Methods and compositions for improving ethanol production

Abstract

The present disclosure generally relates to microbial fermentation, grain processing, and ethanol production. Accordingly, certain embodiments of the present disclosure relate to starch-containing material and microbial fermentation of such starch-containing material for the production of ethanol. More particularly, certain embodiments relate to a novel protease cocktail comprising aspartic proteases and serine proteases for use in processing such starch-containing material in processes for fermenting starch to ethanol as described herein.

Description

Technical Field

[0001] This disclosure generally relates to fields such as biology, molecular biology, genetics, microbial fermentation, grain processing, and ethanol production. Some embodiments of this disclosure relate to starch-containing materials (raw materials) and their microbial fermentation for the bioproduction of ethanol.

[0002] Cross-references to related applications

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 958,149, filed January 7, 2020, which is incorporated herein by reference in its entirety.

[0004] References to sequence lists

[0005] The electronic submission of the text file sequence list named “NB41362-WO-PCT_SequenceListing.txt” was created on December 7, 2020, and is 20KB in size. It is hereby incorporated in its entirety by reference. Background Technology

[0006] Many crops are viable candidates for converting starch into fermentable feedstocks, which can then be fed to various microorganisms to produce a variety of biochemicals (e.g., ethanol). Corn is typically used as the primary starch source for producing such fermentable feedstocks; however, other high-starch sources, such as sorghum, wheat, barley, rye, and cassava, are gaining increasing attention as viable feedstocks for industrial biochemical production. The bioproduction of ethanol from starch-containing materials (feedstocks) is generally well-known in the art.

[0007] For example, methods known in the art as "conventional" processes are commonly used to produce fermentable feedstocks from insoluble starch. This typically involves heating milled whole grains (or starch slurry) to temperatures exceeding 95°C in the presence of α-amylase (this process is referred to as "liquefaction"), followed by cooling, pH adjustment, and subsequent glucosylase hydrolysis (this process is referred to as "saccharification"). Such conventional processes can produce fermentable feedstocks containing, for example, more than 90% glucose (see, for example, U.S. Patent Nos. 3,912,590 and 4,933,279). Another well-known process, often referred to as "raw starch hydrolysis" (or "granular starch hydrolysis"), involves the simultaneous saccharification and fermentation of granular starch below its initial gelatinization temperature in the presence of acidic fungal α-amylase and glucosylase (see, for example, PCT Publications WO 1992 / 20777, WO2003 / 66826, and WO 2007 / 145912).

[0008] While such processes for the bioproduction of ethanol from starch-containing materials (raw materials) are generally known, there remains a persistent and unmet need in the art, including but not limited to methods for increasing the amount of ethanol produced in such processes (e.g., increasing the yield of recovered ethanol), methods for increasing ethanol productivity, methods for reducing the amount of glycerol produced, and methods for improving the ability of host cells to ferment ethanol production. Summary of the Invention

[0009] This disclosure generally relates to methods and compositions for the bioproduction of ethanol. Some embodiments relate to ethanol-producing microbial cells (strains) and their use in fermentation processes. Some embodiments relate to compositions and methods for producing ethanol in a process of fermenting starch into ethanol. Some other embodiments relate to compositions and methods for increasing ethanol production in a process of fermenting starch into ethanol. Other embodiments relate to compositions and methods for reducing the amount of glycerol produced in a process of fermenting starch into ethanol. Some other embodiments relate to identifying a mixture (combination) of proteases that can increase ethanol yield and / or increase ethanol production rate and / or reduce the amount of glycerol produced in a process of fermenting starch into ethanol, wherein the protease mixture (combination) may be added during saccharification and / or fermentation, or be present during saccharification and / or fermentation.

[0010] Some other embodiments relate to compositions and methods for improving the ability of host cell fermentation starch compositions to produce ethanol in a process of starch fermentation to ethanol, wherein a protease mixture (composition) may be added during saccharification and / or fermentation, or may be present during saccharification and / or fermentation.

[0011] Therefore, in some embodiments, this disclosure relates to a method for producing ethanol from a starch-containing material, comprising (a) liquefying the starch-containing material in the presence of α-amylase at a temperature above the initial gelatinization temperature of the starch-containing material, (b) saccharifying the liquefied material obtained in step (a) using a saccharifying enzyme, and (c) producing the material obtained in step (b) by host fermentation with ethanol under conditions suitable for ethanol production, wherein steps (b) and / or step (c) are carried out in the presence of a mixture of aspartic protease and serine protease.

[0012] In some embodiments of these methods, saccharification and fermentation occur simultaneously. In other embodiments of these methods, the produced ethanol is recovered.

[0013] In other embodiments of these methods, the amount of ethanol produced is increased relative to the amount of ethanol produced by the same method except that steps (b) and / or (c) are performed in the presence of aspartic protease or serine protease rather than a mixture thereof. In some other embodiments of these methods, the ethanol production rate is increased relative to the ethanol production rate produced by the same method except that steps (b) and / or (c) are performed in the presence of aspartic protease or serine protease rather than a mixture thereof. In another embodiment of these methods, the amount of glycerol produced is decreased relative to the amount of glycerol produced by the same method except that steps (b) and / or (c) are performed in the presence of aspartic protease or serine protease rather than a mixture thereof. In other embodiments, the amount of supplemental nitrogen required for the ethanol-producing host is reduced relative to the amount of ethanol produced by the same method fermented in the presence of aspartic protease or serine protease rather than a mixture thereof, except that steps (b) and / or (c) are performed in the presence of aspartic protease or serine protease rather than a mixture thereof. In some embodiments, the aspartic protease contains about 60% sequence identity with the aspartic protease of SEQ ID NO: 2 or SEQ ID NO: 6, and the serine protease contains about 60% sequence identity with the serine protease of SEQ ID NO: 4.

[0014] In other embodiments, this disclosure relates to a method for producing ethanol from a starch-containing material, comprising (a) saccharifying the starch-containing material using a saccharifying enzyme at a temperature below the initial gelatinization temperature of the starch-containing material, and (b) producing the material obtained in step (a) from host fermentation with ethanol under conditions suitable for ethanol production, wherein steps (a) and / or (b) are carried out in the presence of a mixture of aspartic proteases and serine proteases. In some embodiments of these methods, the amount of ethanol produced is increased relative to the amount of ethanol produced using the same method except that steps (a) and / or (b) are carried out in the presence of aspartic proteases or serine proteases rather than a mixture thereof. In other embodiments of these methods, the ethanol production rate is increased relative to the ethanol production rate of the same method except that steps (a) and / or (b) are carried out in the presence of aspartic proteases or serine proteases rather than a mixture thereof. In still other embodiments, the amount of glycerol produced is decreased relative to the amount of glycerol produced using the same method except that steps (a) and / or (b) are carried out in the presence of aspartic proteases or serine proteases rather than a mixture thereof. In certain other embodiments of these methods, the ethanol-producing host requires a reduced amount of supplemental nitrogen compared to the same ethanol-producing host fermented using the same method except that steps (a) and / or (b) are performed in the presence of an aspartic protease or a serine protease rather than a mixture thereof. In other embodiments, the aspartic protease comprises approximately 60% sequence identity with the aspartic protease of SEQ ID NO: 2 or SEQ ID NO: 6, and the serine protease comprises approximately 60% sequence identity with the serine protease of SEQ ID NO: 4.

[0015] Therefore, in some other embodiments, this disclosure relates to methods for producing ethanol from starch-containing materials, the methods comprising fermenting the starch-containing material with an ethanol-producing host under conditions suitable for ethanol production, and recovering the produced ethanol, wherein the ethanol-producing host expresses and secretes heterologous aspartic proteases and heterologous serine proteases.

[0016] Therefore, certain other embodiments of this disclosure relate to protease compositions comprising a mixture of aspartic proteases and serine proteases. In preferred embodiments, the protease composition comprising a mixture of aspartic proteases and serine proteases is used for the bioproduction of ethanol from starch-containing materials. Thus, in some embodiments, the aspartic protease comprises about 60% sequence identity with the aspartic protease of SEQ ID NO: 2 or SEQ ID NO: 6, and the serine protease comprises about 60% sequence identity with the serine protease of SEQ ID NO: 4. In some other embodiments, the serine protease excludes enzymes designated as EC 3.4.14 and / or the serine protease excludes enzymes designated as EC 3.4.16. In some embodiments, the protease composition is mixed with starch-containing materials. In another embodiment, the protease composition is mixed with starch-containing materials after liquefaction of the starch-containing materials. In yet another embodiment, the protease composition is mixed with a granular starch composition obtained from starch-containing materials. In other embodiments, the protease composition is mixed with starch-containing materials in a simultaneous saccharification and fermentation (SSF) process.

[0017] Biological sequence description

[0018] SEQ ID NO: 1 is a polynucleotide sequence of *T. reesei* encoding the A10 aspartic protease contained in SEQ ID NO: 2.

[0019] SEQ ID NO: 2 is the amino acid sequence of the A10 aspartic protease encoded by SEQ ID NO: 1.

[0020] SEQ ID NO:3 is a polynucleotide sequence of Aspergillus niger that encodes the aorsin serine protease contained in SEQ ID NO:4.

[0021] SEQ ID NO:4 is the amino acid sequence of the orexin serine protease encoded by SEQ ID NO:3.

[0022] SEQ ID NO: 5 is the aspartic protease encoding SEQ ID NO: 6. The polynucleotide sequence of Trichoderma reesei.

[0023] SEQ ID NO: 6 is The amino acid sequence of aspartic protease. Attached Figure Description

[0024] Figure 1The hydrolytic synergistic effect of a 50 μg / ml combination of aspartic (A10) protease (25 μg / ml) and serine (Aurestin) protease (25 μg / ml) relative to the hydrolytic activity of either aspartic (A10) protease alone (50 μg / ml) or serine (Aurestin) protease alone (50 μg / ml) was demonstrated. Detailed Implementation

[0025] As set forth and described herein, the compositions and methods of this disclosure generally relate to the bioproduction of ethanol. Some embodiments relate to ethanol-producing microbial cells (strains) and their use in fermentation processes. Some embodiments relate to compositions and methods for producing ethanol in a process of starch fermentation to ethanol. Some other embodiments relate to compositions and methods for increasing ethanol production in a process of starch fermentation to ethanol. Other embodiments relate to compositions and methods for reducing the amount of glycerol produced in a process of starch fermentation to ethanol. Some other embodiments relate to protease mixtures (combinations) that can increase ethanol yield and / or increase ethanol production rate and / or reduce the amount of glycerol produced in a process of starch fermentation to ethanol, wherein the protease mixture (combination) may be added during saccharification and / or fermentation, or be present during saccharification and / or fermentation. Some other embodiments relate to compositions and methods for improving the ability of host cells to ferment starch compositions for ethanol production in a process of starch fermentation to ethanol, wherein the protease mixture (combination) may be added during saccharification and / or fermentation, or be present during saccharification and / or fermentation.

