WO2025129043A2 - Procédés de réduction de la viscosité du sirop - Google Patents

Procédés de réduction de la viscosité du sirop Download PDF

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Publication number
WO2025129043A2
WO2025129043A2 PCT/US2024/060099 US2024060099W WO2025129043A2 WO 2025129043 A2 WO2025129043 A2 WO 2025129043A2 US 2024060099 W US2024060099 W US 2024060099W WO 2025129043 A2 WO2025129043 A2 WO 2025129043A2
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seq
protease
amino acid
syrup
fermentation
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WO2025129043A3 (fr
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Jaclyn Diana DEMARTINI
Monica TSE
Kees-Jan GUIJT
Nadia RAMIREZ ANGULO
Bart C. Koops
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Genencor International BV
Danisco US Inc
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Genencor International BV
Danisco US Inc
Genencor International Inc
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    • 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
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
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    • C13—SUGAR INDUSTRY
    • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
    • C13K1/00—Glucose; Glucose-containing syrups
    • C13K1/06—Glucose; Glucose-containing syrups obtained by saccharification of starch or raw materials containing starch
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    • 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)
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    • 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
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
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    • 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
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
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    • 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
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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    • 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)
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    • 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)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
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    • 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)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
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    • 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)
    • C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • C—CHEMISTRY; METALLURGY
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    • 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)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
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    • 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)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
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    • 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)
    • C12Y302/0102—Alpha-glucosidase (3.2.1.20)
    • C—CHEMISTRY; METALLURGY
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    • 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)
    • C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase

Definitions

  • FIELD OF THE INVENTION Provided herein are methods and compositions for reducing the viscosity of syrup produced as a byproduct of fermentation processes.
  • BACKGROUND Processes for producing fermentation products from biomass yield not only desirable fermentation products, e.g., ethanol, but also liquid and solid byproducts from which valuable co- products may be recovered. For example, whole stillage, the liquids and solids remaining after separation of the desired fermentation product (e.g., ethanol), can be further processed into proteinaceous ingredients for animal feed and/or oil for biodiesel production, feed or food additives or products, or other bio-renewable products.
  • Whole stillage consists of both suspended and dissolved solids at various ratios depending on the biomass feedstock, processing, and the fermentation conditions employed.
  • wt weight
  • Dewatered wet cake can be dried to provide “Distillers Dried Grains” (DDG) used as nutrient in animal feed.
  • DDG “Distillers Dried Grains”
  • Thin stillage is typically evaporated to provide condensate and syrup or may alternatively be recycled directly to the slurry tank as backset.
  • Condensate may either be forwarded to a methanator before being discharged or may be recycled to the slurry tank.
  • the syrup may be blended into DDG or added to the wet cake before drying to produce Distillers Dried Grain with Solubles (DDGS). Oil may also be recovered from the thin stillage and/or syrup/evaporated centrate as a co-product for use in biodiesel production or other biorenewable products.
  • IFF10001-WO-PCT [006] Depending on fermentation characteristics and the addition of various compounds and enzymes during fermentation and/or upstream processes, the characteristics of the syrup may be drastically affected, specifically (but not limited to) composition and rheology.
  • Syrup viscosity can be a significant issue for biorefineries, for example, by decreasing flow characteristics, coating walls, damaging pumps, plugging piping networks and centrifuges as well as increasing fouling rates. Increases in viscosity can therefore significantly impact plant efficiency and the quality and amount of co-product produced. [007] Thus, there is a need for methods and compositions capable of reducing syrup viscosity. The methods and compositions provided herein address these and other needs in the art.
  • a viscosity reducing composition including a cellulase, a hemicellulase, a protease, or any combination thereof, is added during step (f).
  • the viscosity reducing composition is added to the thin stillage, to an intermediate product formed during concentrating the thin stillage to produce a syrup, and/or to the syrup produced in step (f).
  • step (b) and step (c) are combined as a simultaneous saccharification and fermentation step (SSF).
  • the cellulase includes a cellobiohydrolase, an endoglucanase, a beta- glucosidase, or any combination thereof.
  • the cellulase includes a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase BGL, or any combination thereof.
  • the cellulase includes an endoglucanase, where the endoglucanase has an amino acid sequence with at least 60, 70, 80, 90, 95, 98, or 100% sequence identity to the sequence set forth by SEQ ID NOs: 4 or 7.
  • the cellulase is derived from a fungus.
  • the cellulase is derived from a strain of Trichoderma. In some embodiments, the cellulase is derived from a strain of Trichoderma reesei. In some embodiments, the hemicellulase includes a xylanase. In some embodiments, the hemicellulase includes a GH10 xylanase. In some embodiments, the hemicellulase includes a sequence having at least 60% sequence identity to the sequence set forth by SEQ ID NO: 1. In some embodiments, the protease includes a sequence having at least 60% sequence identity to the sequence set forth by SEQ ID IFF10001-WO-PCT NO: 3.
  • the protease includes an amino acid sequence having at least 60, 70, 80, 90, 95, 98, or 100% sequence identity to the sequence set forth by SEQ ID NOs: 3 or 9.
  • the viscosity reducing composition further includes an alpha-amylase.
  • the alpha-amylase includes an amino acid sequence having at least 60, 70, 80, 90, 95, 98, or 100% sequence identity to the sequence set forth by SEQ ID NOs: 5 or 6.
  • a composition including a cellulase, a hemicellulase, a protease, or any combination thereof is added during step (a), step (b), and/or step (c); or (ii) where step (b) and step (c) are combined as a simultaneous saccharification and fermentation step (SSF) and a composition including a cellulase, a hemicellulase, a protease, or any combination thereof is added during step (a) and/or the SSF.
  • the composition including a cellulase, a hemicellulase, a protease, or any combination thereof comprises the viscosity reducing composition.
  • the viscosity of the syrup is decreased compared to a syrup not treated with the viscosity reducing composition.
  • the starch-containing material is corn. In some embodiments, the starch-containing material is wheat.
  • FIG. 1 shows the syrup flow rankings (mean ⁇ SD) of Viscous Syrup following either no enzyme treatment (Viscous Syrup – Nil) or treatment with a Trichoderma Whole Cellulase, Engineered Trichoderma Whole Cellulase 1, Engineered Trichoderma Whole Cellulase 2, Thermostable Xylanase, or Thermostable Protease.
  • the Baseline Syrup (Baseline Syrup – Nil) ranking is also shown.
  • DETAILED DESCRIPTION [0011] The use of biomass as feedstock for biorefining has emerged as an environmentally friendly means of producing usable products from sustainable resources.
  • Exemplary beneficial fermentation products produced from biomass include, but are not limited to, alcohols, such as industrial or potable alcohols (e.g., ethanol); biochemicals, such as organic acids (e.g., acetic acid, lactic acid), amino acids (e.g., lysine), and vitamins (e.g., vitamin B, vitamin C); sweeteners, such as sugar, fructose, low calorie sweeteners, syrups (e.g., high fructose corn syrup); and soluble fiber.
  • alcohols such as industrial or potable alcohols (e.g., ethanol); biochemicals, such as organic acids (e.g., acetic acid, lactic acid), amino acids (e.g., lysine), and vitamins (e.g., vitamin B, vitamin C); sweeteners, such as sugar, fructose, low calorie sweeteners, syrups (e.g., high fructose corn syrup); and soluble fiber.
  • alcohols such as industrial or potable alcohols (e.
  • the processes also produce byproducts (fermentation byproducts) such as liquids and solids that contain valuable components, e.g., protein, oil, that can be sold as fermentation co-products, such as ingredients for animal feed IFF10001-WO-PCT and/or oil for biodiesel production, feed or food additives or products, or other bio-renewable products.
  • fermentation byproducts such as liquids and solids that contain valuable components, e.g., protein, oil
  • fermentation co-products such as ingredients for animal feed IFF10001-WO-PCT and/or oil for biodiesel production, feed or food additives or products, or other bio-renewable products.
  • the conversion of biomass to a fermentation product such as alcohol typically involves multiple processing steps, also referred to herein as unit operations, and reagents, each of which is important for maximizing conversion efficiency, throughput, consistency, concentration, and/or yield.
  • a common production process for producing ethanol by fermentation of a biomass feedstock typically involves unit operations including, but not limited to, milling or grinding feedstock, liquefaction, saccharification, fermentation, and distillation, and reagents such as enzymes and additional components, e.g., microorganisms and/or chemicals, may be added during a unit operation to facilitate the reaction.
  • liquefaction includes thinning a mash via partial hydrolysis to lower its viscosity.
  • Enzymes e.g., alpha-amylases, may be used to facilitate the thinning process.
  • complex carbohydrates of the thinned mash are converted to monosaccharides.
  • enzymes e.g., glucoamylases
  • the resulting sugars are then converted by yeasts to ethanol during the fermentation process step.
  • enzymes e.g., glucoamylases, fungal alpha amylase, and trehalase
  • Reagents such as enzymes, may also be used in the steps of milling and distillation to facilitate the process.
  • the saccharification and fermentation steps may be combined into a single simultaneous saccharification and fermentation (SSF) step.
  • SSF simultaneous saccharification and fermentation
  • Other well- known processes include raw starch hydrolysis (RSH), which includes milling the starch- containing material and then performing SSF below the initial gelatinization temperature.
  • Enzymes e.g., acid fungal amylases, glucoamylases, may also be used in such processes to produce fermentation products.
  • Fermentation products from alcohol production processes are typically removed as liquid during a distillation step, which leaves behind other liquids and solids that may be referred to herein as fermentation byproducts.
  • a fermentation byproduct as used herein refers to the remaining liquids and solids that can be further processed to recover a fermentation co-product.
  • a fermentation byproduct is a whole stillage, which includes liquids and solids that can be separated into, e.g., thin stillage and wet cake, respectively.
  • thin stillage and/or wet cake may be further processed to produce fermentation co-products.
  • whole stillage consisting of both suspended and dissolved solids may be separated into wet cake, containing a majority of the suspended solids, and thin stillage containing dissolved solids.
  • Dewatered wet cake can be dried to provide “Distillers Dried Grains” (DDG) used as nutrient in animal feed.
  • DDG “Distillers Dried Grains”
  • Thin stillage is typically evaporated to provide condensate and syrup or IFF10001-WO-PCT may alternatively be recycled directly to the slurry tank as backset.
  • Condensate may either be forwarded to a methanator before being discharged or may be recycled to the slurry tank.
  • the syrup may be blended into DDG or added to the wet cake before drying to produce Distillers Dried Grain with Solubles (DDGS).
  • DDGS Distillers Dried Grain with Solubles
  • Oil from the thin stillage and/or syrup/evaporated centrate may also be recovered as a co-product for use, for example, in biodiesel production or other biorenewable products.
  • various compounds and enzymes may be added during fermentation and/or upstream processes. Such strategies can impact syrup characteristics, such as composition and rheology, dramatically. Indeed, even the characteristics of the fermentation can drastically affect the syrup byproduct.
  • the use of enzymes, such as hemicellulases and cellulases during unit operations prior to distillation, e.g., during saccharification, fermentation, or SSF, caused increases in syrup viscosity.
