Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
  • C12C5/00—Other raw materials for the preparation of beer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
  • C12C11/00—Fermentation processes for beer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
  • C12G1/00—Preparation of wine or sparkling wine
  • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
  • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
• • 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
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
  • C12N1/18—Baker's yeast; Brewer's yeast
• • 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
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
• • 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
• • 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 an improved fermentation process. More specifically the invention relates to a process for producing a fermentation product, in particular ethanol; a composition comprising at least a carbohydrate-source generating enzyme activity and at least an alpha-amylase activity and/or one or more yeast cell wall degrading enzymes, such as an enzyme preparation from Trichoderma, in particular T. harzianum sold under the tradename GLUCANEXTM; and the use of the composition of the invention for saccharification and/or fermentation product, in particular ethanol production.
• a fermentation product in particular ethanol
• a composition comprising at least a carbohydrate-source generating enzyme activity and at least an alpha-amylase activity and/or one or more yeast cell wall degrading enzymes, such as an enzyme preparation from Trichoderma, in particular T. harzianum sold under the tradename GLUCANEXTM
• GLUCANEXTM yeast cell wall degrading enzymes
• Fermentation processes are used for making a vast number of products of big 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; 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, 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 at least comprising a fermenting step.
• Fig. 1 shows schematically an ethanol production process of the invention.
• Fig. 2 shows the indexed CO 2 loss in the fermentation step of an ethanol process of the in- vention without backset of mash.
• Fig. 3 shows the indexed CO 2 loss in the fermentation step of an ethanol process of the invention with backset of mash.
• Fig. 4 shows the fermentation yield based on 69% starch content.
• the present invention relates to an improved process of producing a fermentation product, in particular ethanol, but also for instance the products mentioned in the "Background of the lnvention"-section. Also beverage production, such as beer or wine production is contemplated according to the invention.
• the invention relates to a process for producing a fermentation product, comprising a fermentation step, wherein the fermentation step is carried out in the presence of all of the following enzyme activities: carbohydrate-source generating enzyme activity, alpha-amylase activity, protease activity and debranching enzyme activity and/or one or more yeast cell wall degrading enzymes.
• enzyme activities may according to the invention be added during pre-saccharification, the propagation of fermenting organism cells and/or later on during the actual fermentation.
• Performing the fermentation step in the presence of a yeast cell wall degrading enzyme is advantageous at least partly due to the fact that the fermenting organism cells are hydrolysed during fermentation. This in situ hydrolysis of dead cells generates essential nutrients to the fermenting organism(s). Another advantage is that the yeast cell wall degrading enzyme(s) improve the yeast viability as it maintains a "fresh" population of fermenting cells throughout the fermentation. Further, fermenting in the presence of at least one yeast cell wall degrading enzyme result in increased fermentation rate and ethanol yield.
• GLUCANEXTM has a high effect on the initial fermentation rate: After only 24 hours using AMG E and GLUCANEXTM the ethanol yield was 96-97% as compared to using AMG E only.
• ethanol yield was increased approximately 10%.
• the higher ethanol yields indicate that the corn cell wall glucans are hydrolyzed/converted to glucose.
• the yeast cell wall degrading enzyme(s) may be selected from the group including beta-1 ,3-glucanase, 1 ,3-beta-glucanase, laminarinase, xylanase, chitinase, mannanase, alpha- 1 ,3-glucanase (mutanase).
• the yeast cell wall degrading enzyme is a preparation derived from Trichoderma, in particular Trichderma harzianum, such as the product GLUCANEXTM (available from Novozymes A/S) derived from Trichoderma harzianum.
• yeast cell wall degrading enzymes is made in combination with at least one "carbohydrate-source generating enzyme" or alternatively in the presence of a for the fermenting organism suitable carbohydrate source.
• carbohydrate-source generating enzyme includes glucoamylases (being a glu- cose generator), and beta-amylases and maltogenic amylases (being maltose generators).
• the carbohydrate-source generating enzymes are this way capable of providing energy to the fermenting microorganism(s) in question and/or may be converting directly or indirectly to the desired fermentation product.
• carbohydrate-source generating enzyme includes glucoamylases (being a glucose generator), and beta-amylases and maltogenic amylases (being maltose generators).
• the carbohydrate-source generating enzymes are this way capable of providing energy to the fermenting microorganism(s) in question and/or may be converting directly or indirectly to the desired fermentation product.
• Debranching enzyme include according to the present invention isoamylases and pullulanases.
• Debranching enzymes which can attack amylopectin are divided into two classes: isoamylases (E.C. 3.2.1.68) and pullulanases (E.C. 3.2.1.41), respectively.
• Isoamylase hydrolyses alpha-1 ,6-D-glucosidic branch linkages in amylopectin and beta-limit dextrins and can be distinguished from pullulanases by the inability of isoamylase to attack pullulan, and by the limited action on alpha-limit dextrins.
• the process of the invention may in one embodiment be an ethanol process comprising the below steps, wherein the enzymatic activities are added during pre-saccharification and/or during fermentation.