[0026] More specifically, as described and illustrated herein, a significant finding of this disclosure is that the combined use of a mixture (combination) of aspartic and serine proteases in the starch-to-ethanol fermentation process increases ethanol yield while simultaneously reducing the amount of glycerol produced. Furthermore, as described and illustrated herein, it has been observed that the use of such a combination of aspartic and serine proteases during the starch-to-ethanol fermentation process increases the rate of increase in ethanol yield. Alternatively, another finding of this disclosure is that the combined use of such a mixture of aspartic and serine proteases in the starch-to-ethanol fermentation process improves the fermentation capacity of the ethanol-producing host cell. For example, without wishing to be bound by theory or mechanism, it is believed that the presence or addition of novel aspartic and serine proteases in the starch-to-ethanol fermentation process increases the concentration of peptides and free amino acids present in the feedstock, which are considered excellent sources of amine nitrogen and / or energy and / or nutrients for the ethanol-producing host, as presented and described herein.

[0027] I. Definition

[0028] Before describing the compositions and methods of the present invention in detail, the following terms are defined for clarity. Undefined terms shall conform to their conventional meaning as used in the relevant art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the compositions and methods of the present invention are applied.

[0029] All disclosures and patents referenced in this specification are incorporated herein by reference.

[0030] When a range of values ​​is provided, it should be understood that each intermediate value is a tenth of a unit up to the lower limit (unless the context clearly indicates otherwise), and the range between the upper and lower limits, as well as any other stated or intermediate values ​​within that range, are encompassed within the compositions and methods of the invention. The upper and lower limits of these smaller ranges may be independently included within those smaller ranges and are also encompassed within the compositions and methods of the invention, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both limits, the range excluding any one or both of those included limits is also included in the compositions and methods of the invention.

[0031] Certain ranges are presented herein with the term "approximately" preceding the numerical value. The term "approximately" provides literal support for the exact figures that follow it, as well as figures that are close to or approximate to the figures following the term. In determining whether a figure is close to or approximates a particularly stated figure, an unlisted figure that is close to or approximates can be a figure that is substantially equivalent to the figure specifically stated in the context in which it is presented. For example, with respect to numerical values, the term "approximately" refers to a numerical value of... - 10% to + The range is 10%, unless the term is specifically defined in the context. In another instance, the phrase "approximately 6 pH values" refers to pH values ​​from 5.4 to 6.6, unless pH values ​​are specifically defined otherwise.

[0032] The headings provided herein are not intended to limit the various aspects or embodiments of the compositions and methods of the invention, which can be derived by referring to the specification as a whole. Therefore, when the specification is referred to as a whole, the terms to be defined below are defined more fully.

[0033] According to this specific embodiment, the following abbreviations and definitions apply. It should be noted that the singular forms “a / an” and “the” include a plural of indicators unless the context clearly indicates otherwise. Thus, for example, reference to “enzyme” includes a plurality of such enzymes, and reference to “dosage” includes reference to one or more dosages and their equivalents known to those skilled in the art.

[0034] It should be further noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to be based on the use of exclusive terms such as “alone,” “only,” “excluding,” “not including,” or “negative” limitations related to the description of the claim elements.

[0035] It should be further noted that, as used herein, the term “comprising” means “including, but not limited to, one or more components following the term “comprising”.” The one or more components following the term “comprising” are essential or mandatory, but compositions comprising one or more components may further include other non-mandatory or optional components.

[0036] It should also be noted that, as used herein, the term "composed of" means "including but not limited to" the one or more components following the term "composed of". Therefore, the one or more components following the term "composed of" are essential or mandatory, and one or more other components are not present in the composition.

[0037] Upon reading this disclosure, it will be apparent to those skilled in the art that each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the inventive compositions and methods described herein. Any of the described methods may be performed in the order of the events stated or in any other logically feasible order.

[0038] As used in this article, the term "ethanol" refers to ethanol produced as a result of a bio-fermentation process.

[0039] As used herein, the phrase "recovered ethanol" refers to the purification and / or separation of ethanol. Suitablely, recovery yields ethanol that is substantially free of other components (e.g., contaminants). Therefore, recovery can yield an alcohol that is at least about 90% pure, suitablely at least about 95% pure, and more suitablely at least 99% pure. Preferably, recovery can yield an alcohol that is at least about 99.9% pure.

[0040] As used herein, and as is commonly known to those skilled in the art, the term "Enzyme Committee" (abbreviated as "EC") refers to the enzyme nomenclature recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) (e.g., see Enzyme Nomenclature from NC-IUBMB, 1992 (Academic Press, San Francisco)). Diego, California, including supplements 1-5 published in 1994 (Eur. J. Biochem., 223: 1-5); 1995 (Eur. J. Biochem., 232: 1-6); 1996 (Eur. J. Biochem., 237: 1-5); 1997 (Eur. J. Biochem., 250: 1-6); and 1999 (Eur. J. Biochem., 264: 610-650). Similarly, the nomenclature is regularly supplemented and updated (e.g., see www.chem.qmul.ac.uk / iubmb / enzyme / index.html).

[0041] As used herein, the term "protease" includes any enzyme belonging to the EC3.4 enzyme group (including each of its eighteen subclasses). As described herein, proteins (peptides) having protease activity (i.e., proteases) are also referred to in the art as peptidases, prionases, peptide hydrolases, and proteolytic enzymes.

[0042] As used herein, a protease can be an "exopeptidase" that hydrolyzes the peptide bonds of a protein starting from the N-terminus or C-terminus of the protein chain, or an "endopeptidase" that hydrolyzes the peptide bonds (peptide bonds) at the non-terminus of the protein chain.

[0043] As used herein, the terms “aspartic acid” protease and “aspartic” protease are used interchangeably and refer to any protein (enzyme) belonging to EC 3.4.23 aspartic protease. Typically, aspartic proteases are endopeptidases containing two (2) highly conserved aspartic residues at their active site and exhibit optimal activity at acidic pH.

[0044] As used in this article, Acidic fungal proteases refer to one of a variety of aspartic proteases (e.g., EC 3.4.23) obtained through controlled fermentation of Trichoderma reesei fungi. Encoding Exemplary proteases The polynucleotide sequences are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0045] The S53 family of serine proteases includes several peptidases, including serine carboxypeptidase (EC 3.4.16), tripeptidyl aminopeptidase (exo-type), and serine endopeptidase (Rawlings and Barrett, 1993).

[0046] As used herein, the term "serine" protease can refer to any protein (enzyme) belonging to EC 3.4.21 serine proteases.

[0047] In some embodiments, the term "serine" protease includes proteins (enzymes) belonging to EC 3.4.14 (e.g., exopeptidases such as dipeptidyl-peptidase I, dipeptidyl-peptidase II, tripeptidyl-peptidase I, tripeptidyl-peptidase II, etc.) and / or belonging to EC 3.4.16 (e.g., serine carboxypeptidases such as serine D-Ala-D-Ala carboxypeptidase, carboxypeptidase C, etc.).

[0048] In some embodiments, the term "serine" protease excludes proteins (enzymes) belonging to group EC 3.4.14.

[0049] In some other embodiments, the "serine" protease excludes the "serine-type carboxypeptidase" of EC 3.4.16.

[0050] As used herein, the name “orizin” refers to a serine protease from Aspergillus oryzae (Lee et al., Biochem, 2003) and its homologs from other fungal species that share at least about 40%, 50%, 60%, 70%, 80%, 90%, and 91%–99% sequence identity with Aspergillus oryzae enzyme (orizin).

[0051] As used in this article, the name "sedolisin" refers to the serine carboxypeptidase of EC 3.4.16.

[0052] As used herein, the term "protease activity" refers to proteolytic activity (EC 3.4). Protease activity can generally be measured using any assay employing a substrate that includes peptide bonds specific to the protease in question. Measurements of pH and temperature are equally applicable to the protease in question. Examples of pH measurements are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and examples of 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. Common examples of protease substrates are casein, bovine serum albumin (BSA), and hemoglobin.

[0053] As used herein, "one or more fermentable sugars" refers to sugars that can be metabolized under fermentation conditions. These fermentable sugars typically include glucose, maltose, and maltotriose. In some embodiments, sucrose, galactose, xylose, arabinose, etc., may also be fermentable sugars. Suitably, fermentable sugars can be obtained by hydrolyzing starch and other polysaccharide compositions (e.g., raw materials).

[0054] As used herein, the term "raw material" refers to a composition comprising at least one of the following: starch, cellulose, hemicellulose, lignocellulose, fermentable sugars, or combinations thereof. Raw materials can be starch, cereal-based materials (e.g., cereal crops, wheat, barley, rye, rice, triticale, millet, milo, sorghum, or corn), tubers (e.g., potatoes or cassava), roots, sugars (e.g., sucrose, beet sugar, molasses, or syrup), distillates, wet cakes, DDGS, cellulose biomass, hemicellulose biomass, whey protein, soybean-based materials, lignocellulose biomass, or combinations thereof.

[0055] As used in this article, “raw material fraction” refers to any component of the raw material that is separated during raw material processing.

[0056] As used herein, “starch” refers to any material composed of a complex polysaccharide carbohydrate from plants, which has the formula (C6H) 10 O5)x (where "x" can be any number) is composed of amylose and amylopectin. In particular, "starch" refers to any plant-based material, including but not limited to cereals, cereal crops, grasses, tubers and roots, and more particularly wheat, barley, corn, rye, rice, sorghum, bran, cassava, millet, potato, sweet potato and tapioca.

[0057] As used herein, "starch-containing material" includes starch-containing materials derived from barley, legumes, cassava, cereal crops, corn, sorghum, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, cassava, wheat, and whole grains or any mixture thereof. In some embodiments, the starch-containing material is a granular starch composition. In other embodiments, the starch-containing material is derived from whole grains. Starting materials are typically selected based on the desired fermentation product. The starch-containing material can also be waxy or non-waxy types of corn and barley. In a particular embodiment, the starch-containing material is derived from corn. In another embodiment, the starch-containing material is derived from wheat.