  • Increases in syrup viscosity can be a significant issue for biorefineries, for example, by decreasing flow characteristics, coating walls, damaging pumps, plugging piping networks and centrifuges as well as increasing fouling rates. Increases in viscosity can therefore significantly impact plant efficiency and the quality and amount of co-product produced.
  • a viscosity reduction composition Such enzymes and combinations thereof are referred to generally herein as a viscosity reduction composition.
  • treating the syrup with a viscosity reduction composition improves oil recovery from the syrup.
  • viscosity reduction compositions containing cellulases, hemicellulases, proteases, and combinations thereof that decrease syrup viscosity.
  • the viscosity reducing composition may include or further include an alpha-amylase.
  • ethanol refers to ethanol produced as a result of a biological fermentation process.
  • recovery ethanol refers to the purification and/or isolation of ethanol. Suitably, the recovery results in ethanol that is substantially free of other components (e.g. contaminants).
  • the recovery may result in an alcohol that is at least about 90% pure, at least about 95% pure, at least 99% pure. In some embodiments, the recovery may result in an alcohol that is at least about 99.9% pure.
  • the term “whole stillage” includes the material that remains at the end of the distillation process after recovery of the fermentation product, e.g., ethanol.
  • “thin stillage” refers to the separated liquid fraction of the whole stillage.
  • “wet cake” refers to the separated solid fraction of the whole stillage.
  • the term “oil” refers to a co-product extractable from a byproduct of a biological fermentation process.
  • the phrase “recovering oil” refers to the purification, extraction, and/or isolation of oil.
  • the recovery results in oil that is substantially free of other components (e.g. contaminants). Therefore, the recovery may result in an oil that is at least about 90% pure, at least about 95% pure, at least 99% pure. In some embodiments, the recovery may result in an oil that is at least about 99.9% pure.
  • NC-IUBMB Nomenclature Committee of the International Union of Biochemistry and Molecular Biology
  • cellulase means enzymes that hydrolyze cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof.
  • the two basic approaches for measuring cellulolytic activity include: (1) measuring the total cellulolytic activity, and (2) measuring the individual cellulolytic activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., Outlook for cellulase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24: 452-481.
  • Total cellulolytic activity is usually measured using insoluble substrates, including Whatman No1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc.
  • the most common total cellulolytic activity assay is the filter paper assay using Whatman No1 filter paper as the substrate.
  • beta-glucosidase means a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D-glucose.
  • the term “cellobiohydrolase” includes 1,4-p-D-glucan glucohydrolases (EC 3.2.1.74) as well as 1,4-p-D-glucan cellobiohydrolase (EC 3.2.1.91). Enzymes having activity classified according to EC 3.2.1.176, e.g., cellulose 1,4-beta-cellobiosidase (reducing end), may also be considered cellobiohydrolases herein. Cellobiohydrolases typically cleave cellulose strands to produce cellobiose. In some embodiments, the cellobiohydrolase is a cellobiohydrolase I (CBHI).
  • CBHI cellobiohydrolase I
  • the cellobiohydrolase is a cellobiohydrolase II (CBHII).
  • CBHII cellobiohydrolase II
  • IFF10001-WO-PCT the term “endoglucanase” means an endo-1,4-(1,3;1,4)-beta-D-glucan 4- glucanohydrolase (EC 3.2.1.4) that catalyzes endohydrolysis 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.
  • an endoglucanase may also refer to an enzyme classified according to EC 3.2.1.6.
  • the endoglucanase is an endoglucanase I (EGI).
  • the endoglucanase is an endoglucanase II (EGII).
  • hemicellulase means enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom, D. and Shoham, Y. Microbial hemicellulases. Current Opinion In Microbiology, 2003, 6(3): 219-228. Hemicellulases are key components in the degradation of plant biomass.
  • hemicellulases include, but are not limited to, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a pectate lyase, a xylanase, and a xylosidase.
  • the substrates of these enzymes are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation.
  • the catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups.
  • GHs glycoside hydrolases
  • CEs carbohydrate esterases
  • catalytic modules based on homology of their primary sequence, can be assigned into GH and CE families. Some families, with an overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A). A most informative and updated classification of these and other carbohydrate active enzymes is available in the Carbohydrate- Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitable temperature, e.g., 50°C, 55°C, or 60°C, and pH, e.g., 5.0 or 5.5.
  • a suitable temperature e.g., 50°C, 55°C, or 60°C
  • pH e.g., 5.0 or 5.5.
  • proteases includes any enzyme belonging to the EC 3.4 enzyme group (including each of the eighteen subclasses thereof.
  • proteins polypeptides having protease activity (i.e., proteases)
  • proteases proteins having protease activity
  • proteolytic enzymes are also known in the art as peptidases, proteinases, peptide hydrolases, and proteolytic enzymes.
  • proteolytic activity means proteolytic activity (EC 3.4). Protease activity can generally be measured using any assay, in which a substrate is employed, that includes IFF10001-WO-PCT peptide bonds relevant for the specificity of the protease in question.
  • Assays for pH and assays for temperature are likewise to be adapted to the protease in question.
  • Examples of assay pH-values are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 and examples of assay temperatures are 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 80°C, 90°C, or 95°C.
  • Examples of general protease substrates are casein, bovine serum albumin (BSA) and hemoglobin.
  • proteases may be of the “exo-type” (i.e., exopeptidases) that hydrolyze proteins (peptide bonds) starting at either N-terminal or C-terminal end of the protein chain, or of the “endo-type” (i.e., endopeptidases) that hydrolyze peptide bonds of the non-terminal ends of the protein chain (i.e., internal peptide bonds).
  • exo-type i.e., exopeptidases
  • endopeptidases i.e., endopeptidases
  • “fermentable sugar(s)” refers to saccharides that are capable of being metabolized under fermentation conditions. These fermentable sugars typically refer to glucose, maltose, and maltotriose.
  • sucrose, galactose, xylose, arabinose etc. may also be a fermentable sugar.
  • fermentable sugars may be obtained by the hydrolysis of starch and other polysaccharide compositions (e.g., feedstocks).
  • feedstock refers to a composition including at least one of the following: starch, cellulose, hemicellulose, lignocellulose, fermentable sugars, or a combination thereof.
  • a “fraction of a feedstock” refers to any component of a feedstock that is separated out during the processing of the feedstock.
  • starch refers herein to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (C 6 H 10 O 5 ) x wherein “x” can be any number.
  • starch refers to any plant-based material including, but not limited to grains, cereals, grasses, tubers, and roots, and more particularly wheat, barley, corn, rye, rice, sorghum, brans, cassava, millet, potato, sweet potato, and tapioca.
  • a “starch-containing material” includes starch-containing materials derived from barley, beans, cassava, cereals, corn, milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat, and whole grains, or any mixture thereof.
  • a starch-containing material may also be derived from cobs, sugar cane, sugar beets, triticale, millet, banana, or potatoes.
  • a starch-containing material is a granular starch composition.
  • the starch-containing material is derived from whole grain. The starting material is generally selected based on the desired fermentation product.
  • the starch-containing IFF10001-WO-PCT material may also be a waxy or non-waxy type of corn and/or barley.
  • the starch-containing material is corn or derived from corn.
  • the starch- containing material is wheat or derived from wheat.
  • granular starch refers to uncooked (raw) starch, which has not been subject to gelatinization, where “starch gelatinization” means solubilization of starch molecules to form a viscous suspension.
  • hydrolysis of starch and the like refers to the cleavage of glycosidic bonds with the addition of water molecules.
  • a starch-containing material may be subjected to one or more processing steps either before, during or after fermentation.
  • one or more processing steps includes, but is not limited to, milling, cooking, liquefaction, saccharification, fermentation, and simultaneous saccharification and fermentation (SSF).
  • the term “initial gelatinization temperature” means the lowest temperature at which starch gelatinization commences.
  • milling refers to any milling of a feedstock, including wet milling, dry grinding, or combinations thereof.
  • Milling refers to a process which aids in breaking up the raw material used for the preparation of the feedstock into appropriately sized particles to facilitate downstream processing of the feedstock (e.g., to facilitate the cooking process).
  • wet milling is a process of milling that requires wet steeping of the feedstock (e.g., corn kernel) before processing. Wet steeping is then followed by a series of unit operations carried out in order to recover starch. The grain is typically soaked or “steeped” in water with dilute sulfurous acid for 24 to 48 hours prior to being subjected to a series of grinders.
  • the downstream processes may include removal of oil (e.g., corn oil) followed by further stages to separate out fiber, protein (e.g., gluten) and starch components (e.g., such as the endosperm). This may be achieved by centrifugation, use of screens and hydroclonic separators. The starch and water remaining from this process may then be subjected to fermentation.
  • dry grinding refers to a process in which a starting material, such as a grain, is ground into a flour (e.g., meal) before further processing. Generally, the flour is then slurried with water to form a mash (slum) prior to being processed in downstream steps (e.g., saccharification).
  • a starch-containing material obtained after milling or dry grinding may be subjected to liquefaction and/or saccharification and/or fermentation and/or simultaneous saccharification and fermentation (SSF). This may be with or without a cooking step (e.g., after milling and before either liquefaction or saccharification).
  • SSF simultaneous saccharification and fermentation
  • the starch-containing material is subjected to cooking. Typically, the cooking process may take place post-milling.
  • the cooking process may take place at 90°C-120°C. In some embodiments, the cooking may be carried out prior to liquefaction and/or saccharification. In some embodiments, the cooking process may reduce bacteria levels prior to fermentation. In some embodiments, one or more enzymes may be added at this stage or thereafter. In some embodiments, alpha-amylase may be added following the cooking process (e.g., in a liquefaction process). [0051] In some embodiments, starch-containing material is not subjected to cooking.
  • saccharification and fermentation, or SSF may be carried out on the feedstock (or fraction thereof) containing granular or raw starch (e.g., starch that has been treated at temperatures below gelatinization of the starch).
  • granular or raw starch e.g., starch that has been treated at temperatures below gelatinization of the starch.
  • the term “liquefaction” refers to a process in which the starch is liquefied, usually by increasing the temperature. Liquefaction of the starch results in a significant increase in viscosity. For this reason, amylases may be introduced in order to reduce the viscosity.
  • the temperature at which the starch liquefies varies depending upon the source of the starch.
  • Starch processing can also be carried out at temperatures from about 25°C to just below the liquefaction temperature.
  • starch hydrolysis processes are often referred to as “granular starch hydrolysis,” “direct starch hydrolysis,” “raw starch hydrolysis,” “low temperature starch hydrolysis,” and the like.
  • the starch is pretreated at temperatures below the liquefaction temperatures in order to enhance enzymatic hydrolysis and/or other processes for treatment of starch.
  • liquefaction may be carried out at a lower temperature and/or a “cold cook process” that does not involve complete liquefaction of starch.
  • the starch-containing material may also undergo saccharification. The saccharification may be separate to fermentation, or simultaneously therewith.