• the cell wall degrading enzyme(s) may according to the invention be added during the propagation of yeast cells and/or later on during the actual fermentation. Beverage production, such as beer or wine production is equally contemplated. Alcohol production, in particular ethanol production, from whole grain can be separated into 4 main steps
• the individual process steps of alcohol production may be performed batch wise or as a continuous flow.
• processes where one or more process step(s) is(are) performed batch wise or one or more process step(s) is(are) performed as a continuous flow are equally contemplated.
• processes where the fermentation step is performed as a continuous flow are also contemplated.
• 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 con- templated for the invention. Further information on the cascade process and other ethanol processes can be found in, e.g., "The Alcohol Textbook” Eds. T.P. Lyons, D.R. Kesall and J.E. Murtagh. Nottingham University Press 1995.
• Milling The raw material, such as whole grain, is milled in order to open up the structure and allowing for further processing.
• Two processes are preferred according to the invention: wet and dry milling.
• Preferred for ethanol production is dry milling where the whole kernel is milled and used in the remaining part of the process.
• Wet milling is Wet milling may also be used and gives a good separation of germ and meal (starch granules and protein) and is with a few exceptions applied at locations where there is a parallel production of syrups. Both dry and wet milling is well known in the art of, e.g., ethanol production.
• milled gelatinized (whole) grain raw material is broken down (hydrolyzed) into maltodextrins (dextrins) mostly of a DE higher than 4.
• the hydrolysis may be carried out by acid treatment or enzymatically by al- pha-amylase treatment, in particular with Bacillus alpha-amylases as will be described further below. Acid hydrolysis is used on a limited basis.
• the raw material is in one embodiment of the invention milled whole grain. However, a side stream from starch processing may also be used.
• enzymatic liquefaction is carried out as a three- step hot slurry process.
• the slurry is heated to between 60-95°C, preferably 80-85°C (in the Slurry Tank - see Fig. 1), and the enzyme(s) is(are) added to initiate liquefaction (thinning). Then the slurry is jet-cooked at a temperature between 95-140°C, preferably 105-125°C to complete gelanitization of the slurry. Then the slurry is cooled to 60-95°C and more en- zyme(s) is(are) added to finalize hydrolysis (secondary liquefaction). The liquefaction process is carried out at pH 4.5-6.5, in particular at a pH between 5 and 6. Milled and liquefied whole grains are known as mash.
• the maltodextrin from the liquefaction must be further hydrolyzed.
• the hydrolysis may be done enzymatically and a typically done using a glucoamylase: Alternatively alpha-glucosidases or acid alpha-amylases may be used.
• the carbohydrate source may be supplied by direct addition of, e.g., glucose or maltose.
• a full saccharification step may last up to from 24 to 72 hours, however, it is common only to do a pre-saccharification of typically 40-90 minutes at 30-65°C, typically at about 60°C and then complete saccharification during fermentation (SSF). Saccharification is typi- cally carried out at temperatures from 30-65°C, typically around 60°C, and at a pH between 4 and 5, normally at about pH 4.5.
• the fermenting organism may be yeast, in particular derived from Saccharomyces spp., especially Saccharomyces cere- visiae, which is added to the mash and the fermentation is ongoing for 24-96 hours, such as typically 35-60 hours.
• the temperature is between 26-34°C, in particular about 32°C, and the pH is from pH 3-6, preferably around pH 4-5.
• SSF simultaneous saccharification and fermen- tation
• SSF and SSYPF processes are equally contemplated. Further information on SSF and SSYPF processes may be found in , e.g., "The Alcohol Textbook” Editors. T.P. Lyons, D.R. Kesall and J.E. Murtagh. Nottingham University Press 1995.
• the mash may be distilled to extract the, e.g., alcohol product, in particular ethanol.
• the end product is ethanol, obtained according to the process of the invention, it may be used as, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits or industrial ethanol.
• the saccharification and fermentation may be carried out simultaneously or separately.
• the invention relates to a process for the production of ethanol, comprising the steps of:
• step (a) milling whole grains; (b) liquefying the product of step (a) in the presence of an alpha- amylase, (c) saccharifying the liquefied material obtained in step (b) in the presence of a phytase, (d) fermenting the saccharified material obtained in step (c) using a micro-organism; and optionally (e) distilling of the fermented and saccharified material obtained in step (d), providing two fraction: 1) an alcohol fraction and 2) a Whole Stillage fraction;
• the Thin Stillage is evaporated to provide two fractions: 1) Condensate and 2) Syrup, wherein the steps (c) and/or (d) are carried out in the presence of at least one carbohydrate-source generating enzyme activity and at least one alpha-amylase activity and/or one or more yeast cell wall degrading enzyme, in particular one or more activities as defined as cell wall degrading herein.
• protease activity and/or a debranching enzyme activity is(are) present as well.
• Preferred examples of enzymes are described below in the "ENZYMES" section.