[0058] The phrase “granular starch” refers to ungelatinized, uncooked (raw) starch, where “starch gelatinization” refers to dissolving starch molecules to form a viscous suspension.

[0059] As used in this article, "starch hydrolysis" refers to the cleavage of glycosidic bonds by adding water molecules. Therefore, enzymes with "starch hydrolysis activity" catalyze the cleavage of glycosidic bonds by adding water molecules.

[0060] In some embodiments, the starch-containing material (raw material) may undergo one or more processing steps before, during, or after fermentation. As used herein, the phrase "one or more processing steps" includes, but is not limited to, grinding, cooking, liquefaction, saccharification, fermentation, and simultaneous saccharification and fermentation (SSF).

[0061] As used in this article, the term "initial gelatinization temperature" refers to the lowest temperature at which starch gelatinization begins.

[0062] As used herein, the term "grinding" refers to any grinding of raw materials, including wet grinding, dry milling, or a combination thereof. Grinding refers to a process that helps to break down raw materials to prepare them into particles of appropriate size to facilitate downstream processing of the raw materials (e.g., facilitating cooking processes).

[0063] As used herein, “wet milling” is a milling process in which the raw material (e.g., corn kernels) is wet-soaked before processing. A series of unit operations are then performed after wet soaking to recover starch. Grains are typically soaked or “soaked” in water containing dilute sulfuric acid for 24 to 48 hours before undergoing a series of milling processes. Downstream processes may include oil removal (e.g., corn oil), followed by additional stages of separating fiber, protein (e.g., gluten), and starch components (e.g., endosperm). This can be achieved by centrifugation, using sieves, and hydrocyclones. The remaining starch and water from this process can then undergo fermentation.

[0064] As used herein, “dry milling” refers to the process of milling starting materials (such as grains) into powder (e.g., coarse powder) before further processing. Typically, the powder is then slurried with water to form a mash (slurry) before further processing in downstream steps (e.g., saccharification). Ammonia is typically added to the mash and is used to control pH and provide a nutrient source for the ethanol production host used in fermentation. Therefore, in some embodiments, dry milling is used during the processing of the feedstock (or its fractions).

[0065] Suitable, the starch-containing material (raw material) obtained after milling or dry milling can be subjected to liquefaction and / or saccharification and / or fermentation and / or simultaneous saccharification and fermentation (“SSF” in this document). A cooking step may or may not be performed (e.g., after milling and before liquefaction or saccharification).

[0066] In some embodiments, the starch-containing material (raw material) is subjected to cooking. Typically, the cooking process can be carried out after grinding. Suitably, the cooking process can be carried out at 90°C-120°C. Suitably, the cooking process can be carried out before liquefaction and / or saccharification. Suitably, the cooking process can reduce the bacterial level before fermentation. In some embodiments, one or more enzymes can be added at or after this stage. Suitably, α-amylase can be added after the cooking process (e.g., in the liquefaction process).

[0067] In other embodiments, the starch-containing material (raw material) is not subjected to cooking. In such embodiments, saccharification and fermentation, or SSF, can be carried out on the raw material (or fraction thereof) containing granular or raw starch (e.g., starch treated at temperatures below starch gelatinization temperatures).

[0068] As used herein, the term "liquefaction" refers to the process of liquefying starch, typically by increasing the temperature. Starch liquefaction results in a significant increase in viscosity. Therefore, amylases can be introduced to reduce viscosity. The temperature for starch liquefaction varies depending on the source of the starch.

[0069] Starch processing can also be carried out at temperatures from approximately 25°C to slightly below the liquefaction temperature. These types of processes are often referred to as "granular starch hydrolysis," "direct starch hydrolysis," "raw starch hydrolysis," "low-temperature starch hydrolysis," etc. In some cases, starch is pretreated at temperatures below the liquefaction temperature to enhance enzymatic hydrolysis and / or for other starch processing methods.

[0070] In other embodiments, liquefaction may be carried out at a lower temperature and / or in a “cold cooking process” that does not involve the complete liquefaction of starch.

[0071] Starch-containing materials (raw materials) can also undergo saccharification. Saccharification can be carried out separately from fermentation, or it can be carried out simultaneously with fermentation. Separate saccharification and fermentation is a process in which starch present in the raw material or its fractions is converted into glucose, and subsequently, the host material converts glucose into ethanol.

[0072] As used herein, "simultaneous saccharification and fermentation" or "SSF" is a process in which starch present in a starch-containing material (raw material) is converted into glucose, and simultaneously, in the same reactor, an ethanol production host converts glucose into ethanol. In some embodiments, saccharification may be carried out at low temperatures.

[0073] As used herein, the terms "a saccharifying enzyme" or "multiple saccharifying enzymes" include, but are not limited to, α-amylase (EC 3.2.1.1), glucosylamylase (EC 3.2.1.3), isoamylase (EC 3.2.1.68), β-amylase (EC 3.2.1.2), amylopectinase (EC 3.2.1.41), endoglucanase (EC 3.2.1.4), cellobiase (EC 3.2.1.91), β-glucosidase (EC 3.2.1.21), cellulase (EC 3.2.1.74), licheninase (EC 3.1.1.73), lipase (EC 3.1.1.3), lipid acyltransferases (generally classified as EC 2.3.1.x), and phospholipases (EC 3.1.1.4, EC 3.1.1.32, or EC ...32, EC 3.2.1.4, EC 3.2.1.32, or EC 3.1.1.32, EC 3.2.1.4, EC 3.2.1.32, or EC 3.2.1.32, EC 3.2.1.32, EC 3.2.1.32, EC 3.2. 3.1.1.5), phytases (e.g., 6-phytase (EC 3.1.3.26) or 3-phytase (EC 3.1.3.8), xylanases (e.g., endo-1,4-β-d-xylanase (EC 3.2.1.8) or 1,4β-xylosidase (EC 3.2.1.37) or EC 3.2.1.32, EC 3.1.1.72, EC 3.1.1.73), glucosylamylase (EC 3.2.1.3), hemicellulases (e.g., xylanase), or keratinases (EC 3.4.xx)), debranching enzymes, keratinases, esterases and / or mannanases (e.g., β-mannanase (EC 3.2.1.78)), transferases, glucosidases, and arabinofuranosidases.

[0074] As used herein, the term "mixture" refers to a combination of one or more components and / or enzymes, wherein one or more components or enzymes are added in any order and in any combination. Suitably, mixing may involve mixing one or more components and / or enzymes simultaneously or sequentially.

[0075] As used herein, the phrases “ethanol-producing host,” “ethanol-producing host,” and “ethanol-producing host cell” are used interchangeably and include any microorganism capable of fermenting a fermentable sugar source to produce ethanol.

[0076] In some embodiments, the ethanol-producing host is yeast. Suitably, the yeast may be selected from the group consisting of: *Saccharomyces*, *Kluyveromyces*, *Zygosaccharomyces*, *Issatchenkia*, *Kazachstania*, and *Torulaspora*. In yet another embodiment, the ethanol-producing organism may be bacteria (e.g., *Zymomonas*, *Escherichia*, etc.).

[0077] In some embodiments, an ethanol production host (e.g., yeast) is added before, during, or after the addition of the aspartic protease and serine protease composition to the starch-containing material (raw material).

[0078] As used herein, “lignocellulose biomass” can include cellulose, hemicellulose, and the aromatic polymer lignin. Hemicellulose and cellulose (including insoluble arabinoyl xylan) are also potential energy sources themselves, as they are composed of C5- and C6-sugars.

[0079] Suitablely, lignocellulosic biomass can be any cellulose material, hemicellulose material, or lignocellulosic material, such as agricultural residues, bioenergy crops, industrial solid waste, municipal solid waste, sludge from papermaking, garden waste, wood waste, forestry waste, and combinations thereof. Lignocellulose biomass can be selected from the following groups: corn cobs, crop residues (such as corn husks), corn gluten meal (CGM), corn stalks, corn fiber, grass, beet pulp, wheat straw, wheat husks, oat straw, coarse wheat flour, wheat bran, rice husks, rice bran, oat husks, wet cake, dried distillers grains (DDG), distillers grains with solubles (DDGS), palm kernels, citrus pomace, cotton, lignin, barley straw, hay, rice straw, rice husks, switchgrass, miscanthus, rice straw, reeds, waste paper, bagasse, sorghum residue, sorghum forage, sorghum stalks, soybean stalks, soybeans, components obtained from grinding trees, branches, roots, leaves, wood strips, sawdust, bushes and shrubs, vegetables, fruits and flowers.

[0080] Wet cake, dried grains with solubles (DDG), and DDG with solubles are products obtained by distilling ethanol from the fermentation of grains or grain mixtures using methods employed in the grain distillation industry. The distillate (containing water, grain residue, yeast cells, etc.) is separated into a "solid" portion and a "liquid" portion. The solid portion is called "wet cake" and can be used as animal feed. The liquid portion is (partially) evaporated into a slurry (solubles). The liquid portion is often referred to as dilute distillate. When the wet cake is dried, it becomes DDG. When the wet cake is dried along with the slurry (solubles), it becomes DDGS with solubles.

[0081] As used herein, the term “expression” includes any step involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0082] As used herein, the term “expression vector” refers to a straight or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operatively linked to a control sequence provided for its expression.

[0083] II. Bio-production of ethanol from starch-containing materials

[0084] As generally described above, certain aspects of this disclosure relate to improved methods / processes for producing ethanol from starch-containing materials using a combination of at least one aspartic protease and at least one serine protease described herein. For example, some embodiments relate to a method for producing ethanol from starch-containing materials, comprising (a) saccharifying the starch-containing material using a saccharifying enzyme at a temperature below the initial gelatinization temperature of the starch-containing material, and (b) (under conditions suitable for ethanol production) producing the material obtained in step (a) by host fermentation with ethanol, wherein steps (a) and / or (b) are carried out in the presence of a mixture of aspartic protease and serine protease.

[0085] In another embodiment, a method for producing ethanol from a starch-containing material includes (a) liquefying the starch-containing material in the presence of α-amylase at a temperature above the initial gelatinization temperature of the starch-containing material, (b) saccharifying the liquefied material obtained in step (a) using a saccharifying enzyme, and (c) producing the material obtained in step (b) from host fermentation with ethanol (under conditions suitable for ethanol production), wherein steps (b) and / or (c) are carried out in the presence of a mixture of aspartic protease and serine protease.