  • Separate saccharification and fermentation is a process whereby starch present in a feedstock or a fraction thereof is converted to glucose and subsequently a fermenting microorganism converts the glucose into ethanol.
  • “simultaneous saccharification and fermentation” or “SSF” is a process whereby starch present in a starch-containing material is converted to glucose and, at the same IFF10001-WO-PCT time and in the same reactor, a fermenting microorganism converts the glucose into ethanol.
  • saccharification may be carried out at low temperatures.
  • admixing refers to the mixing of one or more ingredients and/or enzymes, where the one or more ingredients or enzymes are added in any order and in any combination. In some embodiments, admixing may relate to mixing one or more ingredients and/or enzymes simultaneously or sequentially.
  • “fermentation microorganism,” “fermenting organism,” “ethanol production host,” “ethanol producing organism,” “ethanol producing host,” “ethanol producing host cell,” “ethanologen,” and the like may be used interchangeably and include any microorganism that has the ability to ferment a fermentable sugar source to produce ethanol.
  • a fermentation microorganism is a yeast.
  • the yeast may be selected from the group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Issatchenkia, Kazachstania and Torulaspora.
  • an ethanol producing organism may be a bacterium (e.g., Zymomonas, Escherichia, etc.).
  • a “lignocellulosic biomass” may contain cellulose, hemicellulose and the aromatic polymer lignin, hemicellulose, and cellulose (including insoluble arabinoxylans) by themselves are also potential energy sources, as they consist of C5- and C6-saccharides.
  • the lignocellulosic biomass may be any cellulosic, hemicellulosic, or lignocellulosic material, for example agricultural residues, bioenergy crops, industrial solid waste, municipal solid waste, sludge from paper manufacture, yard waste, wood waste, forestry waste, and combinations thereof.
  • the lignocellulosic biomass may be selected from the group consisting of corn cobs, crop residues such as corn husks, corn gluten meal (CGM), corn stover, corn fiber, grasses, beet pulp, wheat straw, wheat chaff, oat straw, wheat middlings, wheat shorts, IFF10001-WO-PCT rice bran, rice hulls, wheat bran, oat hulls, wet cake, DDG, DDGS, palm kernel, citrus pulp, cotton, lignin, barley straw, hay, rice straw, rice hulls, switchgrass, miscanthus, cord grass, reed canary grass, waste paper, sugar cane bagasse, sorghum bagasse, forage sorghum, sorghum stover, soybean stover, soy, components obtained from milling of trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, and flowers.
  • CGM corn gluten meal
  • CGM corn gluten meal
  • Wet-cake, Distillers Dried Grains and Distillers Dried Grains with Solubles are fermentation byproducts obtained after the removal of ethanol by distillation from fermentation of a grain or a grain mixture by methods employed in the grain distilling industry. Stillage coming from the distillation (e.g., comprising water, remainings of the grain, yeast cells, etc.) is separated into a "solid” part and a “liquid” part. The solid part is called “wet-cake” and can be used as animal feed as such. The liquid part is (partially) evaporated into a syrup (solubles). The liquid part is often referred to as the thin stillage. When the wet-cake is dried it is Distillers Dried Grains (DDG).
  • DDG Distillers Dried Grains
  • the terms “recovered,” “isolated,” “extracted,” and “separated,” refer to a compound, protein (polypeptide), cell, nucleic acid, amino acid, oil, or other specified material or component that is removed from at least one other material or component.
  • the at least one other material or component is at least one other material or component with which the compound, protein (polypeptide), cell, nucleic acid, amino acid, oil, or other specified material or component is naturally associated as found in nature.
  • the at least one other material or component is at least one other material or component with which the compound, protein (polypeptide), cell, nucleic acid, amino acid, oil, or other specified material or component is associated with under experimental or production conditions and/or systems.
  • an “isolated” polypeptide includes, but is not limited to, a polypeptide removed from a culture broth containing a heterologous host cell expressing the polypeptide. Recovery, for example, may refer to removal (extraction) of oil from a fermentation byproduct.
  • purified refers to material (e.g., an isolated compound, polypeptide, polynucleotide, or other specified material or component) that is in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99% pure.
  • enriched refers to material (e.g., an isolated compound, polypeptide, polynucleotide, or other specified material or component) that is about 50% pure, at least about 60% pure, at least about 70% pure, or even at least about 80% pure.
  • amino acid sequence is synonymous with the terms “polypeptide,” “protein,” and “peptide,” and are used interchangeably.
  • nucleic acid encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded and may contain chemical modifications. The terms “nucleic acid” and “polynucleotide” are used interchangeably.
  • Hybridized, duplex nucleic acids are characterized by a melting temperature (Tm), where one half of the hybridized nucleic acids are unpaired with the complementary strand. Mismatched nucleotides within the duplex lower the Tm.
  • Tm melting temperature
  • the terms “transformed,” “stably transformed,” and “transgenic,” used with reference to a cell means that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.
  • introduction in the context of inserting a nucleic acid sequence into a cell, encompasses, but is not limited to, “transfection”, “transformation” and “transduction,” as known in the art.
  • Exemplary methods for introducing polynucleotides or polypeptides by transformation into a host cell include, but are not limited to, microinjection, electroporation, stable transformation methods, transient transformation methods (such as induced competence using chemical (e.g. divalent cations such as CaCl2), mechanical (electroporation) means, or methods such as those described in published international applications WO 2018/114983 and WO 2010/149721, which are incorporated herein by reference in their entireties), ballistic particle IFF10001-WO-PCT acceleration (particle bombardment), direct gene transfer, viral-mediated introduction, cell- penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery.
  • microinjection electroporation
  • stable transformation methods such as induced competence using chemical (e.g. divalent cations such as CaCl2)
  • mechanical (electroporation) means or methods such as those described in published international applications WO 2018/114983 and WO 2010/149
  • Introducing a nucleic acid, construct, plasmid, or vector into a host cell may be carried out by conjugation, which is a specific method of natural DNA exchange requiring physical cell-to-cell contact.
  • Introducing a nucleic acid, construct, plasmid, or vector into a host cell may be carried out by transduction, which is the introduction of DNA via a virus (e.g., phage) infection which is also a natural method of DNA exchange.
  • viruses e.g., phage
  • such methods involve incorporating a polynucleotide within a viral DNA or RNA molecule.
  • a “host cell” is an organism into which an expression vector, phage, virus, or other nucleic acid sequence including a polynucleotide encoding a polypeptide of interest (e.g., an epimerase) has been introduced.
  • exemplary host cells are microorganism cells (e.g., bacteria, filamentous fungi, and yeast), mammalian cells, and plant cells capable of expressing the polypeptide of interest.
  • the term “host cell” includes protoplasts created from cells.
  • heterologous with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.
  • endogenous with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.
  • filamentous fungal cells e.g., Aspergillus sp., Trichoderma sp., etc.
  • standard techniques for transformation of filamentous fungi and culturing the fungi which are well known to one skilled in the art are used to transform a fungal host cell of the disclosure.
  • the introduction of a DNA construct or vector into a fungal host cell includes techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection mediated and DEAE-Dextrin mediated transfection), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, gene gun or biolistic transformation, protoplast fusion and the like.
  • General transformation techniques are known in the art (see, e.g., Ausubel et al., 1987, Sambrook et al., 2001 and 2012, and Campbell et al., 1989).
  • Agrobacterium-mediated transfection method such as the one described in U.S. Patent No. 6,255,115.
  • Transformation of Trichoderma sp. cells generally use protoplasts or cells that have been subjected to a permeability treatment, typically at a density of 10 5 to 10 7 /mL, particularly IFF10001-WO-PCT 2 ⁇ 10 6 /mL.
  • a volume of 100 ⁇ L of these protoplasts or cells in an appropriate solution is mixed with the desired DNA.
  • an appropriate solution e.g., 1.2 M sorbitol and 50 mM CaCl2
  • PEG polyethylene glycol
  • expression refers to the process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation.
  • the terms “recombinant” or “non-natural” refer to an organism, microorganism, cell, nucleic acid molecule, vector, polypeptide and the like that has at least one engineered genetic alteration, or has been modified by the introduction of a heterologous nucleic acid molecule; or refer to a cell (e.g., a host cell) that has been altered such that the expression of a heterologous nucleic acid molecule or an endogenous nucleic acid molecule or gene can be controlled.
  • Recombinant also refers to a cell that is derived from a non-natural cell or is progeny of a non-natural cell having one or more such modifications.
  • Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, or other nucleic acid molecule additions, deletions, substitutions, or other functional alteration of a cell’s genetic material.
  • recombinant cells may express genes or other nucleic acid molecules (e.g., polynucleotide constructs) that are not found in identical or homologous form within a native (wild-type) cell or may provide an altered expression pattern of endogenous genes, such as being over-expressed, under-expressed, minimally expressed, or not expressed at all.
  • “Recombination,” “recombining,” or generating a “recombined” nucleic acid is generally the assembly of two or more nucleic acid fragments wherein the assembly gives rise to a chimeric DNA sequence that would not otherwise be found in the genome.
  • a “selective marker” or “selectable marker” refers to a gene capable of being expressed in a host to facilitate selection of host cells carrying the gene. Examples of selectable markers include but are not limited to antimicrobials (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage on the host cell.
  • a “vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types.
  • Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.
  • An “expression vector” refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host.
  • control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a IFF10001-WO-PCT sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.
  • operably linked means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner.
  • a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequences.
  • a “signal sequence” is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell.
  • a cultured cell material refers to a cell lysate or supernatant (including media) that includes a protein of interest as a component.
  • the cell material may be from a cell or host cell that is grown in culture for the purpose of producing the protein.
  • the viscosity reducing composition is a cultured cell material.
  • the cellulases, hemicellulases, and/or protease may be contained in a cultured cell material.
  • “clarified,” when used in reference to cultured cell material, e.g., a whole broth, means a cultured cell material which has been subjected to at least one clarification process to remove cell debris and/or other insoluble components. Clarification processes, as understood in the art include, but are not limited to, centrifugation techniques, cross-flow membrane filtration techniques, solid/liquid filtration techniques, and the like.
  • Default parameters for the CLUSTAL W algorithm are: Gap opening penalty: 10.0 Gap extension penalty: 0.05 Protein weight matrix: BLOSUM series DNA weight matrix: IUB Delay divergent sequences %: 40 Gap separation distance: 8 DNA transitions weight: 0.50 IFF10001-WO-PCT List hydrophilic residues: GPSNDQEKR Use negative matrix: OFF Toggle Residue specific penalties: ON Toggle hydrophilic penalties: ON Toggle end gap separation penalty OFF. [0090] Deletions are counted as non-identical residues, compared to a reference sequence. Deletions occurring at either terminus are included.
  • a method for reducing syrup viscosity including: (a) liquefying a starch-containing material with an alpha-amylase to produce a starch liquefact; (b) saccharifying the starch liquefact with a glucoamylase to produce a saccharification product; (c) fermenting the saccharification product using a fermenting organism to produce a fermentation product; (d) recovering the fermentation product to form a whole stillage; (e) separating the whole stillage into thin stillage and wet cake; and (f) concentrating the thin stillage to produce a syrup; where a viscosity reducing composition including a cellulase and/or a hemicellulase is added during step (f).