• the whole grains in step a) are dry milled, for instance in a hammer mill.
• the DS% (dry solid percentage) in the slurry tank (containing milled whole grains) is in the range from 1-60%, in particular 10-50%, such as 20-40%, such as 25-35%.
• the liquefaction step comprising the following sub-steps: b1) the hot slurry is heated to between 60-95°C, preferably 80-85°C, and at least an alpha-amylase is added; b2) the slurry is jet-cooked at a temperature between 95-140°C, preferably 105- 125°C to complete gelanitization of the slurry; b3) the slurry is cooled to 60-95°C and more alpha-amylase is added to finalize hy- drolysis.
• the liquefaction process is in an embodiment carried out at pH 4.5-6.5, in particular at a pH between 5 and 6.
• Steps (c) and (d) may be carried out either simultaneously or separately/sequential. Further, after step (e) an optional ethanol recovery step n ay be added.
• Raw materials may be any starch-containing raw materials, such as tubers, roots, whole grains, corns, cobs, wheat, barley, rye, milo or cereals, sugar-containing raw materials, such as molasses, fruit materials, sugar, cane or sugar beet, potatoes, cellulose- containing materials, such as wood or plant residues.
• starch-containing raw materials such as tubers, roots, whole grains, corns, cobs, wheat, barley, rye, milo or cereals
• sugar-containing raw materials such as molasses, fruit materials, sugar, cane or sugar beet
• potatoes cellulose- containing materials, such as wood or plant residues.
• the raw material may be the side stream from starch processing, in particular liquefied starch with a DE of 6-20, in particular between 8-10.
• Suitable micro-organisms used for fermentation according to the invention are capable of fermenting sugars or converted sugars, such as glucose or maltose, directly or indirectly into the desired fermentation product.
• contemplated microorganisms include fungal organisms, such as yeast.
• Examples of specific filamentous fungi include strains of Penicillium sp.
• Preferred organisms for ethanol production is yeast.
• Preferred yeast according to the invention is baker's yeast, also known as Saccharomyces cerevisiae.
• the yeast may according to the invention preferably be added before starting the actual fermentation (i.e., during the propagation phase).
• the yeast cells may be added in amounts of 10 5 to 10 12 , preferably from 10 ⁇ to 10 10 , especially 5x10 7 viable yeast count per ml of fermentation broth. During the ethanol producing phase the yeast cell count should preferably be in the range from 10 7 to 10 10 , especially around 2 x 10 8 .
• Example 1 shows a fermentation process of the invention where the yeast is not stressed (yeast count of about 10 10 cells per ml). Further guidance in respect of using yeast for fermentation can be found in, e.g., "The alcohol Textbook” (Editors K. Jacques, T.P. Lyons and D.R.Kelsall, Nottingham University Press, United Kingdom 1999.
• the cell wall degrading enzyme may be any enzyme capable of degrading the cell wall of the fermenting organism(s) used according to the invention (i.e., the organism used in the fermentation step of the invention).
• Contemplated enzyme activities include: beta-1,3-glucanases, 1,3-beta-glucanases, laminarinases, xylanases, chitinases, mannanases, alpha-1 ,3-glucanases (mutanase).
• Beta-1.3-glucanases and Laminarinases include the group of endo-beta-1 ,3-glucanases also called laminarinases (E.C. 3.2.1.39 and E.C. 3.2.1.6, Enzyme Nomenclature, Academic Press, Inc, 1992).
• laminarinases E.C. 3.2.1.39 and E.C. 3.2.1.6, Enzyme Nomenclature, Academic Press, Inc, 1992.
• Pegg et al., Physiol. Plant Pathol., 21 , p. 389-409, 1982 showed that a purified endo-beta-1, 3- glucanase from tomato in combination with an exo-beta-1 ,3-glucanase of fungal origin were capable of hydrolysing isolated cell wall of the fungus Verticillium alboatrum.
• Keen et al., Plant Physiol., 71 , p. 460-465 showed that a purified beta-1
• xylanases The xylanase activity may be derived from any suitable organism, including fungal and bacterial organisms, such as Aspergillus, Disporotrichum, Penicillium, Neurospora, Fusarium and Trichoderma.
• xylanases examples include xylanases derived from H. insolens (WO
• the xylanase is Xylanase II disclosed in WO 94/21785.
• Contemplated commercially available xylanase include SHEARZYME®, BIOFEED WHEAT® (from Novozymes) and SPEZYME® CP (from Genencor Int., USA).
• Chitinases include the groups of exo-chitinases and endochitinases. Exochitinases are also referred to as chitobiosidases or beta-N-acetylhexosaminidases (E.C. 3.2.1.52, Enzyme Nomenclature, Academic Press, Inc., 1992). Endochitinases (E.C. 3.4.1.14) are enzymes, which randomly hydrolyse N-acetyl-beta-D-glucosaminide 1 ,4-beta-linkages of chitin and chito- dextrins.