[0086] As described in the background section, methods / processes for producing ethanol from starch-containing materials are generally well known. "Conventional processes" involve liquefying gelatinized starch at high temperatures, typically using bacterial α-amylase, followed by simultaneous saccharification and fermentation in the presence of glucosylamylase and fermenting organisms. "Raw starch hydrolysis" processes involve simultaneously saccharifying and fermenting granular starch below the initial gelatinization temperature, typically in the presence of acidic fungal α-amylase and glucosylamylase.

[0087] For example, natural starch consists of microscopic particles that are insoluble in water at room temperature. When an aqueous starch slurry is heated, the particles swell and eventually rupture, dispersing starch molecules into the solution. At temperatures as high as approximately 50°C to 75°C, the swelling is reversible. However, at even higher temperatures, irreversible swelling begins, a process known as "gelatinization." During gelatinization, viscosity increases dramatically.

[0088] The granular starch to be processed can have a highly refined starch quality, for example, being at least 90%, at least 95%, at least 97%, or at least 99.5% pure, or it can be a coarse starch-containing material comprising (e.g., milled) whole grains, which include non-starch fractions such as germ residues and fiber. Raw materials (such as whole grains) can have their particle size reduced (e.g., by milling to open up the structure and allow for further processing). In dry milling, the whole grain is milled and used. Wet milling effectively separates the germ and coarse flour (starch granules and protein) and is commonly used in places where starch hydrolysates are used, for example, in the production of syrups. Both dry and wet milling are well known in the field of starch processing and can be used in the processes disclosed herein. Methods for reducing the particle size of starch-containing materials are well known to those skilled in the art.

[0089] In some embodiments, the starch must be diluted or "liquefied" to make it suitable for processing. In current commercial practice, this reduction in viscosity is primarily achieved through enzymatic degradation. Liquefaction typically occurs in the presence of α-amylases (e.g., bacterial α-amylases and / or acidic fungal α-amylases). In some embodiments, phytase is also present during liquefaction. In some other embodiments, viscosity-reducing enzymes such as xylanase and / or β-glucanase are also present during liquefaction.

[0090] During liquefaction, long-chain starch is degraded into branched and shorter linear units (maltodextrin) by α-amylase. Liquefaction can be carried out as a three-step hot slurry process. The slurry is heated to between 60°C and 95°C, and α-amylase is added to initiate liquefaction (dilution). The slurry can be jet-cooked between 95°C and 140°C (e.g., 105°C to 125°C for about 1–15 minutes, e.g., about 3–10 minutes, especially about 5 minutes). The slurry is then cooled to 60°C to 95°C, and more α-amylase is added to achieve final hydrolysis (secondary liquefaction). The jet-cooking process is carried out at a pH of 4.5–6.5, typically between pH 5 and 6. α-amylase can be added in a single dose, for example, before jet-cooking. The liquefaction process is carried out between 70°C and 95°C, for example, 80°C-90°C, or around 85°C, for approximately 10 minutes to 5 hours, typically for 1-2 hours. The pH is between 4 and 7, for example, between 4.5 and 5.5. To ensure optimal enzyme stability under these conditions, calcium may optionally be added (to provide 1-60 ppm of free calcium ions, for example, about 40 ppm of free calcium ions). After such treatment, the liquefied starch will typically have a "dextrose equivalent" (DE) of 10-15. Generally, such liquefaction and liquefaction conditions are well known in the art.

[0091] The α-amylase used in liquefaction can be an acid-stable bacterial α-amylase, such as α-amylase from Bacillus species, such as Bacillus stearothermophilus or Bacillus licheniformis.

[0092] Similarly, saccharification can be performed using saccharifying enzymes (e.g., glucoamylase, β-amylase) under conditions well known in the art. For example, the complete saccharification step can last from about 24 hours to about 72 hours. Alternatively, a pre-saccharification step of about 40-90 minutes can be performed at a temperature of 30°C-65°C (typically about 60°C), followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation (SSF) process. Saccharification is typically performed at temperatures in the range of 20°C-75°C (e.g., 25°C-65°C and 40°C-70°C, typically around 60°C) and at a pH between about 4 and 5 (e.g., at about pH 4.5).

[0093] The saccharification and fermentation steps can be performed sequentially or simultaneously. In the examples, saccharification and fermentation are performed simultaneously (referred to as "SSF"). However, typically, a pre-saccharification step is performed at a temperature of 30°C to 65°C, typically around 60°C, lasting approximately 30 minutes to 2 hours (e.g., 30 to 90 minutes), followed by complete saccharification during fermentation (i.e., simultaneous saccharification and fermentation; SSF). In the simultaneous saccharification and fermentation (SSF) process, there is no saccharification hold-up phase; instead, ethanologens and enzymes are added together, and the process is then carried out at temperatures of 25°C to 40°C (e.g., between 28°C and 35°C, such as between 30°C and 34°C, such as around 32°C). The SSF process can be carried out at pH values ​​ranging from about 3 to 7, typically from pH 4.0 to 6.5, or from pH 4.5 to 5.5.

[0094] In other embodiments, ethanol is produced from starch-containing materials without gelatinization (i.e., without cooking) (e.g., a "raw starch hydrolysis" process). For example, ethanol fermentation products can be produced without liquefying an aqueous slurry comprising starch-containing materials and water. Thus, in some embodiments, the process includes saccharifying (e.g., milled) starch-containing materials (e.g., granular starch) at a temperature below the initial gelatinization temperature in the presence of a saccharifying enzyme (e.g., α-amylase) to produce sugars that can be fermented into ethanol by a suitable fermentation organism.

[0095] In certain embodiments, the saccharification and fermentation steps are performed simultaneously, wherein the saccharifying enzyme and the fermentation organism (e.g., yeast strain) are added together, and fermentation is carried out at a temperature of 25°C–40°C. The SSF process can be performed at pH values ​​from about 3 to 7 (e.g., pH 4.0 to 6.5, or pH 4.5 to 5.5). In some embodiments, fermentation takes about 6 to 120 hours.

[0096] As generally defined above, the initial gelatinization temperature refers to the lowest temperature at which starch gelatinization begins. Typically, starch heated in water begins to gelatinize between approximately 50°C and 75°C; the exact gelatinization temperature depends on the specific starch and can be readily determined by those skilled in the art. Therefore, the initial gelatinization temperature can vary depending on the plant species, the specific variety of the plant species, and the growing conditions. In some embodiments, temperatures below the initial gelatinization temperature are typically in the range of 30°C to 75°C, preferably between 45°C and 60°C. For example, in some embodiments, the process is carried out at temperatures ranging from 25°C to 40°C, such as 28°C to 35°C, such as 30°C to 34°C, preferably around 32°C.

[0097] A. α-Amylase present and / or added during liquefaction

[0098] Starch can be hydrolyzed into simpler carbohydrates by acids, various enzymes, or combinations thereof. For example, the main enzymes used to hydrolyze starch into simpler carbohydrates are endometrial amylases, exometrial amylases, and debranching enzymes, which typically hydrolyze both amylose and amylopectin. Amylose is primarily hydrolyzed by amylases, while amylopectin requires debranching enzymes such as amylopectin (EC 3.2.1.41) for complete hydrolysis.

[0099] An exemplary endoamylase is α-amylase (EC 3.2.1.1), which specifically targets the α-1,4-bonds of both amylose and amylopectin. Exoamylases have the ability to hydrolyze both the α-1,4-bonds and α-1,6-bonds of both amylose and amylopectin. An exemplary exoamylase is amylase glucosidase (commonly known as glucosylamylase; EC 3.2.1.20). β-amylase is an enzyme capable of hydrolyzing the α-1,4-bonds of amylose. Debranching enzymes (e.g., amylopectin (EC 3.2.1.41)) hydrolyze the α-1,6-bonds in amylopectin, with the hydrolysis products of debranching enzymes primarily being maltotriose and maltose.

[0100] In some embodiments, the α-amylase is thermophilic Bacillus α-amylase or a variant thereof.

[0101] B. Glucoamylase for use in saccharification and / or fermentation processes

[0102] In some embodiments, the carbohydrate-producing enzyme present during saccharification is glucosylamylase. Therefore, in some embodiments, glucosylamylase is present and / or added to the saccharification and / or fermentation processes. In specific embodiments, glucosylamylase is present and / or added to the simultaneous saccharification and fermentation (SSF) process.

[0103] For example, in some embodiments, the glucosylamylase present and / or added in the saccharification and / or fermentation and / or SSF processes is of fungal origin. In some embodiments, the glucosylamylase is derived from strains of the genus *Aspergillus* (e.g., *Aspergillus niger*, *Aspergillus awamori*, *Aspergillus oryzae*). In some other embodiments, the glucosylamylase is derived from strains of the genus *Trichoderma* (e.g., *Trichoderma reesei*). In other embodiments, the glucosylamylase is derived from the genus *Talaromyces* (e.g., *T. emersonii*), the genus *Trametes* (e.g., *T. cingulate*), etc. In other embodiments, the glucosylamylase is a variant glucosylamylase derived from natural glucosylamylase.

[0104] Glucoamylase can be added to saccharification and / or fermentation in the following amounts: 0.0001-20 AGU / g DS, such as, between 0.001-10 AGU / g DS, or 0.01-5 AGU / g DS (e.g., 0.1-2 AGU / g DS or 0.1-0.5 AGU / g DS).

[0105] Commercially available compositions containing glucosylamylase are generally known to those skilled in the art. Therefore, in some other embodiments, glucosylamylase is present and / or added in combination with α-amylase during saccharification and / or fermentation, as described below.

[0106] C. α-amylase present and / or added during saccharification and / or fermentation

[0107] In some embodiments, α-amylase is present and / or added in the saccharification and / or fermentation processes of this disclosure. In some embodiments, the α-amylase is of fungal or bacterial origin. For example, in some embodiments, the α-amylase is an acid-stable fungal α-amylase. An acid-stable fungal α-amylase is an α-amylase that is active in a pH range of 3.0 to 7.0, and preferably in a pH range of 3.5 to 6.5 (including activity at pH values ​​of about 4.0, 4.5, 5.0, 5.5, and 6.0).

[0108] In certain embodiments, the α-amylases of this disclosure are derived from the genus *Aspergillus*, such as *Aspergillus terreus*, *Aspergillus niger*, *Aspergillus oryzae*, *Aspergillus avocado*, and *Aspergillus kawachi*. In another embodiment, the α-amylases of this disclosure are derived from the genus *Rhizomucor* (e.g., *Rhizomucor pusillus*) or the genus *Meripilus* (e.g., *Meripilus giganteus*).