  • a method for reducing syrup viscosity including: (a) liquefying a starch-containing material with an alpha-amylase to produce a starch liquefact; (b) saccharifying the starch liquefact with a glucoamylase to produce a saccharification product; (c) fermenting the saccharification product using a fermenting organism to produce a fermentation product; (d) recovering the fermentation product to form a whole stillage; (e) separating the whole stillage into thin stillage and wet cake; and (f) concentrating the thin stillage to produce a syrup; where a viscosity reducing composition including a protease, is added during step (f).
  • a method for reducing IFF10001-WO-PCT syrup viscosity including: (a) liquefying a starch-containing material with an alpha-amylase to produce a starch liquefact; (b) saccharifying the starch liquefact with a glucoamylase to produce a saccharification product; (c) fermenting the saccharification product using a fermenting organism to produce a fermentation product; (d) recovering the fermentation product to form a whole stillage; (e) separating the whole stillage into thin stillage and wet cake; and (f) concentrating the thin stillage to produce a syrup; where a viscosity reducing composition including an alpha-amylase, is added during step (f).
  • the “conventional process” includes liquefying gelatinized starch at high temperature typically using an alpha-amylase, e.g., a bacterial alpha- amylase, followed by simultaneous saccharification and fermentation (SSF) carried out in the presence of a glucoamylase and a fermenting microorganism.
  • the “raw starch hydrolysis” process includes simultaneously saccharifying and fermenting granular starch below the initial gelatinization temperature, typically in the presence of an acid fungal alpha-amylase and a glucoamylase.
  • the fermentation product is then recovered, e.g., by distillation, leaving a collection of liquids and solids commonly referred to as whole stillage.
  • the whole stillage fermentation byproduct may be further processed to produce valuable co-products.
  • the methods for recovery include separating (dewatering) the whole stillage to form a thin stillage and wet cake.
  • the thin stillage is processed to produce a syrup.
  • oil is recovered from the syrup.
  • the syrup is added to DDG to produce DDGS.
  • viscosity reducing compositions may be added to the thin stillage, during the production of syrup from the thin stillage, and/or to the syrup to decrease syrup viscosity.
  • the starch-containing material is or is derived from corn, cobs, sugar cane, sugar beets, wheat, barley, rye, triticale, milo, sago, millet, cassava, tapioca, sorghum, rice, peas, bean, banana, or potatoes.
  • the starch-containing material is or is derived from corn.
  • the starch-containing material is or is derived from wheat.
  • Starch from a grain may be ground or whole and may include solids, such as corn kernels, bran and/or cobs.
  • the starch may also be highly refined raw starch or feedstock from starch refinery processes.
  • Native starch consists of microscopic granules, which are insoluble in water at room temperature. When an aqueous starch slurry is heated, the granules swell and eventually burst, IFF10001-WO-PCT dispersing the starch molecules into the solution. At temperatures up to about 50°C to 75°C, the swelling may be reversible. However, with higher temperatures, an irreversible swelling called “gelatinization” begins. During “gelatinization” there is a dramatic increase in viscosity.
  • Granular starch to be processed may be a highly refined starch quality (e.g., at least 90%, at least 95%, at least 97%, or at least 99.5% pure), or it may be a crude starch-containing material including (e.g., milled) whole grains including non-starch fractions such as germ residues and fibers.
  • the raw material e.g., feedstock
  • whole grains may be reduced in particle size (e.g., by milling, in order to open up the structure and allowing for further processing). In dry milling, whole kernels are milled and used.
  • starch hydrolysate is used in the production of, for example, syrups. Both dry and wet milling are well known in the art of starch processing and may be used in a process of the instant disclosure. Methods for reducing the particle size of the starch-containing material are well known to those skilled in the art.
  • the starch e.g., of the starch-containing material
  • liquefying a starch- containing material produces a starch liquefact.
  • Liquefaction is generally carried out in the presence of an alpha-amylase (e.g., a bacterial alpha-amylase and/or acid fungal alpha- amylase).
  • an alpha-amylase e.g., a bacterial alpha-amylase and/or acid fungal alpha- amylase.
  • liquefying a starch-containing material with an alpha-amylase produces a starch liquefact.
  • a phytase is also present during liquefaction.
  • enzymes such as a xylanase and/or beta-glucanase are also present during liquefaction.
  • the long-chained starch is degraded into branched and linear shorter units (maltodextrins) by an alpha-amylase.
  • Liquefaction may be carried out as a three-step hot slurry process.
  • the slurry is heated to between 60-95°C and an alpha-amylase is added to initiate liquefaction (thinning).
  • the slurry may be jet-cooked at between 95-140°C (e.g., 105- 125°C, for about 1-15 minutes, e.g., about 3-10 minutes, especially about 5 minutes).
  • the slurry is then cooled to 60-95 °C and more alpha-amylase is added to obtain final hydrolysis (secondary liquefaction).
  • the jet-cooking process is carried out at pH 4.5-6.5, typically at a pH between 5 and 6.
  • the alpha-amylase may be added as a single dose, for example, before jet cooking.
  • the liquefaction process is carried out at between 70-95°C, such as 80-90°C, such as about 85°C, for about 10 minutes to 5 hours, typically for 1-2 hours.
  • the pH is between 4 and 7, such as between 4.5 and 5.5.
  • calcium may be IFF10001-WO-PCT added (to provide 1-60 ppm free calcium ions, such as about 40 ppm free calcium ions).
  • the liquefied starch will typically have a “dextrose equivalent” (DE) of 10-15.
  • Starch can be hydrolyzed into simpler carbohydrates by acids, various enzymes, or a combination thereof.
  • the primary enzymes used to hydrolyze starch into simpler carbohydrates are endo-amylases, exo-amylases, and debranching enzymes, which generally hydrolyze amylose and amylopectin.
  • Amylose is hydrolyzed mainly by amylases, while amylopectin also requires debranching enzymes such as pullulanases (EC 3.2.1.41) for complete hydrolysis.
  • An exemplary endo-amylase is an alpha-amylase (EC 3.2.1.1), specific for ⁇ -1,4- linkages of amylose and amylopectin. Exo-amylases have the ability to hydrolyze both ⁇ -1,4- linkages and ⁇ -1,6-linkages of amylose and amylopectin.
  • An exemplary exo-amylase is amyloglucosidase (often referred to as glucoamylase; EC 3.2.1.20).
  • Beta-amylase is an enzyme that has the ability to hydrolyze the ⁇ -1,4-linkages of amylose.
  • Alpha-amylases for use in liquefaction can be bacterial acid stable alpha-amylases, for example an alpha-amylase is from a Bacillus sp. such as Bacillus stearothermophilus or Bacillus lichenformis. In some embodiments, the alpha-amylase is Bacillus stearothermophilus alpha- amylase, or a variant thereof.
  • addition of an enzyme during liquefaction may facilitate an increase in co-product production.
  • adding an enzyme during liquefaction increases the amount of oil in a fermentation byproduct.
  • certain enzymes added during liquefaction may increase the amount and/or accessibility of oil in a fermentation byproduct.
  • adding an enzyme during liquefaction increases the amount of protein in a fermentation byproduct.
  • certain enzymes added during liquefaction may increase the amount and/or accessibility of protein in a fermentation byproduct.
  • the methods for reducing syrup viscosity include adding enzymes during liquefaction.
  • a cellulase, a hemicellulase, a protease, or any combination thereof may be added during liquefaction. In some embodiments, a cellulase is added during liquefaction. In some embodiments, the cellulase includes a cellobiohydrolase (EC 3.2.1.91 and/or EC 3.2.1.74), an endoglucanase (EC 3.2.1.4), beta-glucosidase (EC 3.2.1.21), or any combination thereof.
  • a cellobiohydrolase EC 3.2.1.91 and/or EC 3.2.1.74
  • an endoglucanase EC 3.2.1.4
  • beta-glucosidase EC 3.2.1.21
  • the cellulase includes a cellobiohydrolase (EC 3.2.1.91, EC IFF10001-WO-PCT 3.2.1.74, and/or EC 3.2.1.176), an endoglucanase (EC 3.2.1.4 and/or EC 3.2.1.6), beta-glucosidase (EC 3.2.1.21), or any combination thereof.
  • the cellulase includes a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase (BGL), or any combination thereof.
  • the cellulase is derived from Trichoderma reesei. In some embodiments, a hemicellulase is added during liquefaction. In some embodiments, the hemicellulase includes a xylanase. In some embodiments, the hemicellulase includes a GH10 xylanase.
  • the hemicellulase includes a xylanase having an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 1.
  • the hemicellulase includes a xylanase having an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the hemicellulase includes a xylanase having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the hemicellulase includes a xylanase having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the hemicellulase includes a xylanase having an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1.
  • SEQ ID NO: 2 MNRPSTRRTVRALLTAALAATALTAPAAPSLAQEASQEAALTRDLDLTGTEVALLRAA ESEAMDREEELSAVLGSDFGGVYLAPETGEVTVAVTDPAAVPVVEQSGATAQVVTFGE IFF10001-WO-PCT TALNDFVDSLNAVADRADEQITGWYTDLAADTVVITAFPGGSAAAEELAALAGVDER AVRVTESAARPQLFADVIGGNPYYFGGYRCSIGFSVRKGSDTGFATAGHCGETGTLTRS PEGVVAGSYFPGRDMGWVRLTGADTVTPLVNRYDGGTVTVTGSQEAVTGSSVCRSGS TTGWRCGIIQSKNQTVRYAEGTVTGLTRTTACAEAGDSGGPWLTGSQAQGVTSGGSGN CRTGGITYFQPINPLLSYFGLELVTG [00112]
  • the protease has an amino acid sequence with at least 60% sequence identity to SEQ ID NO:
  • the protease has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 2.
  • SEQ ID NO: 3 ADVIGGNPYYFGGYRCSIGFSVRKGSDTGFATAGHCGETGTLTRSPEGVVAGSYFPGRD MGWVRLTGADTVTPLVNRYDGGTVTVTGSQEAVTGSSVCRSGSTTGWRCGIIQSKNQT VRYAEGTVTGLTRTTACAEAGDSGGPWLTGSQAQGVTSGGSGNCRTGGITYFQPINPLL SYFGLELVTG [00115]
  • the protease has an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 3.
  • the protease has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 3.
  • the protease has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 9. In some embodiments, the protease has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 9. In some embodiments, the protease has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 9. In some embodiments, the protease has an amino acid sequence with at least IFF10001-WO-PCT 95% sequence identity to SEQ ID NO: 9. In some embodiments, the protease has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 9. In some embodiments, the protease has the amino acid sequence of SEQ ID NO: 9.
  • a pre-saccharification step of approximately 40-90 minutes at a temperature between 30-65°C (typically about 60°C) may be used, followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation (SSF) process.
  • Saccharification is generally performed at a temperature in the range of 20-75°C (e.g., 25-65°C and 40-70°C, typically about 60°C), and at a pH between about 4 and 5 (e.g., at about pH 4.5).
