 BGU/g and 217 FXU/g .
  • Composition 3 commercially available from Genencor Int. as "Laminex BG” comprising 479 FBG/g, 810 BGU/g, 70 EGU/g and 450 FXU
  • Composition 4 a bacterial alpha-amylase available from Novozymes A/S comprising 263 KNU/g.
  • Non-starch degrading enzyme compositions were tested in a slurry of 24% DS milled barley at pH 5.5 portioned into vials each holding 4Og. All treatments were performed in duplicate and received a dosage of bacterial alpha-amylase (Composition 4, 0.15 kg/t DS) and a dosage of a non-starch degrading enzyme composition (Compositions 1 , 2, 3 or negative control, 0.15 kg/t DS). The treatments were placed in a Rapid ViscoTM Analyzer (RVA) and subjected to a temperature profile comprising a mixing step at 50 0 C for 15 min, ramp up to 90 0 C and stay for approx. 35 min, ramp-down to 50 0 C and stay for approx. 10 min. Data are presented in table 1.
  • RVA Rapid ViscoTM Analyzer
  • Stop heating 23 90 256 991 1014 1286
  • composition 1 reduced viscosity slightly more than compositions or 3.
  • composition 1 resulted in a viscosity in the range of 200 cPs, while compositions 2 or 3 were in the range of 700-900 cP.
  • composition 1 resulted in the lowest viscosity level (200 cP) and similar to the values measured before the liquefaction.
  • Compositions 2 or 3 resulted in much higher viscosity (1 ,500 cP), and the negative control was even higher (2,500 cP).

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Abstract

L'invention concerne un procédé de fabrication d'un produit de fermentation, selon lequel la viscosité de la trempe est réduite par application d'un bêta-glucanase thermostable.
PCT/EP2007/058821 2006-08-25 2007-08-24 Procédé de fermentation WO2008023060A1 (fr)

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WO2009148945A1 (fr) * 2008-05-29 2009-12-10 Danisco Us Inc., Genencor Division Procédé de fabrication d'alcool et de co-produit à partir de sorgho grain
WO2010128140A1 (fr) * 2009-05-07 2010-11-11 Danisco A/S Complexe enzymatique à partir d'enzymes de trichoderma reesei et p. funiculosum
CN101948881A (zh) * 2010-08-10 2011-01-19 宜兴协联生物化学有限公司 一种混合原料发酵生产柠檬酸的方法
US7943363B2 (en) 2008-07-28 2011-05-17 University Of Massachusetts Methods and compositions for improving the production of products in microorganisms
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CN102918160A (zh) * 2010-03-30 2013-02-06 诺维信北美公司 产生发酵产物的方法
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EP3017706A1 (fr) 2014-11-05 2016-05-11 Dupont Nutrition Biosciences ApS Enzymes de maltage
WO2016106432A3 (fr) * 2014-12-22 2016-10-06 Novozymes A/S Variants d'endoglucanase et polynucléotides codant pour ceux-ci
EP3156495A1 (fr) * 2008-08-11 2017-04-19 DSM IP Assets B.V. Dégradation de matière ligno-cellulosique
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US20180118794A1 (en) * 2012-05-09 2018-05-03 The University Of Akron Enzyme-based protein separation and enrichment from soy meal, wheat meal, and other protein-rich materials derived from plant seeds, fruits and other biomass
CN108669431A (zh) * 2018-05-18 2018-10-19 北京万霖星云生物科学研究院 一种小米高值化利用工艺方法
US10227613B2 (en) 2012-03-30 2019-03-12 Novozymes A/S Processes for producing fermentation products
WO2019200054A1 (fr) * 2018-04-11 2019-10-17 Locus Ip Company, Llc Production de micro-organismes anaérobies à l'aide d'un milieu nutritif rendu visqueux
CN115747262A (zh) * 2021-09-03 2023-03-07 国投生物科技投资有限公司 利用小麦生产乙醇的方法

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