[0109] In some embodiments, the ratio between glucosylamylase and α-amylase present and / or added during saccharification and / or fermentation may preferably be in the range of 500:1 to 1:1, for example from 250:1 to 1:1, for example from 100:1 to 1:1, for example from 100:2 to 100:50, for example from 100:3 to 100:70.

[0110] For example, suitable enzyme preparations for use in saccharification include SSF (obtained from DuPont N&B) contains α-amylase (EC 3.2.1.1), glucosylamylase (EC 3.2.1.3), isoamylase (EC 3.2.1.68), β-amylase (EC 3.2.1.2), amylopectin (EC 3.2.1.41), and Aspergillopepsin 1 (EC 3.4.23.18).

[0111] III. Protease Composition and Dosage

[0112] As generally described above, certain embodiments of this disclosure relate to compositions and methods for the bioproduction of ethanol. Certain other embodiments of this disclosure relate to methods for producing ethanol comprising specific combinations of proteases and the use of these protease combinations in one or more fermentation processes described herein. For example, in some embodiments, a method for producing ethanol from a starch-containing material includes (a) liquefying the starch-containing material in the presence of α-amylase at a temperature above the initial gelatinization temperature of the starch-containing material, (b) saccharifying the liquefied material obtained in step (a) using a saccharifying enzyme, and (c) (under conditions suitable for ethanol production) producing the material obtained in step (b) by host fermentation with ethanol, wherein steps (b) and / or (c) are carried out in the presence of a mixture of aspartic proteases and serine proteases.

[0113] In another embodiment, a method for producing ethanol from a starch-containing material includes (a) saccharifying the starch-containing material at a temperature below the initial gelatinization temperature of the starch-containing material using a saccharifying enzyme, and (b) producing the material obtained in step (a) from host fermentation with ethanol (under conditions suitable for ethanol production), wherein steps (a) and / or (b) are carried out in the presence of a mixture of aspartic protease and serine protease.

[0114] For example, U.S. Patent No. 5,231,017 describes the use of acidic fungal proteases in a process that typically involves liquefying gelatinized starch with α-amylase during ethanol fermentation. PCT Publication No. WO 2003 / 066826 discloses a process for hydrolyzing raw starch in the presence of fungal glucosylamylase, α-amylase, and fungal protease. PCT Publication No. WO 2010 / 008841 describes a process for producing fermentation products from gelatinized starch and / or ungelatinized starch-containing materials by saccharifying starch material using at least glucosylamylase and metalloproteinases (i.e., derived from *Thermoascus aurantiacus*) and fermenting it using yeast strains. PCT Publication No. WO 2015 / 078372 describes serine proteases derived from *Grifola frondosa*, *Trametes versicolor*, and *Dichomitus squalens* for use in starch wet milling processes. PCT Publication No. WO 2016 / 065238 describes the use of tripeptidyl peptidase (exopeptidase) for the production of alcohol from starch-containing raw materials. PCT Publication No. WO 2019 / 046232 describes a process for the production of ethanol from starch-containing materials using a combination of M35 family endopeptidase and S53 family serine protease.

[0115] More specifically, as illustrated in Example 1 of this disclosure, in the determination of dimethylcasein, the applicant of this invention surprisingly identified a hydrolytic synergistic effect when combining aspartic protease and serine protease. For example, Figure 1 Results are presented on the use of dimethylcasein assays to evaluate the proteolytic activity of each protease individually (i.e., aspartic protease or serine protease) and in combination (i.e., aspartic protease and serine protease) to assess whether the mixture of combinations has any synergistic effect. Figure 1 As shown, the results demonstrate that the mixture of 25 μg / ml serine protease (named "Aurest") and 25 μg / ml aspartic protease (named "A10") is more efficient in hydrolyzing dimethyl casein than either 50 μg / ml serine protease (Aurest) or 50 μg / ml aspartic protease (A10) alone.

[0116] Example 2 of this disclosure further presents the results of adding different combinations of proteases during yeast cell fermentation for ethanol production. For example, as generally described in this example, saccharified corn liquefaction was treated with (i) a “control” treatment or (ii) an “experimental” treatment. The control treatment contained an aspartic protease. Urea (600 ppm) was used as the nitrogen source for yeast cells. Experimental treatments included combinations of aspartic protease and serine protease with urea (200 or 600 ppm) as the nitrogen source for yeast cells. As presented in Table 1 of Example 2, the combination of aspartic protease and serine protease outperformed commercial products in both the presence and absence of sufficient urea (600 ppm) and in the absence of sufficient urea (200 ppm). Protease.

[0117] Furthermore, the data presented in Table 2 (Example 2) demonstrate that, in the presence of sufficient urea (600 ppm), compared to the presence of aspartic protease... The control treatment, using a combination of proteases (aspartic protease + serine protease), decreased glycerol concentration and increased ethanol concentration. Finally, as shown in Table 3 of Example 2, the treatment with aspartic protease was used in the presence of high urea concentration (600 ppm) to reduce glycerol concentration and increase ethanol concentration. The control treatment, in the presence of low urea concentration (200 ppm), included an experimental treatment containing a combination of aspartic and serine proteases, which increased the ethanol concentration (14.91) and decreased the glycerol concentration (1.19).

[0118] As shown in Table 4 of Example 3, experimental treatments containing a combination of aspartic and serine proteases outperformed commercial products in terms of initial fermentation rates (e.g., 16 h, 24 h). (Aspartic) protease, which helps to convert carbon into ethanol more quickly in the fermentation process.

[0119] Example 4 of this disclosure further presents and describes the results of protease combinations in the fermentation process of granular starch yeast. For example, the results presented in Table 5 show that the experimental treatment containing the combined protease (aspartic protease + serine protease) performed better than the control in the fermentation process of granular starch yeast. Protease treatment. More specifically, as shown in Table 15, the fermentation rate was improved throughout the granular starch fermentation process, demonstrating that carbon is converted to ethanol more quickly in the granular starch fermentation process.

[0120] Therefore, certain embodiments of this disclosure relate to novel protease compositions for use in ethanol production. In some embodiments, novel protease compositions are used in granular starch hydrolysis processes. For example, in some embodiments, this disclosure relates to a method for producing ethanol from starch-containing materials, comprising (a) saccharifying the starch-containing material using a saccharifying enzyme at a temperature below the initial gelatinization temperature of the starch-containing material, and (b) (under conditions suitable for ethanol production) producing the material obtained in step (a) by host fermentation with ethanol, wherein steps (a) and / or (b) are carried out in the presence of a mixture of aspartic protease and serine protease.

[0121] In some embodiments, novel protease compositions are used in conventional starch hydrolysis processes. For example, some embodiments relate to a method for producing ethanol from starch-containing materials, comprising (a) liquefying the starch-containing material in the presence of α-amylase at a temperature above the initial gelatinization temperature of the starch-containing material, (b) saccharifying the liquefied material obtained in step (a) using a saccharifying enzyme, and (c) producing the material obtained in step (b) by host fermentation with ethanol (under conditions suitable for ethanol production), wherein steps (b) and / or step (c) are carried out in the presence of a mixture of aspartic protease and serine protease.

[0122] Therefore, in some embodiments, this disclosure relates to a protease composition comprising at least (i) a combination of an aspartic protease and (ii) a serine protease.

[0123] As generally stated in the definition section above, aspartic proteases refer to any protein (enzyme) belonging to EC 3.4.23 aspartic proteases. For example, aspartic proteases include, but are not limited to, pepsin A, pepsin B, gastricsin, rennet, cathepsin D, cathepsin E, Nepenthesin, renin, Aspergillus pepsin I, and Penicillopepsin.

[0124] As generally stated in the definition section above, and as used herein, "serine protease" refers to any protein (enzyme) belonging to the serine proteases of EC 3.4.21. For example, serine proteases of EC 3.4.21 include, but are not limited to, chymotrypsin, chymotrypsin C, trypsin, thrombin, oxaliplatin, plasmin, kexin, subtilisin, granzyme B, oryzin, trypsin-like enzymes, acrosome protein, etc.

[0125] In some embodiments, the serine proteases of this disclosure include proteins (enzymes) classified as exopeptidases (exopeptidases). Therefore, in some embodiments, the serine proteases of this disclosure include enzymes classified as exopeptidases (exopeptidases), such as EC 3.4.14.

[0126] In some other embodiments, the serine proteases of this disclosure are limited to endopeptidases (endopeptidases). For example, in certain embodiments, the serine proteases of this disclosure exclude proteins (enzymes) classified as exopeptidases (exopeptidases). Therefore, in other embodiments, the serine proteases of this disclosure exclude any proteins (enzymes) belonging to EC 3.4.14 (e.g., excluding exopeptidases such as dipeptidyl peptidase I, dipeptidyl peptidase II, tripeptidyl peptidase I, tripeptidyl peptidase II, etc.).

[0127] In some embodiments, the aspartic protease of this disclosure comprises about 50% to 100% sequence identity with the aspartic protease of SEQ ID NO: 2. For example, in some embodiments, the aspartic protease of this disclosure comprises about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 99% sequence identity with the aspartic protease of SEQ ID NO: 2.

[0128] In other embodiments, the serine protease of this disclosure comprises about 50% to 100% sequence identity with the serine protease of SEQ ID NO: 4. For example, in some embodiments, the serine protease of this disclosure comprises about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 99% sequence identity with the serine protease of SEQ ID NO: 4.

[0129] In certain embodiments, the novel aspartic protease and serine protease composition of this disclosure is mixed with a starch-containing material. In some embodiments, the aspartic protease and serine protease composition is mixed with the starch-containing material after liquefaction. In another embodiment, the aspartic protease and serine protease composition is mixed with the starch-containing material after liquefaction and saccharification. In some embodiments, the aspartic protease and serine protease composition is used in a granular starch hydrolysis process. In certain embodiments, the aspartic protease and serine protease composition is mixed with a granular starch composition and used in a simultaneous saccharification and fermentation (SSF) process.

[0130] As described herein, the aspartic protease and serine protease compositions (mixtures) and methods disclosed herein are particularly useful for increasing the ethanol production rate of their fermentation processes, and / or increasing the ethanol yield of their fermentation processes, and / or reducing the glycerol yield of their fermentation processes, and / or reducing the supplemental nitrogen requirements (e.g., urea) of their fermentation processes, etc.