  • the saccharification and fermentation steps may be carried out either sequentially or simultaneously. In some embodiments, saccharification and fermentation are performed simultaneously (SSF).
  • a pre-saccharification step for about 30 minutes to 2 hours (e.g., 30 to 90 minutes) at a temperature of 30 to 65°C, typically about 60°C which is followed by a complete saccharification during fermentation (i.e., simultaneous saccharification and fermentation; SSF).
  • SSF simultaneous saccharification and fermentation
  • the ethanologen and enzymes are added together, and the process is then carried out at a temperature of 25-40°C (such as between 28°C and 35°C, such as between 30°C and 34°C, such as about 32°C).
  • the SSF process may be carried out at a pH from about 3 and 7, generally from pH 4.0 to 6.5, or pH 4.5 to 5.5.
  • ethanol is produced from starch-containing material without gelatinization (i.e., without cooking) of the starch-containing material (e.g., a "raw starch hydrolysis" process).
  • the ethanol fermentation product can be produced without liquefying the aqueous slurry containing the starch-containing material and water.
  • the process includes saccharifying a (e.g., milled) starch-containing material (e.g., granular starch) below the initial gelatinization temperature, in the presence of saccharifying enzymes (e.g., an alpha-amylase) to produce sugars that can be fermented into ethanol by a suitable fermenting organism.
  • saccharifying enzymes e.g., an alpha-amylase
  • the saccharification and fermentation steps are performed simultaneously, where the saccharifying enzymes and fermenting organisms (e.g., a yeast strain) are added together and fermentation is carried out at a temperature of 25-40°C.
  • the SSF process may be carried out at a pH from about 3 to 7 (e.g., pH 4.0 to 6.5, or pH 4.5 to 5.5). In some embodiments, fermentation is performed for about 6 to 120 hours.
  • the initial gelatinization temperature means the lowest temperature at which starch gelatinization commences. In general, starch heated in water begins to gelatinize between about 50°C and 75°C; the exact temperature of gelatinization depends on the specific starch and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the feedstock, to the particular variety of the feedstock as well as with the growth conditions.
  • a temperature below the initial gelatinization temperature means that the temperature typically lies in the range between 30-75°C, preferably between 45-60°C.
  • the process is carried at a temperature from 25°C to 40°C, 28°C to 35°C, 30°C to 34°C, or about 32°C.
  • Fermentation conditions are generally determined based on the type of starch- containing material used, the available fermentable sugars and/or the fermenting organism(s) used in the fermentation process. One skilled in the art can readily determine suitable fermentation conditions. The fermentation may be carried out at conventionally used conditions. In some embodiments, fermentation processes are anaerobic processes.
  • fermentations may be carried out at temperatures as high as 75°C (e.g., between 40-70°C or 50-60°C).
  • bacteria with a significantly lower temperature optimum down to about room temperature (about 20°C) are also known. Examples of suitable fermenting organisms can be found in Section I-C-3 below.
  • the fermentation may go on for 24 to 96 hours. In some embodiments, the fermentation may go on for 35 to 60 hours. In some embodiments, the fermentation is carried out at a temperature between 20°C to 40°C. In some embodiments, the fermentation is carried out at a temperature between 26°C to 34°C. In some embodiments, the fermentation is carried out at a temperature of about 32°C.
  • the fermentation may include, in addition to a fermenting microorganism (e.g., yeast), nutrients, and additional enzymes, including phytases.
  • a fermenting microorganism e.g., yeast
  • additional enzymes including phytases.
  • yeast in fermentation is well known in the art.
  • fermentation is carried out at a pH in the range between 3.0 and 7.0 (e.g., pH 3.5 to 6, or pH 4 to 5).
  • fermentations are ongoing for 6-96 hours.
  • the fermentation processes of the disclosure may be performed as a batch process or as a continuous process.
  • Fermentations may be conducted in an ultrafiltration system wherein the IFF10001-WO-PCT retentate is held under recirculation in the presence of solids, water, and the fermenting microorganism, and where the permeate is the desired fermentation product containing liquid.
  • contemplated are methods/processes conducted in continuous membrane reactors with ultrafiltration membranes and where the retentate is held under recirculation in presence of solids, water, and the fermenting organism(s) and where the permeate is the fermentation product containing liquid.
  • the fermenting organism may be separated from the fermented slurry and recycled.
  • the fermentation product may be separated from the fermentation medium.
  • the fermentation product e.g., ethanol
  • alcohol is separated from the fermented starch-containing material and purified by conventional methods of distillation.
  • the method includes distillation to obtain the fermentation product, e.g., ethanol.
  • the fermentation and the distillation may be carried out simultaneously and/or separately/sequentially.
  • the distillation is followed by one or more process steps for further refinement of the fermentation product.
  • the fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques.
  • Ethanol with a purity of up to about 96 vol. % can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.
  • the remaining material may be further processed to produce fermentation co-products.
  • Glucoamylases for Use in Saccharification and/or Fermentation Processes [00135]
  • a carbohydrate source generating enzyme present during saccharification is a glucoamylase.
  • a glucoamylase is present and/or added in a saccharification process and/or a fermentation process.
  • a glucoamylase is present and/or added in a simultaneous saccharification and fermentation (SSF) process.
  • SSF simultaneous saccharification and fermentation
  • a glucoamylase present and/or added in a saccharification process and/or a fermentation process and/or in an SSF process is of fungal origin.
  • the glucoamylase is derived from a strain of Aspergillus (e.g., A. niger, A. awamori, A. oryzae).
  • the glucoamylase is derived from a strain of Trichoderma, e.g., T. reesei.
  • the glucoamylase is derived from IFF10001-WO-PCT Talaromyces (e.g., T. emersonii), Trametes (e.g., T. cingulate), and the like.
  • the glucoamylase is a variant glucoamylase derived from native glucoamylase.
  • Glucoamylases can be added to the saccharification and/or fermentation in an amount of 0.0001-20 AGU/g DS, such as, for example, 0.001-10 AGU/g DS, or between 0.01-5 AGU/g DS (e.g., 0.1-2 AGU/g DS or 0.1-0.5 AGU/g DS).
  • compositions comprising glucoamylase are generally known to one skilled in the art.
  • a glucoamylase is present and/or added in saccharification and/or fermentation in combination with an alpha-amylase, described below.
  • Alpha-Amylase Present and/or Added in Saccharification, Fermentation, or SSF
  • an alpha-amylase is present and/or added in a saccharification process and/or a fermentation process described herein.
  • the alpha-amylase is of a fungal origin or a bacterial origin.
  • the alpha-amylase is a fungal acid stable alpha-amylase.
  • a fungal acid stable alpha-amylase is an alpha-amylase that has activity in the pH range of 3.0 to 7.0 and preferably in the pH range from 3.5 to 6.5, including activity at a pH of about 4.0, 4.5, 5.0, 5.5, and 6.0.
  • an alpha-amylase of the disclosure is derived from the genus Aspergillus, such as A. terreus, A. niger, A. oryzae, A. awamori, and A. kawachi.
  • the alpha-amylase is derived from the genus Rhizomucor (e.g., R. pusillus), or the genus Meripilus (e.g., M.
  • the ratio between glucoamylase and alpha-amylase present and/or added during saccharification and/or fermentation may be in the range from 500:1 to 1:1, 250:1 to 1:1, 100:1 to 1:1, 100:2 to 100:50, or 100:3 to 100:70.
  • a saccharifying enzyme may be used.
  • an enzyme preparation for use in saccharification may be used.
  • the enzyme preparation includes one or more saccharifying enzymes.
  • the enzyme preparation includes alpha-amylase (EC 3.2.1.1), glucoamylase (E.C.
  • the enzyme preparation includes one or more of an alpha- amylase (EC 3.2.1.1), a glucoamylase (EC 3.2.1.3), an isoamylase (EC 3.2.1.68), a beta-amylase (EC 3.2.1.2), a pullulanase (EC 3.2.1.41) and an Aspergillopepsin 1 (EC 3.4.23.18). 3.
  • a fermenting organism may be a bacterium from one or more genus selected from the group consisting of Zymomonas, Arthrobacter, Bacillus, Clostridium, IFF10001-WO-PCT Erwinia, Escherichia, Klebsiella, Lactobacillus, Pseudomonas, Streptomyces, and Thermoanaerobacter.
  • the bacterium is Zymomonas mobilis or Escherichia coli.
  • the fermenting organism may be a fungus.
  • the fungus for use in accordance with the present invention may be any ascomycetous fungus (e.g., an Ascomycete).
  • the fermenting organism is a yeast.
  • the yeast may be selected from the group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Issatchenkia, Kazachstania, and Torulaspora.
  • the yeast may be one or more selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces carlsbergensis, Saccharomyces kudriavtsevii, Saccharomyces kudriavzevii, and Saccharomyces pasforianus.
  • the fermenting organism is a Saccharomyces cerevisiae.
  • the fermenting organism is a Saccharomyces cerevisiae var. diastaticus yeast.
  • the fermenting organism may be a non-naturally occurring microorganism.
  • the fermenting organism is a yeast including one or more genetic modifications, where the one or more genetic modifications increase the amount and/or rate of ethanol production and/or reduce the amount of an undesirable fermentation product, such as glycerol and/or acetate.
  • an enzyme during saccharification, fermentation, or SSF may facilitate an increase in co-product production.
  • adding an enzyme during saccharification, fermentation, or SSF increases the amount of oil in a fermentation byproduct.
  • certain enzymes added during saccharification, fermentation, or SSF may increase the amount and/or accessibility of oil (co-product) in a fermentation byproduct.
  • adding an enzyme during saccharification, fermentation, or SSF increases the amount of protein in a fermentation byproduct.
  • certain enzymes added during saccharification, fermentation, or SSF may increase the amount and/or accessibility of protein in a fermentation byproduct.
  • the method for reducing syrup viscosity includes adding enzymes during saccharification, fermentation, or SSF.
  • the method for reducing syrup viscosity includes adding enzymes during saccharification.
  • the method for reducing syrup viscosity includes adding enzymes during fermentation.
  • the method for reducing syrup viscosity includes adding enzymes during saccharification and fermentation.
  • the method for reducing syrup viscosity includes adding enzymes during SSF.
  • a cellulase, a hemicellulase, a protease, or any combination thereof may be added during saccharification, fermentation, or SSF.
  • a cellulase, a hemicellulase, a protease, or any combination thereof may be added during saccharification.
  • a cellulase, a hemicellulase, a protease, or any combination thereof may be added during fermentation.
  • a cellulase, a hemicellulase, a protease, or any combination thereof may be added during SSF.
  • a cellulase is added during saccharification, fermentation, or SSF. In some embodiments, a cellulase is added during saccharification and/or fermentation. In some embodiments, a cellulase is added during SSF. In some embodiments, a cellulase is present during saccharification, fermentation, or SSF. In some embodiments, a cellulase is present during saccharification and/or fermentation. In some embodiments, a cellulase is present during SSF.