• Fungal chitinases include the ones described by Harman et al., (1993), Mol. Plant Pathology 83, 313-318; Blaiseau and Lafay, (1992), Elsevier science publisher B.V., 243-248; and Gracia, (1994), Current Genetics 27, 83-89.
• chitinases include the ones described in WO 92/22314 (Cornell Research Foundation, INC) describes two chitinases from Trichoderma harzianum P1 (ATCC 74058); WO 94/24288 and WO 94/02598 (Cornell Research Foundation, INC) disclosing two chitinases from Trichoderma harzianum P1 (ATCC 74058); and EP 440.304 which concerns plants exhibiting a relative overexpression of at least one gene encoding intracellular chitinase and intra- or extracellular beta-1 ,3 glucanase.
• Mannanases have been identified in several Bacillus organisms. For example, Talbot et al., Appl. Environ. Microbiol., Vol.56, No. 11, pp. 3505-3510 (1990) describes a beta- mannanase derived from Bacillus stearothermophilus in dimer form having molecular weight of 162 kDa and an optimum pH of 5.5-7.5. Mendoza et al., World J. Microbiol. Biotech., Vol.
• pp. 551-555 (1994) describes a beta-mannanase derived from Bacillus subtilis having a molecular weight of 38 kDa, an optimum activity at pH 5.0 and 55°C and a pi of 4.8.
• JP-03047076 discloses a beta-mannanase derived from Bacillus sp., having a molecular weight of 37+3 kDa measured by gel filtration, an optimum pH of 8-10 and a pi of 5.3-5.4.
• JP-63056289 describes the production of an alkaline, thermostable beta-mannanase which hydrolyses beta-1 ,4-D-mannopyranoside bonds of e.g. mannans and produces manno- oligosaccharides.
• JP-63036774 relates to the Bacillus microorganism FERM P-8856, which produces beta-mannanase and beta-mannosidase at an alkaline pH.
• JP-08051975 discloses alkaline beta-mannanases from alkalophilic Bacillus sp. AM-001.
• WO 97/11164 A purified mannanase from Bacillus amyloliquefaciens useful in the bleaching of pulp and paper and a method of preparation thereof is disclosed in WO 97/11164.
• WO 94/25576 discloses an enzyme from Aspergillus aculeatus, CBS 101.43, exhibiting mannanase activity, which may be useful for degradation or modification of plant or algae cell wall material.
• WO 93/24622 discloses a mannanase isolated from Trichoderma reseei useful for bleaching lignocellulosic pulps.
• Mutanases are alpha-1 ,3-glucanases (also known as ⁇ -1 ,3-glucanohydrolases), which degrade the alpha-1 , 3-glycosidic linkages in mutan. Mutanases have been described from two species of Trichoderma (Hasegawa et al., (1969), Journal of Biological Chemistry 244, p. 5460-5470; Guggenheim and Haller, (1972), Journal of Dental Research 51, p. 394-402) and from a strain of Streptomyces (Takehara et al., (1981), Journal of Bacteriology 145, p. 729- 735), Cladosporium resinae (Hare et al.
• Preferred cell wall degrading enzymes have an optimum activity within the pH and temperature of the fermentation step, i.e., at acidic pH, in particular at a pH between 3-6, preferably between pH 4-5 and a temperature between 26-34°C, in particular about 32°C.
• Carbohydrate-source generating enzymes include any enzyme capable of generating a carbohydrate source, which the fermenting organism can use as energy source, for the fermentation or directly or indirectly converting into the desired fermentation product.
• Specifically contemplated carbohydrate-source generating enzymes are glucoamylase, beta-amylase, and maltogenic amylase.
• Glucoamylase derived from a microorganism or a plant.
• a glucoamylase of fungal or bacterial origin selected from the group consisting of Aspergillus glucoamylases, in particular A. niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102), or variants thereof, such as disclosed in WO 92/00381 and WO 00/04136; the A. awamori glucoamylase (WO 84/02921), A. oryzae (Ag- ric. Biol. Chem. (1991), 55 (4), p. 941-949), or variants or fragments thereof.
• variants include variants to enhance the thermal stability: G137A and G139A (Chen et al. (1996), Prot. Engng. 9, 499-505); D257E and D293E/Q (Chen et al. (1995), Prot. Engng. 8, 575-582); N182 (Chen et al. (1994), Bio- chem. J. 301, 275-281); disulphide bonds, A246C (Fierobe et al. (1996), Biochemistry, 35, 8698-8704; and introduction of Pro residues in position A435 and S436 (Li et al. (1997), Protein Engng. 10, 1199-1204.
• glucoamylases include Tala- romyces glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus, Talaromyces duponti (US 32,153), Talaromyces thermophilus (US 4,587,215).
• Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP and C. thermohydrosulfu- ncum (WO 86/01831).