[0131] Therefore, the aspartic protease and serine protease mixture described herein used in the methods of this disclosure can be administered in any suitable amount. In some embodiments, the aspartic protease and serine protease composition (mixture) can be administered in amounts of about 5 mg to 3 g of aspartic protease and about 5 mg to 3 g of serine protease per kg of starch-containing material (raw material). In other embodiments, the aspartic protease and serine protease composition (mixture) can be administered in amounts of about 25 mg to 1000 mg of aspartic protease and 25 mg to 1000 mg of serine protease per kg of starch-containing material (raw material). Therefore, in some other embodiments, the aspartic protease and serine protease composition (mixture) can be administered in amounts of about 0.01 mg to 100 mg, 0.5 mg to 100 mg, 1 mg to 50 mg, 5 mg to 100 mg, 5 mg to 20 mg, 10 mg to 100 mg, 0.05 mg to 50 mg, or 0.10 mg to 10 mg of enzyme per kg of raw material.

[0132] For example, as in the following example 1 (see, for example, see...) Figure 1 As described in [reference needed], the combination of aspartic protease (25 μg / ml) and serine protease (25 μg / ml) (i.e., equal amounts) exhibits a synergistic effect in hydrolyzing the substrate (i.e., dimethyl casein) relative to either aspartic (A10) protease alone (50 μg / ml) or serine (orretin) protease alone (50 μg / ml). Therefore, in some embodiments, the aspartic protease and serine protease composition (mixture) may be administered in approximately equal amounts of aspartic protease and serine protease. For example, equal amounts of aspartic protease and serine protease may be based on equal aspartic protease and serine protease concentrations, equal aspartic protease and serine protease activities, etc. The exact amount will generally depend on the specific type of starch-containing material used and on the specific protease activity / mg protein.

[0133] In other embodiments, the aspartic protease and serine protease composition can be administered in amounts based on the grams of dry solids present in the raw materials.

[0134] IV. Fermentation of starch-containing materials to produce ethanol

[0135] As described herein, fermentation conditions are typically determined based on the type of plant (starch-containing) material used, the available fermentable sugars, and / or one or more fermentation organisms used in the fermentation process. Those skilled in the art can readily determine suitable fermentation conditions. Fermentation can be carried out under conventionally used conditions. An anaerobic fermentation process is preferred.

[0136] For example, fermentation can be carried out at temperatures as high as 75°C (e.g., between 40°C and 70°C, or between 50°C and 60°C). However, it is also known that bacteria have a significantly lower optimum temperature, down to around room temperature (around 20°C). Examples of suitable fermentation organisms can be found in the ethanol production host section below.

[0137] For the production of ethanol using yeast, fermentation can last from 24 to 96 hours, particularly from 35 to 60 hours. In some embodiments, fermentation is carried out at temperatures between 20°C and 40°C, preferably between 26°C and 34°C, particularly around 32°C. In addition to fermenting microorganisms (e.g., yeast), fermentation may also include nutrients and additional enzymes (including phytase). The use of yeast in fermentation is well known in the art.

[0138] Fermentation typically takes place at a pH range between 3 and 7 (e.g., pH 3.5 to 6, or pH 4 to 5). Fermentation typically lasts from 6 to 96 hours. The fermentation process disclosed herein can be carried out as a batch process or as a continuous process. Fermentation can be carried out in an ultrafiltration system, wherein the permeate is kept under recirculation in the presence of solids, water, and fermentation organisms, and wherein the permeate is a liquid containing the desired fermentation product. Similarly, a method / process in a continuous membrane reactor with an ultrafiltration membrane is contemplated, wherein the permeate is kept under recirculation in the presence of solids, water, and one or more fermentation organisms, and wherein the permeate is a liquid containing the fermentation product. After fermentation, the fermentation organisms can be separated from the fermented slurry and recovered.

[0139] V. Ethanol production host

[0140] In some embodiments, the ethanol production host may be bacteria from one or more genera selected from the group consisting of: *Zymomonas mobilis*, *Arthrobacter*, *Bacillus*, *Clostridium*, *Erwinia*, *Escherichia*, *Klebsiella*, *Lactobacillus*, *Pseudomonas*, *Streptomyces*, and *Thermoanaerobacter*. In a particular embodiment, the bacteria are *Zymomonas mobilis* or *Escherichia coli*.

[0141] In other embodiments, the ethanol production host can be a fungus. The fungus used according to the invention can be any ascomycete (e.g., Ascomycete).

[0142] In some embodiments, the ethanol production host is yeast. Suitably, the yeast may be selected from the group consisting of: *Saccharomyces*, *Kluyveromyces*, *Zygosaccharomyces*, *Issarum*, *Kazakhstansaccharomyces*, and *Cyclophorus*. In other embodiments, 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 pastorianus*. In another embodiment, the ethanol production host is *Saccharomyces cerevisiae var. diastaticus*.

[0143] In some embodiments, an ethanol production host (e.g., active yeast) is added before, during, or after the addition of the aspartic protease and serine protease composition to the starch-containing material (raw material).

[0144] For example, *Saccharomyces cerevisiae* is commonly used in the commercial production of ethanol, where it is adept at fermenting glucose into ethanol (e.g., often to concentrations greater than 20% w / v). However, *Saccharomyces cerevisiae* has a limited ability to generate nitrogen sources, which can slow fermentation or require the addition of exogenous nitrogen sources such as urea. More specifically, as generally explained above and described in the Examples section herein, the novel aspartic and serine protease mixtures (combinations) of this disclosure improve the ability of the ethanol production host to ferment starch-containing materials in such starch-to-ethanol fermentation processes. For example, it has been observed herein that the addition of the aspartic and serine protease mixtures (combinations) of this disclosure increases the fermentation rate by providing free amino acids (via the hydrolysis of proteins found in starch-containing materials, such as corn). Therefore, using such aspartic and serine protease mixtures (combinations) of this disclosure advantageously reduces the amount of exogenous nitrogen (e.g., urea) that needs to be added during fermentation, thereby reducing the overall cost of such starch-to-ethanol fermentation processes.

[0145] In some embodiments, the improvement in the fermentation capacity of the ethanol production host in the presence of a combination of aspartic and serine proteases can be measured by the following: the increase in the amount of glucose consumed by the ethanol production host during fermentation compared to the level of glucose consumed by the ethanol production host during fermentation in the presence of aspartic proteases and / or the increase in the amount of glucose consumed by the ethanol production host during fermentation compared to the level of glucose consumed by the ethanol production host during fermentation in the presence of serine proteases.

[0146] Modified ethanol production host

[0147] In some embodiments, this disclosure relates to methods for producing ethanol from starch-containing materials, methods comprising (under conditions suitable for ethanol production) fermenting the starch-containing material with ethanol-producing host, and recovering the produced ethanol, wherein the ethanol-producing host expresses and secretes heterologous aspartic proteases and heterologous serine proteases. In other embodiments, this disclosure relates to methods for producing ethanol from starch-containing materials, methods comprising fermenting the starch-containing material with at least two types of ethanol-producing host under conditions suitable for ethanol production, and recovering the produced ethanol, wherein a first ethanol-producing host expresses and secretes heterologous aspartic proteases and a second ethanol-producing host expresses and secretes heterologous serine proteases.

[0148] Therefore, in some embodiments, heterologous nucleic acid molecules (i.e., nucleic acid sequences) are introduced into the host cells of this disclosure, and these heterologous nucleic acid molecules may be codon-optimized for the intended recipient host cell. Methods for introducing polynucleotides (e.g., expression cassettes) into microbial host cells (strains) are well known to those skilled in the art.

[0149] More specifically, in some embodiments, the heteronucleotide sequence of this disclosure encodes an aspartic protease. In other embodiments, the heteronucleotide sequence of this disclosure encodes a serine protease. In some embodiments, the heteronucleotide sequence encoding an aspartic protease, or the heteronucleotide sequence encoding a serine protease, comprises a gene or its open reading frame (ORF) encoding an aspartic protease or a serine protease operably linked to an upstream (5′) promoter sequence and optionally operably linked to a downstream (3′) terminator sequence.

[0150] VI. Combination with other components

[0151] The proteases disclosed herein can be formulated in any manner known in the art. In some embodiments, the aspartic proteases and / or serine proteases used in this disclosure can be formulated as liquids, powders, or granules. Thus, in some embodiments, the aspartic proteases and / or serine proteases are formulated as liquid preparations. In another embodiment, the aspartic proteases and / or serine proteases are formulated as powders.

[0152] In some embodiments, additional components may be mixed with aspartic proteases and / or serine proteases, such as salts (e.g., Na₂SO₄), maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat components, starch, talc, PVA, polyols (e.g., sorbitol and glycerol), benzoates, sorbates, sugars (e.g., sucrose and glucose), propylene glycol, 1,3-propanediol, parabens, sodium chloride, citrates, acetates, sodium acetate, phosphates, calcium, metabisulfite, formates, or mixtures thereof.

[0153] In one embodiment, the salt may be selected from the group consisting of: Na2SO4, NaH2PO4, Na2HPO4, Na3PO4, (NH4)H2PO4, K2HPO4, KH2PO4, K2SO4, KHSO4, ZnSO4, MgSO4, CuSO4, Mg(NO3)2, (NH4)2SO4, sodium borate, magnesium acetate, sodium citrate, or combinations thereof.

[0154] Dry powder or granules can be prepared by means known to those skilled in the art, such as in a top-spray fluidized bed coating machine, drum granulation (e.g., high-shear granulation), extrusion, pot coating, etc.

[0155] Suitablely, the aspartic protease and / or serine protease can be dried together with the ethanol production host. In some embodiments, the aspartic protease and / or serine protease can be coated, for example, encapsulated. Such coating can, for example, protect the aspartic protease and / or serine protease from heat inactivation and can be considered a heat protectant.

[0156] In other embodiments, aspartic proteases and / or serine proteases may be diluted using a diluent (such as starch powder, limestone, etc.).

[0157] In another embodiment, aspartic proteases and / or serine proteases can be formulated by applying (e.g., spraying) one or more enzymes onto a carrier substrate (e.g., milled wheat).