  • the cellulase includes a cellobiohydrolase (EC 3.2.1.91 and/or EC 3.2.1.74), an endoglucanase (EC 3.2.1.4), beta-glucosidase (EC 3.2.1.21), or any combination thereof.
  • the cellulase includes a cellobiohydrolase (EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176), an endoglucanase (EC 3.2.1.4 and/or EC 3.2.1.6), beta-glucosidase (EC 3.2.1.21), or any combination thereof.
  • the cellulase includes a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase (BGL), or any combination thereof.
  • the cellulase is derived from Trichoderma reesei.
  • a hemicellulase is added during saccharification, fermentation, or SSF.
  • a hemicellulase is added during saccharification and/or fermentation.
  • a hemicellulase is added during SSF.
  • a hemicellulase is present during saccharification, fermentation, or SSF. In some embodiments, a hemicellulase is present during saccharification and/or fermentation. In some embodiments, a hemicellulase is present during SSF. In some embodiments, the hemicellulase includes a xylanase. In some embodiments, the hemicellulase includes a GH10 xylanase.
  • the hemicellulase includes a xylanase having an amino acid sequence set forth by SEQ ID NO: 1, or a sequence having at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to SEQ ID NO: 1.
  • the hemicellulase includes a xylanase having an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 1.
  • the hemicellulase includes a xylanase having an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 1.
  • the hemicellulase includes a xylanase having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the hemicellulase includes a xylanase having an amino acid sequence with at IFF10001-WO-PCT least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the hemicellulase includes a xylanase having an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the hemicellulase includes a xylanase having an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 1.
  • a protease is added during saccharification, fermentation, or SSF. In some embodiments, a protease is added during saccharification and/or fermentation. In some embodiments, a protease is added during SSF. In some embodiments, a protease is present during saccharification, fermentation, or SSF. In some embodiments, a protease is present during saccharification and/or fermentation. In some embodiments, a protease is present during SSF.
  • the protease has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence set forth by SEQ ID NO: 3, or a sequence having at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 3.
  • the protease has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has the amino acid sequence of SEQ ID NO: 3.
  • the protease has an amino acid sequence set forth by SEQ ID NO: 8, or a sequence having at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to SEQ ID NO: 8.
  • the protease has an amino acid IFF10001-WO-PCT sequence with at least 60% sequence identity to SEQ ID NO: 8.
  • the protease has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 8.
  • the protease has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 8.
  • the protease has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, the protease has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, the protease has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 8. In some embodiments, the protease has the amino acid sequence of SEQ ID NO: 8. In some embodiments, the protease has an amino acid sequence set forth by SEQ ID NO: 9, or a sequence having at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to SEQ ID NO: 9.
  • the protease has the amino acid sequence of SEQ ID NO: 9.
  • any combination of cellulase, hemicellulase, or protease described herein is added during saccharification and/or fermentation or SSF.
  • any combination of cellulase, hemicellulase, or protease described herein is present during saccharification and/or fermentation or SSF.
  • Other enzymes for potential use during the saccharification and/or fermentation, or SSF include, but are not limited to, trehalase and pullulanase.
  • Separating whole stillage into thin stillage and wet IFF10001-WO-PCT cake to remove a significant portion of the liquid/water may be accomplished using any suitable separation technique, including, but not limited to, centrifugation, pressing, and filtration.
  • the separation (dewatering) is carried out by centrifugation.
  • industrial centrifuges may be used, such as, for example, decanter type centrifuges or high-speed decanter type centrifuges.
  • the separation (dewatering) is carried out using other conventional separation equipment such as a plate/frame filter presses, belt filter presses, screw presses, gravity thickeners and deckers, or similar equipment.
  • the enzymes added are a viscosity reducing composition including a cellulase and a hemicellulase. In some embodiments, the enzymes added are a viscosity reducing composition including a cellulase and a protease. In some embodiments, the enzymes added are a viscosity reducing composition including a hemicellulase and a protease. In some embodiments, the enzymes added are a viscosity reducing composition including an alpha-amylase.
  • the viscosity reducing composition includes an endoglucanase.
  • the endoglucanase has the amino acid sequence set forth by SEQ ID NO: 4, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 4.
  • the endoglucanase has an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 4.
  • the endoglucanase has an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 4.
  • the cellulase is derived from a strain of Penicillium. In some embodiments, the strain of Penicillium is a strain of Penicillium emersonii or Penicillium oxalicum. In some embodiments, the cellulase is derived from a strain of Talaromyces. In some embodiments, the strain Talaromyces is a strain of Talaromyces aurantiacus or Talaromyces emersonii. In some embodiments, the cellulase is derived from a strain of Trichoderma. In some embodiments, the strain of Trichoderma is a strain of Trichoderma reesei.
  • the expressed or over- expressed cellulase is a heterologous cellulase.
  • the over-expressed cellulase is a native cellulase.
  • native, heterologous, and/or recombinant cellulase are derived from the fungus.
  • the cellulase is contained in a cultured cell material, e.g., a cultured cell material from a fungus.
  • the cellulase is contained in a cultured IFF10001-WO-PCT cell material, e.g., a cultured cell material from a fungus where the fungus is a host cell (e.g., a recombinant fungus).
  • the cultured cell material is a whole broth.
  • the cultured cell material is clarified.
  • the fungus from which the cellulase is derived also expresses a hemicellulase.
  • the hemicellulase is a xylanase, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a pectate lyase, a xylosidase, or any combination thereof.
  • the hemicellulase includes a xylanase.
  • the hemicellulase includes an esterase.
  • the esterase is a feruloyl esterase (EC 3.1.1.73).
  • the esterase is an acetylxylan esterase (EC 3.1.1.72).
  • the hemicellulase includes an arabinofuranosidase (EC 3.2.1.55).
  • the hemicellulase is a native hemicellulase, e.g., a native xylanase, esterase, arabinofuranosidase.
  • the hemicellulase is a recombinant hemicellulase, e.g., a recombinant xylanase, esterase, arabinofuranosidase.
  • the fungus from which the cellulase is derived is a host cell that expresses, over-expresses, or does not express a hemicellulase.
  • the expressed or over-expressed hemicellulase is a recombinant hemicellulase.
  • the over-expressed hemicellulase is a native hemicellulase.
  • the cellulase includes one or more hemicellulases.
  • the one or more hemicellulases include native and/or recombinant hemicellulases.
  • the viscosity reducing composition includes a hemicellulase.
  • the enzyme composition includes a hemicellulase that is not or is in addition to a hemicellulase that may be part of a cultured cell material including cellulases.
  • the hemicellulase includes an arabinofuranosidase (EC 3.2.1.55). In some embodiments, the hemicellulase includes an esterase. In some embodiments, the esterase is a feruloyl esterase (EC 3.1.1.73). In some embodiments, the esterase is an acetylxylan esterase (EC 3.1.1.72). In some embodiments, the hemicellulase is a native hemicellulase.
  • the hemicellulase is a recombinant hemicellulase, e.g., a recombinant xylanase, esterase, arabinofuranosidase.
  • IFF10001-WO-PCT [00171]
  • the hemicellulase is not natively or recombinantly expressed by a fungus from which the cellulases are derived.
  • the enzyme composition includes a hemicellulase that is not contained in a cultured cell material.
  • the enzyme composition includes a hemicellulase, as described herein, that is native to a fungus from which the cellulase is derived and a hemicellulase that is heterologous to the fungus from which the cellulases are derived.
  • the hemicellulase is a heterologous xylanase.
  • the hemicellulase is a recombinant xylanase.
  • the hemicellulase includes a GH10 xylanase.
  • the hemicellulase includes a xylanase having the amino acid sequence set forth by SEQ ID NO: 1, or a sequence having at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to SEQ ID NO: 1.
  • the hemicellulase includes a xylanase having an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 1.
  • the hemicellulases includes a xylanase having an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 1.
  • the hemicellulase includes a xylanase having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the hemicellulase includes a xylanase having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the hemicellulase includes a xylanase having an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the hemicellulase includes a xylanase having an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 1.
  • the hemicellulase includes a xylanase having the amino acid sequence of SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellulase and/or a GH10 xylanase.
  • the viscosity reducing composition includes a cellulase derived from a strain of Trichoderma reesei and a GH10 xylanase.
  • the viscosity reducing composition includes a cellulase and a xylanase having at least 60, 70, 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellulase and a xylanase having at least 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the viscosity reducing composition includes a cellulase and a xylanase having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the viscosity reducing composition includes a cellulase and a xylanase having at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the viscosity reducing composition includes a cellulase and a xylanase having at least 99% sequence identity to SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellulase and a IFF10001-WO-PCT xylanase having the sequence set forth by SEQ ID NO: 1.
  • the cellulase of the viscosity reducing composition is derived from Trichoderma reesei.
  • the viscosity reducing composition includes one or more of a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase (BGL), or a GH10 xylanase.
  • CBHI cellobiohydrolase I
  • CBHII cellobiohydrolase II
  • EGI endoglucanase I
  • EGIII endoglucanase II
  • BGL beta-glucosidase
  • the viscosity reducing composition includes one or more of a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta- glucosidase (BGL), or a xylanase having at least 60, 70, 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1.
  • CBHI cellobiohydrolase I
  • CBHII cellobiohydrolase II
  • EGI endoglucanase I
  • EGII endoglucanase II
  • BGL beta- glucosidase
  • the viscosity reducing composition includes a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase (BGL), and a xylanase having at least 60% sequence identity to SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase (BGL), and a xylanase having at least 80% sequence identity to SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase (BGL), and a xylanase having at least 85% sequence identity to SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase (BGL), and a xylanase having at least 90% sequence identity to SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase (BGL), and a xylanase having at least 95% sequence identity to SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase (BGL), and a xylanase having the sequence set forth by SEQ ID NO: 1.
  • any of the viscosity reducing compositions described herein further include a protease.
  • the viscosity reducing composition includes one or more of a cellobiohydrolase (EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176), an endoglucanase (EC 3.2.1.4 and/or EC 3.2.1.6), a beta-glucosidase (EC 3.2.1.21), or a GH10 xylanase.
  • a cellobiohydrolase EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176
  • an endoglucanase EC 3.2.1.4 and/or EC 3.2.1.6
  • a beta-glucosidase EC 3.2.1.21
  • GH10 xylanase GH10 xylanase
  • the viscosity reducing composition includes one or more of a cellobiohydrolase (EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176), an endoglucanase (EC 3.2.1.4 and/or EC 3.2.1.6), a beta- glucosidase (EC 3.2.1.21), or a xylanase having at least 60, 70, 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1.
  • a cellobiohydrolase EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176
  • an endoglucanase EC 3.2.1.4 and/or EC 3.2.1.6
  • a beta- glucosidase EC 3.2.1.21
  • a xylanase having at least 60, 70, 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellobiohydrolase (EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176), an endoglucanase (EC 3.2.1.4 and/or EC 3.2.1.6), a beta-glucosidase (EC 3.2.1.21), and a xylanase having at least 60% sequence identity to SEQ ID NO: 1.