• Glucoamylases may in an embodiment be added in an amount of 0.02-2 AGU/g DS, preferably 0.1-1 AGU/g DS, such as 0.2 AGU/g DS
• the ratio between acidic fungal alpha-amylase activity (AFAU) per glucoamylase activ- ity (AGU) (AFAU per AGU) may in one embodiment be at least 0.1, in particular at least 0.16, such as in the range from 0.12 to 0.30.
• AMG 200L AMG 300 L; SANTM SUPER and AMGTM E (from Novozymes); OPTIDEXTM 300 (from Genencor Int.); AMIGASETM and AMIGASETM PLUS (from DSM); G-ZYMETM G900 (from Enzyme Bio-Systems); G-ZYMETM G990 ZR (A. niger glucoamylase and low protease content).
• Beta-amylase Beta-amylase (E.C 3.2.1.2) is the name traditionally given to exo-acting maltogenic amylases, which catalyze the hydrolysis of 1 ,4-alpha-glucosidic linkages in amylose, amy- lopectin and related glucose polymers. Maltose units are successively removed from the non-reducing chain ends in a step-wise manner until the molecule is degraded or, in the case of amylopectin, until a branch point is reached. The maltose released has the beta anomeric configuration, hence the name beta-amylase.
• Beta-amylases have been isolated from various plants and microorganisms (W.M. Fo- garty and C.T. Kelly, Progress in Industrial Microbiology, vol. 15, pp. 112-115, 1979). These beta-amylases are characterized by having optimum temperatures in the range from 40°C to 65°C and optimum pH in the range from 4.5 to 7.
• a commercially available beta-amylase from barley is SPEZYMETM BBA 1500 from Genencor Int., USA.
• 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. Furthermore, 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
• 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).
• Phytases can be classified according to their specificity in the initial hydrolysis step, viz. according to which phosphate-ester group is hydrolyzed first.
• the phytase to be used in the invention may have any specificity, e.g., be a 3-phytase (EC 3.1.3.8), a 6-phytase (EC 3.1.3.26) or a 5-phytase (no EC number).
• the phytase has a temperature optimum in the range from 25-70°C, preferably 28-50°C, especially 30-40°C. This is advantageous when the phytase is added during fermentation.
• the phytase has a temperature optimum above 50°C, such as in the range from 50-70°C. This is advantageous when the phytase is added during pre-saccharification.
• a preferred suitable dosage of the phytase is in the range from 0.005- 25 FYT/g DS, preferably 0.01-10 FYT/g, such as 0.1-1 FYT/g DS.
• the phytase activity is determined FYT units, one FYT being the amount of enzyme that liberates 1 micromole inorganic ortho-phosphate per min. under the following conditions: pH 5.5; temperature 37°C; substrate: sodium phytate (C 6 H 6 O 24 P 6 Na 12 ) at a concentration of 0.0050 mole/I.
• the phytase may be derived from plants or microorganisms, such as bacteria or fungi, e.g., yeast or filamentous fungi.
• the plant phytase may be from wheat-bran, maize, soy bean or lily pollen. Suitable plant phytases are described in Thomlinson et al, Biochemistry, 1 (1962), 166-171; Barrien- tos et al, Plant. Physiol., 106 (1994), 1489-1495; WO 98/05785; WO 98/20139.
• a bacterial phytase may be from genus Bacillus, Pseudomonas or Escherichia, specifically the species ⁇ . subtilis or E. coli.
• Suitable bacterial phytases are described in Paver and Jagannathan, 1982, Journal of Bacteriology 151 :1102-1108; Cosgrove, 1970, Australian Journal of Biological Sciences 23:1207-1220; Greiner et al, Arch. Biochem. Biophys., 303, 107-113, 1993; WO 98/06856; WO 97/33976; WO 97/48812.
• a yeast phytase or myo-inositol monophosphatase may be derived from genus Saccharomyces or Schwanniomyces, specifically species Saccharomyces cerevisiae or Schwanniomyces occidentalis.
• the former enzyme has been described as a Suitable yeast phytases are described in Nayini et al, 1984, Strukturtician und Technologie 17:24-26; Wodzinski et al. Adv. Appl. Microbiol., 42, 263-303; AU-A-24840/95;
• Phytases from filamentous fungi may be derived from the fungal phylum of Ascomy- cota (ascomycetes) or the phylum Basidiomycota, e.g., the genus Aspergillus, Thermomyces (also called Humicola), Myceliophthora, Manascus, Penicillium, Peniophora, Agrocybe, Pax- illus, or Trametes, specifically the species Aspergillus terreus, Aspergillus niger, Aspergillus niger var. awamori, Aspergillus ficuum, Aspergillus fumigatus, Aspergillus oryzae, T.
• Ascomy- cota ascomycetes
• Basidiomycota e.g., the genus Aspergillus, Thermomyces (also called Humicola), Myceliophthora, Manascus, Penicillium,
• lanugi- nosus also known as H. lanuginosa
• Myceliophthora thermophila Peniophora lycii
• Agrocybe pediades Manascus anka, Paxillus involtus, or Trametes pubescens.
• Suitable fungal phytases are described in Yamada et al., 1986, Agric. Biol. Chem.