[0158] In some other embodiments, the methods and / or compositions of this disclosure may further comprise one or more enzymes, which include cellulase activity, hemicellulase activity, and combinations thereof. Suitably, one or more enzymes comprising cellulase activity and / or hemicellulase activity are selected from the group consisting of: endoglucanase (EC 3.2.1.4); cellobiase (EC 3.2.1.91), β-glucosidase (EC 3.2.1.21), cellulase (EC 3.2.1.74), licheninase (EC 3.1.1.73), lipase (EC 3.1.1.3), lipid acyltransferase (generally classified as EC 2.3.1.x), phospholipase (EC 3.1.1.4, EC 3.1.1.32 or EC 3.1.1.5), phytase (e.g., 6-phytase (EC 3.1.3.26) or 3-phytase (EC 3.1.3.8), acid phosphatase, amylase, α-amylase (EC 3.1.3.8), etc. 3.2.1.1) Xylanases (e.g., endo-1,4-β-d-xylanase (EC 3.2.1.8) or 1,4β-xylosidase (EC 3.2.1.37) or EC 3.2.1.32, EC 3.1.1.72, EC 3.1.1.73), glucosylamylase (EC 3.2.1.3), amylopectinase, hemicellulase, keratinase (EC 3.4.xx), debranching enzymes, keratinases, esterases and / or mannanases (e.g., β-mannanase (EC 3.2.1.78)), transferases, glucosidases, arabinofuranases and phytases (e.g., 6-phytase (EC 3.1.3.26) or 3-phytase (EC 3.1.3.8)).

[0159] VII. Exemplary Embodiment

[0160] Non-limiting examples of the compositions and methods disclosed herein are as follows:

[0161] 1. A method for producing ethanol from a starch-containing material, comprising (a) liquefying the starch-containing material in the presence of α-amylase at a temperature above the initial gelatinization temperature of the starch-containing material, (b) saccharifying the liquefied material obtained in step (a) using a saccharifying enzyme, and (c) producing the material obtained in step (b) by host fermentation with ethanol under conditions suitable for ethanol production, wherein steps (b) and / or (c) are carried out in the presence of a mixture of aspartic protease and serine protease.

[0162] 2. A method for producing ethanol from a starch-containing material, comprising (a) saccharifying the starch-containing material at a temperature below the initial gelatinization temperature of the starch-containing material using a saccharifying enzyme, and (b) producing the material obtained in step (a) by host fermentation with ethanol under conditions suitable for ethanol production, wherein steps (a) and / or (b) are carried out in the presence of a mixture of aspartic protease and serine protease.

[0163] 3. The method as described in Example 1 or Example 2, wherein saccharification and fermentation are carried out simultaneously.

[0164] 4. The method as described in Example 1 or Example 2, further comprising recovering the produced ethanol.

[0165] 5. The method as described in Example 1, wherein the α-amylase in step (a) is a thermostable α-amylase.

[0166] 6. The method as described in Example 1 or Example 2, wherein α-amylase is present or added during saccharification and / or fermentation.

[0167] 7. The method as described in Example 1 or Example 2, wherein the saccharifying enzyme is selected from the group consisting of: glucosidase, α-glucosidase, maltose amylase, amylopectinase, and β-amylase.

[0168] 8. The method as described in Example 1 or Example 2, wherein the serine protease excludes enzymes designated as EC 3.4.14.

[0169] 9. The method as described in Example 1 or Example 2, wherein the serine protease excludes enzymes designated as EC 3.4.16.

[0170] 10. The method as described in Example 1 or Example 2, wherein the mixture of aspartic protease and serine protease comprises, on the basis of total protease, about 5% (w / w) aspartic protease and about 95% (w / w) serine protease, or 10% (w / w) aspartic protease and 90% (w / w) serine protease, or 20% (w / w) aspartic protease and 80% (w / w) serine protease, or 30% (w / w) aspartic protease and 70% (w / w) serine protease, or 40% (w / w) aspartic protease and 60% (w / w) serine protease, or The total protease contains 50% (w / w) aspartic protease and 50% (w / w) serine protease, or 60% (w / w) aspartic protease and 40% (w / w) serine protease, or 70% (w / w) aspartic protease and 30% (w / w) serine protease, or 80% (w / w) aspartic protease and 20% (w / w) serine protease, or 90% (w / w) aspartic protease and 10% (w / w) serine protease, or approximately 95% (w / w) aspartic protease and 5% (w / w) serine protease.

[0171] 11. The method as described in Example 1, wherein the amount of ethanol produced is increased relative to the amount of ethanol produced by the same method except that steps (b) and / or (c) are performed in the presence of an aspartic protease or a serine protease rather than a mixture thereof.

[0172] 12. The method as described in Example 2, wherein the amount of ethanol produced is increased relative to the amount of ethanol produced by the same method except that steps (a) and / or (b) are performed in the presence of an aspartic protease or a serine protease rather than a mixture thereof.

[0173] 13. The method as described in Example 1, wherein the ethanol production rate is increased relative to the ethanol production rate of the same method except that steps (b) and / or (c) are performed in the presence of an aspartic protease or a serine protease rather than a mixture thereof.

[0174] 14. The method as described in Example 2, wherein the ethanol production rate is increased relative to the ethanol production rate of the same method except that steps (a) and / or (b) are performed in the presence of an aspartic protease or a serine protease rather than a mixture thereof.

[0175] 15. The method as described in Example 1, wherein the amount of glycerol produced is reduced relative to the amount of glycerol produced by the same method except that steps (b) and / or (c) are performed in the presence of an aspartic protease or a serine protease rather than a mixture thereof.

[0176] 16. The method as described in Example 2, wherein the amount of glycerol produced is reduced relative to the amount of glycerol produced using the same method except that steps (a) and / or (b) are performed in the presence of an aspartic protease or a serine protease rather than a mixture thereof.

[0177] 17. The method as described in Example 1, wherein the ethanol production host requires a reduced amount of supplemental nitrogen compared to the same ethanol production host fermented using the same method except that steps (b) and / or (c) are performed in the presence of aspartic protease or serine protease rather than a mixture thereof.

[0178] 18. The method as described in Example 2, wherein the ethanol production host requires a reduced amount of supplemental nitrogen compared to the same ethanol production host fermented using the same method except that steps (a) and / or (b) are performed in the presence of aspartic protease or serine protease rather than a mixture thereof.

[0179] 19. The method as described in Example 1 or Example 2, wherein the starch-containing material is derived from barley, beans, cassava, cereal crops, corn, sorghum, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, cassava, wheat, whole grains, or any combination thereof.

[0180] 20. The method as described in Example 1 or Example 2, wherein the ethanol production host is a yeast cell.

[0181] 21. The method as described in Example 1 or Example 2, wherein the aspartic protease contains about 60% sequence identity with the aspartic protease of SEQ ID NO: 2 or SEQ ID NO: 6, and the serine protease contains about 60% sequence identity with the serine protease of SEQ ID NO: 4.

[0182] 22. A method for producing ethanol from a starch-containing material, the method comprising fermenting the starch-containing material with an ethanol-producing host under conditions suitable for ethanol production, and recovering the produced ethanol, wherein the ethanol-producing host expresses and secretes heterologous aspartic protease and heterologous serine protease.

[0183] 23. A method for producing ethanol from a starch-containing material, the method comprising, under conditions suitable for ethanol production, fermenting the starch-containing material with at least two types of ethanol-producing hosts, and recovering the produced ethanol, wherein the first ethanol-producing host expresses and secretes a heterologous aspartic protease and the second ethanol-producing host expresses and secretes a heterologous serine protease.

[0184] 24. The method as described in Example 22 or Example 23, wherein the starch-containing material is derived from barley, beans, cassava, cereal crops, corn, sorghum, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, cassava, wheat, whole grains, or any combination thereof.

[0185] 25. The method as described in Example 22 or Example 23, wherein the starch-containing material has been liquefied and saccharified.

[0186] 26. The method as described in Example 22 or Example 23, wherein the starch-containing material is a raw starch composition.

[0187] 27. The method as described in Example 22 or Example 23, wherein the ethanol production host is a yeast cell.

[0188] 28. The method as described in Example 22, wherein the ethanol production host comprises an expression cassette encoding the introduction of a heterologous aspartic protease and an expression cassette encoding the introduction of a heterologous serine protease.

[0189] 29. The method of Example 23, wherein the first ethanol production host comprises an expression cassette encoding the introduction of a heterologous aspartic protease and the second ethanol production host comprises an expression cassette encoding the introduction of a heterologous serine protease.

[0190] 30. The method as described in Example 22 or Example 23, wherein the serine protease excludes enzymes designated as EC3.4.14.

[0191] 31. The method as described in Example 22 or Example 23, wherein the serine protease excludes enzymes designated as EC3.4.16.

[0192] 32. A protease composition comprising a mixture of aspartic protease and serine protease.

[0193] 33. The protease composition as described in Example 32, for use in the production of ethanol from starch-containing materials.

[0194] 34. The protease composition as described in Example 32, wherein the aspartic protease contains about 60% sequence identity with the aspartic protease of SEQ ID NO: 2 or SEQ ID NO: 6, and the serine protease contains about 60% sequence identity with the serine protease of SEQ ID NO: 4.

[0195] 35. The protease composition as described in Example 32, wherein the serine protease excludes enzymes designated as EC 3.4.14.

[0196] 36. The protease composition as described in Example 32, wherein the serine protease excludes enzymes designated as EC 3.4.16.

[0197] 37. The protease composition as described in Example 32, mixed with a starch-containing material.

[0198] 38. The protease composition as described in Example 32, wherein the protease composition is mixed with the starch-containing material after the starch-containing material has been liquefied.

[0199] 39. The protease composition as described in Example 32, mixed with a granular starch composition for obtaining starch-containing material.

[0200] 40. The protease composition as described in Example 32, mixed with a starch-containing material in a simultaneous saccharification and fermentation (SSF) process.

[0201] 41. The protease composition as described in Example 30, wherein the mixture of aspartic protease and serine protease comprises, on the basis of total protease, about 5% (w / w) aspartic protease and about 95% (w / w) serine protease, or 10% (w / w) aspartic protease and 90% (w / w) serine protease, or 20% (w / w) aspartic protease and 80% (w / w) serine protease, or 30% (w / w) aspartic protease and 70% (w / w) serine protease, or 40% (w / w) aspartic protease and 60% (w / w) serine protease, or The total protease contains 50% (w / w) aspartic protease and 50% (w / w) serine protease, or 60% (w / w) aspartic protease and 40% (w / w) serine protease, or 70% (w / w) aspartic protease and 30% (w / w) serine protease, or 80% (w / w) aspartic protease and 20% (w / w) serine protease, or 90% (w / w) aspartic protease and 10% (w / w) serine protease, or approximately 95% (w / w) aspartic protease and 5% (w / w) serine protease.

[0202] 42. The protease composition as described in Example 32, wherein the starch-containing material is derived from barley, beans, cassava, cereal crops, corn, sorghum, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, cassava, wheat, whole grains, or any combination thereof.

[0203] Example

[0204] Certain aspects of this disclosure may be further understood from the following examples, which should not be construed as limiting. Modifications to the materials and methods will be apparent to those skilled in the art.