  • a cellobiohydrolase EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176
  • an endoglucanase EC 3.2.1.4 and/or EC 3.2.1.6
  • a beta-glucosidase EC 3.2.1.21
  • a xylanase having at least 60% sequence identity to SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellobiohydrolase (EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176), an endoglucanase (EC 3.2.1.4 and/or EC 3.2.1.6), a beta-glucosidase (EC 3.2.1.21), and a xylanase having at least 70% sequence identity to SEQ ID NO: 1.
  • a cellobiohydrolase EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176
  • an endoglucanase EC 3.2.1.4 and/or EC 3.2.1.6
  • a beta-glucosidase EC 3.2.1.21
  • a xylanase having at least 70% sequence identity to SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellobiohydrolase (EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176), an endoglucanase (EC 3.2.1.4 and/or EC 3.2.1.6), a beta-glucosidase (EC 3.2.1.21), and a xylanase having at least 80% sequence identity to SEQ ID NO: 1.
  • a cellobiohydrolase EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176
  • an endoglucanase EC 3.2.1.4 and/or EC 3.2.1.6
  • a beta-glucosidase EC 3.2.1.21
  • a xylanase having at least 80% sequence identity to SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellobiohydrolase (EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176), an endoglucanase (EC 3.2.1.4 and/or EC 3.2.1.6), a beta-glucosidase (EC 3.2.1.21), and a xylanase having at least 85% sequence identity to SEQ ID NO: 1.
  • a cellobiohydrolase EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176
  • an endoglucanase EC 3.2.1.4 and/or EC 3.2.1.6
  • a beta-glucosidase EC 3.2.1.21
  • a xylanase having at least 85% sequence identity to SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellobiohydrolase (EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176), an endoglucanase (EC 3.2.1.4 and/or EC 3.2.1.6), a beta-glucosidase (EC 3.2.1.21), and a xylanase having at least 90% sequence identity to SEQ ID NO: 1.
  • a cellobiohydrolase EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176
  • an endoglucanase EC 3.2.1.4 and/or EC 3.2.1.6
  • a beta-glucosidase EC 3.2.1.21
  • a xylanase having at least 90% sequence identity to SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellobiohydrolase (EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176), an endoglucanase (EC 3.2.1.4 and/or EC 3.2.1.6), a beta-glucosidase (EC 3.2.1.21), and a xylanase having at least 95% sequence identity to SEQ ID NO: 1.
  • a cellobiohydrolase EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176
  • an endoglucanase EC 3.2.1.4 and/or EC 3.2.1.6
  • a beta-glucosidase EC 3.2.1.21
  • a xylanase having at least 95% sequence identity to SEQ ID NO: 1.
  • the viscosity reducing composition includes a cellobiohydrolase (EC 3.2.1.91, EC 3.2.1.74, and/or EC 3.2.1.176), an endoglucanase (EC 3.2.1.4 and/or EC 3.2.1.6), a beta-glucosidase (EC 3.2.1.21), and a xylanase having the sequence set forth by SEQ ID NO: 1.
  • any of the viscosity reducing compositions described herein further include a protease.
  • the viscosity reducing composition includes or further includes a protease.
  • the protease has an amino acid sequence with at least 80% IFF10001-WO-PCT sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the protease has the amino acid sequence of SEQ ID NO: 2.
  • the protease has an amino acid sequence set forth by SEQ ID NO: 3, or a sequence having at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to SEQ ID NO: 3.
  • the protease has an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 3.
  • the protease has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 3.
  • the protease has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 3.
  • the protease has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 3. In some embodiments, the protease has the amino acid sequence of SEQ ID NO: 3. [00178] In some embodiments, the protease has an amino acid sequence set forth by SEQ ID NO: 8, or a sequence having at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to SEQ ID NO: 8.
  • the protease has an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 8. In some embodiments, the protease has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 8. In some embodiments, the protease has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 8. In some embodiments, the protease has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, the protease has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, the protease has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 8.
  • the protease has the amino acid sequence of SEQ ID NO: 8. [00179] In some embodiments, the protease has an amino acid sequence set forth by SEQ ID NO: 9, or a sequence having at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to SEQ ID NO: 9. In some embodiments, the protease has an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 9. In some embodiments, the protease has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 9.
  • the protease has an amino acid sequence with at least 80% sequence identity IFF10001-WO-PCT to SEQ ID NO: 9. In some embodiments, the protease has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 9. In some embodiments, the protease has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 9. In some embodiments, the protease has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 9. In some embodiments, the protease has the amino acid sequence of SEQ ID NO: 9. [00180] In some embodiments, the viscosity reducing composition includes or further includes an alpha-amylase.
  • any of the foregoing cellulase, hemicellulase, or protease combinations may further include an alpha-amylase.
  • the alpha-amylase has an amino acid sequence set forth by SEQ ID NO: 5, or a sequence having at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to SEQ ID NO: 5.
  • the alpha-amylase has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 5. In some embodiments, the alpha-amylase has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, the alpha-amylase has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, the alpha-amylase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the alpha-amylase has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 5. In some embodiments, the alpha-amylase has the amino acid sequence of SEQ ID NO: 5.
  • the alpha-amylase has an amino acid sequence set forth by SEQ ID NO: 6, or a sequence having at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to SEQ ID NO: 6.
  • SEQ ID NO: 6 IFF10001-WO-PCT AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTSQADVGY GPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGDVVMNHKAGADYT ENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTYSNWKWQWFHFDGTDWDQS RSLSRIFKFHGKAWDWPVSSENGNYDYLMYADYDYDHPDVVNEMKKWGVWYANEV GLDGYRLDAVKHIKFSFLKDWVDNARAATGKEMFTVGEYWQNDLGALNNYLAKVNY NQSLFDAPLHYNFYAASTGGGAYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLES TVQPWFKPLAYAFILTRSGGYPAVFYGDMYGTKGTTTYEIPALKSKIEPLLKARKDY
  • the alpha-amylase has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 6. In some embodiments, the alpha-amylase has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, the alpha-amylase has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the alpha-amylase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the alpha-amylase has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, the alpha-amylase has the amino acid sequence of SEQ ID NO: 6.
  • the cellulases of the viscosity reducing composition are derived from Trichoderma reesei.
  • the viscosity reducing composition and the compositions including a cellulase, a hemicellulase, a protease, or any combination thereof include the same cellulases, hemicellulases, and/or proteases.
  • the viscosity reducing composition and the compositions including a cellulase, a hemicellulase, a protease, or any combination thereof include different cellulases, hemicellulases, and/or proteases.
  • the viscosity reducing composition and the compositions including a cellulase, a hemicellulase, a protease, or any combination thereof include the same and different cellulases, hemicellulases, and/or proteases.
  • the viscosity reducing composition is added to any one or more of the thin stillage, an intermediate product formed during concentrating the thin stillage to a produce a syrup, or the syrup produced.
  • the viscosity reducing composition is added to the thin stillage.
  • the viscosity reducing composition is added to an intermediate product formed during concentrating the thin stillage to a produce a syrup.
  • the viscosity reducing composition is added to the syrup.
  • IFF10001-WO-PCT [00189]
  • the viscosity reducing composition as described in any of these embodiments may be incubated with the thin stillage, intermediate product, and/or syrup for a period of time sufficient to decrease the viscosity of the syrup.
  • the incubation period with the viscosity reducing composition is at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 40 hours, at least 41 hours, at least 42 hours, at least 43 hours, at least 44 hours, at least 45 hours, at least 46 hours, at least 47 hours, at least 48 hours, at least 50 hours, at least 55 hours, at least 60 hours, at least 62 hours, at least 64 hours, at least 68 hours, at least 70 hours, or at least 72 hours.
  • the incubation period is 48 hours. In some embodiments, the incubation period is at least or about 30 minutes. In some embodiments, the incubation period is in the range of 30 minutes to 6 hours. In some embodiments, the incubation period is in the range of 1 to 6 hours. In some embodiments, the incubation period is in the range of 2 to 6 hours. In some embodiments, the incubation period is in the range of 3 to 6 hours. In some embodiments, the incubation period is in the range of 4 to 6 hours. In some embodiments, the incubation period is in the range of 5 to 6 hours. Those skilled in the art will appreciate that to achieve longer incubation periods, containers, such as holding tanks, may be used for the desired time period.
  • the conditions may be adjusted to further optimize the reduction in syrup viscosity.
  • the temperature may be adjusted in a holding tank to a set point or within a range that is optimal for the enzymes of the viscosity reducing composition.
  • the temperature range may be from 20°C to 90°C.
  • the temperature range may be from 30°C to 85°C.
  • the viscosity reducing composition is dosed at a concentration of at least about 0.00001% weight (wt) enzyme/wt.
  • the viscosity reducing composition is dosed at a concentration of at least about 0.0001% wt enzyme/wt. In some embodiments, the viscosity reducing composition is dosed at a concentration of at least about 0.001% wt enzyme/wt. In some embodiments, the viscosity reducing composition is dosed at a concentration of at least about 0.005% wt enzyme/wt. In some embodiments, the viscosity reducing composition is dosed at a concentration of at least about 0.01% wt enzyme/wt. In some embodiments, the viscosity reducing composition is dosed at a concentration of at least about 0.05% wt enzyme/wt.
  • the viscosity reducing composition is dosed at a concentration of at least about 0.1% wt enzyme/wt. In some embodiments, the viscosity IFF10001-WO-PCT reducing composition is dosed at a concentration of at least about 0.5% wt enzyme/wt. In some embodiments, the viscosity reducing composition is dosed at a concentration of about 0.00001% wt enzyme/wt to about 1% wt enzyme/wt. In some embodiments, the viscosity reducing composition is dosed at a concentration of about 0.0001% wt enzyme/wt to about 1% wt enzyme/wt.
  • the viscosity reducing composition is dosed at a concentration of about 0.001% wt enzyme/wt to about 1% wt enzyme/wt. In some embodiments, the viscosity reducing composition is dosed at a concentration of about 0.005% wt enzyme/wt to about 1% wt enzyme/wt. In some embodiments, the viscosity reducing composition is dosed at a concentration of about 0.01% wt enzyme/wt to about 1% wt enzyme/wt. In some embodiments, the viscosity reducing composition is dosed at a concentration of about 0.05% wt enzyme/wt to about 1% wt enzyme/wt.
  • the viscosity reducing composition is dosed at a concentration of about 0.1% wt enzyme/wt to about 1% wt enzyme/wt. In some embodiments, the viscosity reducing composition is dosed at a concentration of about 0.25% wt enzyme/wt to about 1% wt enzyme/wt. In some embodiments, the viscosity reducing composition is dosed at a concentration of about 0.5% wt enzyme/wt to about 1% wt enzyme/wt. In some embodiments, the viscosity reducing composition is dosed at a concentration of about 0.75% wt enzyme/wt to about 1% wt enzyme/wt.