• Modified phytases or phytase variants are obtainable by methods known in the art, in particular by the methods disclosed in EP 897010; EP 897985; WO 99/49022; WO 99/48330.
• BIO- FEED PHYTASETM, PHYTASE NOVOTM CT or L all available from Novozymes A/S
• NATUPHOSTM NG 5000 available from DSM.
• Alpha-amylases The liquefaction step may be performed in the presence of an alpha-amylase derived from a microorganism or a plant.
• Preferred alpha-amylases are of fungal or bacterial origin.
• Bacillus alpha-amylases (often referred to as "Termamyl-like alpha-amylases"), variant and hybrids thereof, are specifically contemplated according to the invention.
• Well-known Termamyl-like alpha-amylases include alpha-amylase derived from a strain of S. licheniformis (commercially available as TermamylTM), B. amyloliquefaciens, and B. stearothermophilus al- pha-amylase (BSG).
• Termamyl-like alpha-amylases include alpha-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO 95/26397, and the alpha-amylase described by Tsukamoto et al., Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31.
• a Termamyl-like alpha-amylase is an alpha-amylase as defined in WO 99/19467 on page 3, line 18 to page 6, line 27.
• Contemplated variants and hybrids are de- scribed in WO 96/23874, WO 97/41213, and WO 99/19467.
• Contemplated alpha-amylase derived from a strain of Aspergillus includes Aspergillus oryzae and Aspergillus niger - amylases.
• alpha-amylase products and products containing alpha- amylases include TERMAMYLTM SC, FUNGAMYLTM, LIQUOZYMETM and SANTM SUPER.
• Fungal alpha-amylases may be added in an amount of 0.001-1.0 AFAU/g DS, preferably from 0.002-0.5 AFAU/g DS, preferably 0.02-0.1 AFAU/g DS.
• Bacillus alpha-amylases may be added in effective amounts well known to the person skilled in the art.
• protease(s) has generally been attributed, to an increase in the FAN (Free amino nitrogen) level and thereby an increase in the rate of metabolism of the yeast.
• Proteases in particular acidic proteases, including especially Rhizomucor miehei protease, has according to the invention been demon- strated to reduce flocculation of yeast cells and attachment of yeast cells to insoluble material in high gravity fermentation of ethanol. This reduced flocculation itself will result in higher fermentation efficiency (productivity) and a higher immediate alcohol yield.
• Suitable proteases include 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, Rhizomucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra, Irpex, Penicillium, Scle- rotium and 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.
• a preferred embodiment of the invention includes the use of Rhizomucor miehei (Mucor miehei) acidic protease encoded by the sequence shown in figure 4a-4b in EP238023. Another preferred embodiment comprises the use of R.
• a third preferred embodiment comprises the use of a fermentation product from transformed A. oryzae in which the R. miehei protease is co-expressed together with the native A. oryzae protease.
• the R. miehei protease may according to the invention preferably be added in amounts of 0.01-1.0 mg enzyme protein per g of dry substance in the fermenting medium, preferably 0.1 - 0.5 mg enzyme protein per g of dry substance in the fermenting medium.
• the use of protease, in particular acidic proteases, especially Rhizomucor miehei protease may according to the invention be applicable to batch as well as to continued fermentation processes.
• Bacterial proteases which are not acidic proteases, such as Bacillus proteases, in- elude the commercially available products ALCALASE® and NEUTRASE® (available from Novozymes A/S).
• ALCALASETM is a Bacillus licheniformis protease (subti ⁇ sin, Carlsberg). ALCALASETM may according to the invention preferably be added is amounts of 10 "7 to 10 "3 gram active protease protein/g DS, in particular 10 "6 to 10 "4 gram active protease protein/g DS, or in amounts of 0.1-0.0001 AU/g DS, preferably 0.00025-0.001 AU/g DS.
• FLAVOURZYMETM is a protease preparation derived from Aspergillus oryzae.
• FLA- VOURZYMETM may according to the invention preferably be added in amounts of 0.01-1.0 LAPU/g DS, preferably 0.05-0.5 LAPU/g DS)
• protease(s) may in one embodiment be added in an amount of 10 "7 to 10 "3 gram active protease protein/g DS, in particular 10 "6 to 10 "4 gram active protease protein/g DS
• Pullulanase Contemplated pullulanases include the thermostable pullulanase from, e.g., Pyrococcus or
• Bacillus sp. including protein engineered pullulanases from, e.g., a Bacillus strain such as Bacillus acidopullulyticus (e.g, the one described in FEMS Mic. Let. (1994) 115, 97-106), Bacillus deramificans (e.g., the Bacillus deramificans pullulanase with GeneBank accession number Q68699), or Bacillus naganoensis.
• Contemplated commercially available pullulanases include PROMOZYMETM D and
• Glucoamylase/pullulanase Combination products Further, commercially available combination products include DEXTROZYMETM E and
• DEXTROZYMETM E ULTRA comprising glucoamylase from A. niger and pullulanase (from
• DEXTROZYMETM D which is a balanced mixture of glucoamylase derived from Aspergillus niger and a pullulanase
• DEXTROZYMETM 225/75 L which is a balanced mixture of glucoamylase derived from Aspergillus niger and pullulanase from Novozymes
• OPTI-D a balanced mixture of glucoamylase derived from Aspergillus niger and pullulanase from Novozymes
• MAXTM 7525 which is a blend of glucoamylase and heat stable pullulanase.
• the ration between glucoamylase and pullulanase determined as, e.g., AGU/PUN may be from 5:1 to 1:5, preferably 4:1 to 1:4, such as 1:1 or 2:1 or 3:1.
• Isoamylase Contemplated isoamylase according to the invention include Pseudomonas amylod- eramosa Biochim. Biophys. Acta, 1087, p.309-315 (1990); Pseudomonas sp. (EP 0 302 838 A2); Flavobacterium sp. (WO 96/03513), in particular Flavobacterium sp. IFO 14590 (shown as SEQ ID NO: 11 in WO 99/01545); Flavobacterium odoratum (JP08023981-A); Sulfolobus acidocaldarius Biochim. Biophys. Acta, 1291 , p.177-181 (1996); Rhodothermus marinus
• Rhodothermus marinus DSM 4252 shown as SEQ ID NO: 4 in
• Flavobacterium devorans ATCC 10829 shown as SEQ ID NO: 12 in WO 99/01545
• Xanthomonas campestris ATCC 31922 shown as SEQ ID NO: 13 in WO
• Rhodothermus obamensis JCM 9785 shown as SEQ ID NO: 14 in WO
• composition comprising all of the following enzyme ac- tivities: carbohydrate-source generating enzyme activity (as defined), alpha-amylase activity, protease activity and debranching enzyme activity (as defined).
• the composition may further comprise a phytase and/or protease, in particular an acid protease, such as an acid fungal protease.
• Glucoamylase and acid alpha-amylase Aspergillus niger glucoamylase (available as
• AMG E from Novozymes A/S) (SE-2000-00034, 382 AGU/g).
• FLAVOURZYMETM is a protease/peptidase complex derived from Aspergillus oryzae (Available from Novozymes A/S).
• Protease derived from Rhizomucor miehei was produced in Aspergillus oryzae as described in Example 7 in EP 238023.
• An experimental enzyme product with an enzyme protein content of 48.8 mg/g was prepared.
• PROMOZYME® is derived from Bacillus acidopullulyticus and described in EP 63.909 (available from Novozymes).
• Alpha-amylase BSG ( ⁇ . stearothermophilus alpha-amylase which is available from Novozymes as TERMAMYLTM SC).
• Liquefied whole corn mash Liquefied whole corn mash was prepared by a hot slurry process and Termamyl SC treatment. The mash has a DE of about 17 and a dry substance of about 28% (SS-99-00007).
• Liquefied whole corn mash Liquefied whole corn mash was prepared by a hot slurry process and Termamyl SC treatment. The mash has a DE of about 12 and a dry substance of about 30% (SS-00-00012).
• the yeast applied was a Saccharomyces cervisiae (S-00640-2) Determination of Beta-Glucanase units
• Beta Glucanase Unit corresponds to the quantity of enzyme required to produce 1 micromole of reducing sugars per minute under standard conditions. A detailed description of Novozymes's analytic method is available on request.
• Phadebas® tablets Purgesebas® Amylase Test, supplied by Pharmacia Diagnostic
• con- tain a cross-linked insoluble blue-colored starch polymer which has been mixed with bovine serum albumin and a buffer substance and tabletted.
• test time is in the range of 0.2 to 2.0 absorbance units at 620 nm. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law). The dilution of the enzyme must therefore be adjusted to fit this criterion.
• a specified set of conditions temperature, pH, reaction time, buffer conditions
• 1 mg of a given alpha-amylase will hydrolyze a certain amount of substrate and a blue colour will be produced.
• the colour intensity is measured at 620 nm.
• the measured absorbance is directly proportional to the specific activity (activ- ity/mg of pure alpha-amylase protein) of the alpha-amylase in question under the given set of conditions.
• Alpha-amylase activity is determined by a method employing the PNP-G7 substrate.
• PNP-G7 which is a abbreviation for p-nitrophenyl-alpha,D-maltoheptaoside is a blocked oli- gosaccharide which can be cleaved by an endo-amylase.
• Kits containing PNP-G7 substrate and alpha-GIucosidase is manufactured by Boehringer-Mannheim (cat. No. 1054635).
• BM 1442309 To prepare the substrate one bottle of substrate (BM 1442309) is added to 5 ml buffer (BM1442309).
• BM 1462309 To prepare the alpha-GIucosidase one bottle of alpha-GIucosidase (BM 1462309) is added to 45 ml buffer (BM1442309).
• the working solution is made by mixing 5 ml alpha-GIucosidase solution with 0.5 ml substrate.