  • the enzyme wt/wt is enzyme wt/wt syrup. In some embodiments, the enzyme wt/wt is enzyme wt/wt thin stillage. In some embodiments, the enzyme wt/wt is enzyme wt/wt intermediate product formed during concentrating the thin stillage to a produce a syrup. In some embodiments, the enzyme wt is the weight of the viscosity reducing composition. [00191]
  • the pH of the incubation may range from about 3.0 to about 7. In some embodiments, the pH is in a range of about 3.0 to 6.0. In some embodiments, the pH is in a range of about 3.5 to 6.0. In some embodiments, the pH is in a range of about 3.5 to 5.
  • the pH is about 3.0. In some embodiments, the pH is about 3.1. In some embodiments, the pH is about 3.2. In some embodiments, the pH is about 3.3. In some embodiments, the pH is about 3.4. In some embodiments, the pH is about 3.5. In some embodiments, the pH is about 3.6. In some embodiments, the pH is about 3.7. In some embodiments, the pH is about 3.8. In some embodiments, the pH is about 3.9. In some embodiments, the pH is about 4.0. In some embodiments, the pH is about 4.1. In some embodiments, the pH is about 4.2. In some embodiments, the pH is about 4.3. In some embodiments, the pH is about 4.4. In some embodiments, the pH is about 4.5.
  • the pH is about 6.1. In some embodiments, the pH is about 6.2. In some embodiments, the pH is about 6.3. In some embodiments, the pH is about 6.4. In some embodiments, the pH is about 6.5. In some embodiments, the pH is about 6.6. In some embodiments, the pH is about 6.7. In some embodiments, the pH is about 6.8. In some embodiments, the pH is about 6.9. In some embodiments, the pH is about 7.0. [00192] In some embodiments, the viscosity of the syrup is less than the viscosity of a syrup not treated with a viscosity reducing composition as described herein.
  • the decrease in syrup viscosity is at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50% or more compared to the viscosity of a syrup not treated with a viscosity reducing composition as described herein. In some embodiments, the decrease in syrup viscosity is at least 2% or more compared to the viscosity of a syrup not treated with a viscosity reducing composition as described herein. In some embodiments, the decrease in syrup viscosity is at least 5% or more compared to the viscosity of a syrup not treated with a viscosity reducing composition as described herein.
  • an increased amount of oil is recovered from the syrup compared to a syrup not treated with a viscosity reducing composition as described herein.
  • the increase in oil recovered is at least 1, 2, 3, 4, 5, 10, 20% or more compared to the oil recovered from a syrup not treated with a viscosity reducing composition as described herein. It will be appreciated that all comparisons are made between syrups produced under identical conditions with the only difference being the presence or absence of the viscosity reducing composition.
  • a method for reducing syrup viscosity comprising: (a) liquefying a starch-containing material with an alpha-amylase to produce a starch liquefact; (b) saccharifying the starch liquefact with a glucoamylase to produce a saccharification product; (c) fermenting the saccharification product using a fermenting organism to produce a fermentation product; (d) recovering the fermentation product to produce a whole stillage; (e) separating the whole stillage into thin stillage and wet cake; and (f) concentrating the thin stillage to produce a syrup; wherein a viscosity reducing composition comprising a cellulase, a hemicellulase, a protease, or any combination thereof, is added during step (f).
  • step (f) 2. The method of embodiment 1, wherein the viscosity reducing composition is added to the thin stillage, to an intermediate product formed during concentrating the thin stillage to a produce a syrup, and/or to the syrup produced in step (f). 3. The method of embodiment 1 or embodiment 2, wherein step (b) and step (c) are combined as a simultaneous saccharification and fermentation step (SSF). 4. The method of any one of embodiments 1-3, wherein the cellulase comprises a cellobiohydrolase, an endoglucanase, a beta-glucosidase, or any combination thereof. 5.
  • the cellulase comprises a cellobiohydrolase I (CBHI), a cellobiohydrolase II (CBHII), an endoglucanase I (EGI), an endoglucanase II (EGII), a beta-glucosidase BGL, or any combination thereof.
  • CBDHI cellobiohydrolase I
  • CBHII cellobiohydrolase II
  • EGI endoglucanase I
  • EGII endoglucanase II
  • beta-glucosidase BGL beta-glucosidase BGL
  • the hemicellulase comprises a xylanase.
  • the hemicellulase comprises a GH10 xylanase.
  • the hemicellulase comprises a sequence having at least 60% sequence identity to the sequence set forth by SEQ ID NO: 1.
  • IFF10001-WO-PCT 11.
  • the method of any one of embodiments 1-10, wherein the protease comprises a sequence having at least 60% sequence identity to the sequence set forth by SEQ ID NO: 3. 12.
  • step (i) a composition comprising a cellulase, a hemicellulase, a protease, or any combination thereof is added during step (a), step (b), and/or step (c); or (ii) wherein step (b) and step (c) are combined as a simultaneous saccharification and fermentation step (SSF) and a composition comprising a cellulase, a hemicellulase, a protease, or any combination thereof is added during step (a) and/or the SSF. 13.
  • SSF simultaneous saccharification and fermentation step
  • Example 1 Screening enzyme classes on syrup for flowability improvement
  • High and low viscosity syrups were collected from a commercial dry grind ethanol plant and frozen, prior to testing in a laboratory-scale experiment.
  • the high viscosity syrup (termed “Viscous Syrup”) was collected from the plant during a cellulase and xylanase SSF trial, during which an engineered Trichoderma whole cellulase and a thermostable xylanase were added into SSF.
  • the low viscosity syrup was collected prior to the cellulase and xylanase SSF trial.
  • No-enzyme control vials were also prepared for the Viscous and Baseline Syrups, IFF10001-WO-PCT with only water added to the syrups, instead of enzyme. Vials were placed into a forced air incubator set to 65°C, shaking at 200 rpm, for 2 hours.
  • Table E1 shows the resulting viscosity for the 15 enzymes tested, as well as the Viscous and Baseline syrups without enzyme added.
  • the enzymes that improved syrup viscosity the most (Low-Moderate category) were Trichoderma Whole Cellulase, Engineered Trichoderma Whole Cellulase 1, Engineered Trichoderma Whole Cellulase 2, and Thermostable Xylanase. Following this category, three additional enzymes provided some benefit and resulted in syrups in the Moderate category, including Engineered Whole Cellulase 3, Xylanase-Amylase-Protease Blend, and Thermostable Protease. All other enzymes tested had either no or very little impact on syrup viscosity. Table E1.
  • Example 2 Enzyme treatment to improve syrup flowability
  • High and low viscosity syrups were collected and frozen as described in Example 1.
  • IFF10001-WO-PCT [00202] The frozen syrups were thawed and incubated at 70°C for around 3 hours to pasteurize the syrups and allow for the heated syrups to be transferred to reaction vessels.4.0 grams of heated Viscous Syrup were weighed into 15 mL polypropylene tubes. To each tube, enzyme was added at a dose of 0.1% volume of enzyme per weight of syrup.
  • Trichoderma Whole Cellulase Trichoderma Whole Cellulase
  • Engineered Trichoderma Whole Cellulase 1 Engineered Trichoderma Whole Cellulase 2
  • Thermostable Xylanase SEQ ID NO: 1
  • No-enzyme control tubes were also prepared for the Viscous and Baseline Syrups, with only water added to the syrups, instead of enzyme. Tubes were placed into a forced air incubator set to 80°C, shaking at 200 rpm, for 2 hours.
  • FIG.1 shows the average speed rankings and corresponding standard deviations of the four observations.
  • Tubes were IFF10001-WO-PCT incubated in a water bath at either 80°C for 2 hours (for Viscous Syrup) or 88°C for 0.7 hours (for Baseline Syrup) with orbital shaking at 100 rpm. Following incubation, viscosity was measured on a Brookfield LVDVE Viscometer. Percent viscosity reduction over the no-enzyme control, by syrup type, was calculated for each sample, and listed in Table E2. Table E2. Enzyme treatments to reduce syrup viscosities.
  • Enzymes were added to Viscous or Baseline Syrups and incubated prior to viscosity measurement and calculation of r nt r d ti n v r th n nz m ntr l p ase, and a blend of both products reduce syrup viscosity.
  • Example 4 Viscosity reduction in syrup from corn [00208] Syrups from two commercial corn ethanol plants were obtained. The syrups were evaporated thin stillage at the dry solid (DS) content that was obtained at the plant. Syrup 1 had a DS of 42% and Syrup 2 had a DS of 27%.
  • enzymes that consistently showed viscosity reduction included cellulase (e.g., endoglucanase), xylanase, protease, and alpha-amylase.
  • cellulase e.g., endoglucanase
  • xylanase e.g., xylanase
  • protease e.g., alpha-amylase
  • alpha-amylase e.g., alpha-amylase
  • Enzyme blends for example, xylanase, endoglucanase, and protease, also provided viscosity reduction.
  • Table E3 Viscosity reduction by syrup and enzyme treatment
  • Syrup Enzyme Treatment Enzyme dose
  • Viscosity IFF10001-WO-PCT Thermostable Protease SEQ ID NO: 3
  • Endoglucanase EC 3.2.1.4
  • SEQ ID NO: 7 [00212]
  • Syrups from commercial wheat ethanol plants were obtained. The syrup was evaporated thin stillage at the DS that was obtained at the plant. Syrup 1 had a DS of 37.6%, Syrup 2 had a DS of 41%, and Syrup 3 had a DS of 32.1%.
  • Syrup 3 was obtained from a blend of mainly wheat with barley, rye, and triticale included.
  • Viscosity reduction by enzymes was tested as described in Example 4 above. [00213] The results shown in Table E4 demonstrate that the enzymes had a substantial impact on syrups from wheat. Although the effects varied by syrup, enzymes that consistently showed good viscosity reduction on syrups from wheat included cellulase (e.g., endoglucanase), xylanase, protease, and alpha-amylase. Blends of enzymes also performed well in reducing syrup viscosity.
  • cellulase e.g., endoglucanase
  • xylanase xylanase
  • protease and alpha-amylase
  • Table E4 Viscosity reduction by syrup and enzyme treatment Syrup Enzyme Treatment Enzyme dose Viscosity IFF10001-WO-PCT Trichoderma Whole Cellulase (cellobiohydrolase, 0.3;0.4 15% endoglucanase, and beta-glucosidase); [00 ] e presen nven on s no n ended o be m ed n scope o e par cu ar d sclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

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Abstract

L'invention concerne des procédés et des compositions destinés à réduire la viscosité d'un sirop obtenu comme sous-produit de processus de fermentation.
PCT/US2024/060099 2023-12-13 2024-12-13 Procédés de réduction de la viscosité du sirop Pending WO2025129043A2 (fr)

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WO2010149721A1 (fr) 2009-06-23 2010-12-29 Danisco A/S Procédé de transformation d'une bactérie appartenant au genre streptococcus par compétence naturelle
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WO2015057517A1 (fr) * 2013-10-17 2015-04-23 Danisco Us Inc. Utilisation d'hémicellulases pour améliorer la production d'éthanol
CN107250163A (zh) * 2015-02-06 2017-10-13 嘉吉公司 修饰的葡糖淀粉酶和具有增强的生物产物产生的酵母菌株

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