• the assay is performed by transforming 20 micro I enzyme solution to a 96 well mi- crotitre plate and incubating at 25°C. 200 micro I working solution, 25°C is added. The solution is mixed and pre-incubated 1 minute and absorption is measured every 15 sec. over 3 minutes at OD 405 nm.
• the slope of the time dependent absorption-curve is directly proportional to the specific activity (activity per mg enzyme) of the alpha-amylase in question under the given set of conditions.
• FAU Fungal Alpha-Amylase Unit
• Novozymes standard method for determination of alpha-amylase based upon the following standard conditions:
• Acid alpha-amylase activity is measured in AFAU (Acid Fungal Alpha-amylase Units), which are determined relative to an enzyme standard.
• AMG 300 L wild type A. niger G1 AMG sold by Novozymes.
• the neutral alpha-amylase in this AMG falls after storage at room temperature for 3 weeks from approx. 1 FAU/mL to below 0.05 FAU/mL.
• the acid alpha-amylase activity in this AMG standard is determined in accordance with AF 9 1/3 (available from Novozymes A/S method for the determination of fungal alpha- amylase). In this method, 1 AFAU is defined as the amount of enzyme, which degrades 5.260 mg starch dry matter per hour under standard conditions. Iodine forms a blue complex with starch but not with its degradation products. The intensity of colour is therefore directly proportional to the concentration of starch.
• Amylase activity is determined using reverse col- orimetry as a reduction in the concentration of starch under specified analytic conditions.
• Enzyme concentration 0.025 AFAU/mL
• Enzyme working range 0.01-0.04 AFAU/mL
• the Novo Amyloglucosidase Unit is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute at 37°C and pH 4.3.
• a detailed description of the analytical method (AEL-SM-0131) is available on request from Novozymes.
• 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.
• 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 soluble product
• AU Anson Unit
• LAPU Protease assay methods
• LAPU 1 Leucine Amino Peptidase Unit
• Beta-amylase activity (DP°)
• SPEZYME® BBA 1500 The activity of SPEZYME® BBA 1500 is expressed in Degree of Diastatic Power (DP 0 ). It is the amount of enzyme contained in 0.1 ml of a 5% solution of the sample enzyme preparation that will produce sufficient reducing sugars to reduce 5 ml of Fehling's solution when the sample is incubated with 100 ml of substrate for 1 hour at 20°C.
• Pullulanase activity (New Pullulanase Unit Novo (NPUN)
• NPUN Pullulanase Unit Novo
• Pullulanase activity may be determined relative to a pullulan substrate.
• Pullulan is a linear D-glucose polymer consisting essentially of maltotriosyl units joined by 1 ,6-alpha-links. Endopullulanases hydrolyze the 1 ,6-alpha-links at random, releasing maltotriose, 6 3 -alpha- maltotriosyl-maltotriose, 6 3 -alpha-(6 3 -alpha-maltotriosyl-maltotriosyl)-maltotriose, etc. the number of links hydrolyzed is determined as reducing carbohydrate using a modified Somogyi-
• PUN pullulanase unit
• One pullulanase unit is the amount of enzyme which, under standard conditions (i.e. after 30 minutes reaction time at 40°C and pH 5.0; and with 0.2% pullulan as substrate) hydrolyzes pullulan, liberating reducing carbohydrate with a reducing power equivalent to 1 micro mol glucose per minute.
• This example demonstrates how the presence of R. miehei protease reduces flocculation.
• 2.5 g of washed yeast was suspended in 100 mL of ion-exchanged water at room temperature. The suspension was stirred on a magnetic stirrer for 15 minutes. 15 mL samples were transferred to centrifuge tubes with volume indication. NaCI, CaCI 2 and Miehei protease was added to create the solutions given in table 1. Incubation of solutions was made at room temperature for 15 minutes in a rotary shaker, which turned the closed tubes end-over-end at 20 rpm. Hereafter the tubes were left in vertical position for 60 minutes after which the volume of the sediment was measured. The results are shown in table 1.
• This example demonstrates how the presence of R. miehei protease increases fermentation rate and ethanol yield.
• the bottles were cooled in a water bath for 40 minutes to 30°C and dry yeast was added at a dosage of 0.8 g/bottle (in order to reach 30°C within 40 minutes it was necessary to add ice to the water bath).
• the dry yeast was added in excess, meaning that the yeast nutrition and viability is the limiting factor for the fermentation rate.
• the bottles were closed using a yeast-lock filled with concentrated H 2 SO 4 .
• the fermentation was continued for 72 hours and by weighing the bottle at regular intervals the CO 2 loss was monitored. Every 24, 48 and 72 hours samples for HPLC analysis were taken out.
• AMG E 0.2 AG/g dry matter + GLUCANEXTM: 2,4 BGXU/g dry matter
• GLUCANEXTM 2.4 BGXU/g dry matter
• GLUCANEXTM had a positive effect on the fermentation rate and ethanol yields.

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