[0205] Example 1

[0206] Synergistic hydrolytic effect between aspartic proteases and serine proteases

[0207] The aspartic protease from *Trichoderma reesei*, named “A10”, is produced by expressing a cloned *Trichoderma reesei* gene (SEQ ID NO: 1) in a *Trichoderma reesei* strain under the expression control of a strong cbhI promoter. Methods for overexpressing genes in *Trichoderma reesei* under the control of a cbhI promoter are well known in the art, and *Trichoderma reesei* strains suitable for such expression (e.g., RUT-C30 or RL-P37) are available from public collections of strains. The recombinant A10 protease (SEQ ID NO: 2) is expressed in high yield relative to the background protease activity in *Trichoderma reesei*. For example, by the methods described herein, the proteolytic activity of parental *Trichoderma reesei* strains lacking the recombinant cassette for expressing the A10 protease is undetectable and therefore can be considered negligible. Similarly, the serine protease from *Aspergillus niger*, named "orestrin" (active at acidic pH), was produced by expressing a cloned *Aspergillus niger* gene (SEQ ID NO: 3) in *Trichoderma reesei* strains under the expression control of a strong cbhI promoter, using the same method described for the A10 protease. Similar to the A10 protease, the orestrin protease (SEQ ID NO: 4) was expressed at a high level, such that any proteolytic activity in the culture supernatant other than orestrin (active) was considered negligible. The culture supernatants of *Trichoderma reesei* strains expressing A10 and orestrin were concentrated by ultrafiltration and used without further purification.

[0208] like Figure 1 As shown, dimethylcasein was used to determine the proteolytic activity of each protease individually (aspartic acid or serine) and in combination (aspartic acid + serine) to assess whether the mixture of combinations has any synergistic effect. This is demonstrably the most universal protease assay because it measures the total amount of free N-termini generated during proteolysis.

[0209] The assay was performed in 0.2 M sodium acetate buffer, pH 4.5 (Sigma Aldrich C9801) containing 5 mg / ml dimethyl casein. The reaction was initiated by adding 50 μg / ml of the protease and run at 37 °C. In samples using a mixture of two (2) proteases, 25 μg / ml of each protease (25 μg / ml aspartic acid + 25 μg / ml serine) was added. Twenty (20) μl aliquots were removed at specified time intervals (0–120 min) and frozen on dry ice until the end of the run. The aliquots were mixed with 200 μl of 0.05% trinitrobenzenesulfonic acid (pH 8.6) in 0.2 M sodium borate buffer. The reagents were mixed thoroughly and incubated at 37 °C for 15 min. Twenty (20) μl aliquots of these reaction mixtures were transferred to clear MTP plates and diluted with 100 μl of water. Optical density (OD) was measured using a microtiter plate reader. 410nm The results are presented in Figure 1 The study clearly indicated that a mixture of 25 μg / ml ozorelin (serine) protease and 25 μg / ml A10 (aspartic) protease was more efficient at hydrolyzing dimethyl casein than either 50 μg / ml ozorelin (serine) protease or 50 μg / ml A10 (aspartic) protease alone.

[0210] Example 2

[0211] Effects of proteases during yeast fermentation

[0212] This example presents the results of adding different combinations of proteases during yeast cell fermentation for ethanol production. Therefore, the data presented here typically pertain to yeast fermentation rate, ethanol concentration, and / or its glycerol concentration. More specifically, novel combinations of serine and aspartic proteases were used with commercial proteases (DuPont). A comparison was made.

[0213] For example, corn liquefaction from a commercial dry-milled ethanol plant was collected and frozen for this experiment. The frozen liquefaction was thawed at 65°C for three (3) hours for pasteurization. The liquefaction was cooled to room temperature and the pH was adjusted to 4.8 with ammonium hydroxide and sulfuric acid. Aminoglycosides equivalent to 0.325 GAU / g ds were added while continuously mixing. The liquefaction was aliquoted into 125 mL Erlenmeyer flasks (100 g each). For the control treatment, [the following was added] Protease and urea (600 ppm) as a nitrogen source for yeast. For experimental protease treatment, urea was added at levels of 200 ppm and 600 ppm, along with a combination of aspartic and serine proteases. Active dry yeast was added to all flasks at a dose of 0.01% w / w, and the flasks were sealed with rubber stoppers with small holes.

[0214] The initial weight of all flasks was recorded, and the flasks were placed in an air-heated incubator at 32°C and 200 rpm for 62 (62) hours. The weight of the flasks was recorded periodically (i.e., at hours 16, 24, 40, 48, and 62 of incubation) to track weight loss due to the CO2 produced concurrently with the production of ethanol from glucose. At the end of the 62 (62) hour fermentation, samples were removed for processing and prepared for HPLC to measure the concentrations of ethanol, glycerol, sugars, and organic acids.

[0215] The results are presented in Tables 1-3 below, and show that the addition of a novel combination of aspartic and serine proteases improved the fermentation rate, final ethanol concentration, and reduced glycerol byproducts, which improved the carbon conversion fermentation efficiency of yeast ethanol production.

[0216] Table 1

[0217] Measurement of weight loss (g) during SSF incubation with and without sufficient urea.

[0218]

[0219] As shown in Table 1 above, the novel combination of aspartic and serine proteases outperformed commercial products in both cases with and without sufficient urea (600 ppm). Protease.

[0220] Table 2

[0221] Ethanol and glycerol concentrations (% weight / volume) in the presence of high urea concentration (600 ppm)

[0222]

[0223] As shown in Table 2 above, in the presence of sufficient urea (600 ppm), with the addition of a novel protease combination, compared to the control protease... The glycerol concentration decreased and the ethanol concentration increased.

[0224] Table 3

[0225] Ethanol and glycerol concentrations at 62 hours in the presence of low urea concentration (200 ppm)

[0226]

[0227] As shown in Table 3 above, compared to the control protease in the presence of a high urea concentration (600 ppm), In the presence of low urea concentration (200 ppm), the addition of a combination of aspartic protease and serine protease increased the ethanol concentration (14.91) while simultaneously decreasing the glycerol concentration (1.19).

[0228] Example 3

[0229] Effects of proteases during yeast fermentation

[0230] This example presents the results of adding different combinations of aspartic protease and serine protease (i.e., sedolisin) during yeast cell fermentation for ethanol production. Therefore, the data presented herein generally pertain to yeast fermentation rates. More specifically, novel combinations of serine and aspartic proteases were used in conjunction with commercial aspartic protease (DuPont). A comparison was made.

[0231] For example, corn liquefaction from a commercial dry-milled ethanol plant was collected and frozen for this experiment. The frozen liquefaction was thawed at 65°C for three (3) hours for pasteurization. The liquefaction was cooled to room temperature and the pH was adjusted to 4.8 with ammonium hydroxide and sulfuric acid. Aminoglycosides equivalent to 0.325 GAU / g ds were added while continuously mixing. The liquefaction was aliquoted into 125 mL Erlenmeyer flasks in 100 g portions. For the control treatment, [the following was added] Protease and urea (200 ppm) as a nitrogen source for yeast. For experimental protease treatment, urea was added at a level of 200 ppm, along with a combination of aspartic protease and serine protease (sedolisin). Active dry yeast was added to all flasks at a dose of 0.01% w / w, and the flasks were sealed with rubber stoppers with small holes.

[0232] Record the initial weight of all flasks and place them in an air-heated incubator at 32°C and 200 rpm for 62 (62) hours. Record the weight of the flasks periodically (i.e., at the 16th, 24th, 40th, 48th and 62nd hours of incubation) to track weight loss due to the CO2 produced concurrently with the production of ethanol from glucose.

[0233] The results are presented in Table 4 below, which shows that the addition of a novel combination of aspartic and serine proteases improved the fermentation rate at 16 and 24 hours, which improved the carbon conversion fermentation efficiency and the fermentation time to complete yeast ethanol production.

[0234] Table 4

[0235] Measurement of weight loss (g) during SSF incubation

[0236]

[0237] Therefore, as shown in Table 4 above, the novel combination of aspartic protease and serine protease (sedolisin) outperforms commercial products in terms of initial fermentation rate. Proteases help to convert carbon into ethanol more quickly in fermentation processes.

[0238] Example 4

[0239] The effect of proteases during yeast fermentation of granular starch

[0240] This example presents the results of fermentation of granular starch yeast with different combinations of aspartic and serine proteases during ethanol production. Therefore, the data presented herein generally pertain to yeast fermentation rates. More specifically, novel combinations of serine and aspartic proteases were used in conjunction with commercial aspartic proteases (DuPont). A comparison was made.

[0241] For example, milled corn flour was collected for this experiment. The milled corn flour was mixed with filtered tap water, and the pH was adjusted to 4.8 with ammonium hydroxide and sulfuric acid. Saccharifying enzyme equivalent to 1.0 GAU / g ds was added while continuously mixing. The milled corn pulp was divided into 100g portions and placed into 125mL Erlenmeyer flasks. For the control treatment, [further details needed]. Protease and urea (600 ppm) as a nitrogen source for yeast. For experimental protease treatment, urea was added at a level of 600 ppm, along with a combination of aspartic and serine proteases. Active dry yeast was added to all flasks at a dose of 0.01% w / w, and the flasks were sealed with rubber stoppers with small holes.

[0242] Record the initial weight of all flasks and place them in an air-heated incubator at 32°C and 200 rpm for 62 (62) hours. Record the weight of the flasks periodically (i.e., at the 17th, 24th, 41st, 62nd, and 86th hours of incubation) to track weight loss due to the CO2 produced concurrently with the production of ethanol from glucose.

[0243] The results are presented in Table 5 below, showing that the addition of a novel combination of aspartic and serine proteases improved the fermentation rate throughout the granular starch fermentation process, which improved the carbon conversion fermentation efficiency and the fermentation time to complete yeast ethanol production.

[0244] Table 5

[0245] Measurement of weight loss (g) during fermentation of granular starch

[0246] Hour

[0247]

[0248] As shown in Table 5 above, the novel combination of aspartic and serine proteases outperformed commercial products in terms of fermentation rate throughout the incubation process. Proteases help to convert carbon into ethanol more quickly in granular starch fermentation processes.

[0249] References

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[0265] Wilson,“Proteins of the kernel”In:Corn:Chemistry and Technology,1987,S.A.Watson,and P.E.Ramstad,eds.AACC International:St.Paul,MN.

[0266] Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), NC-IUBMB, 1992 (Academic Press, San Diego, California), including supplements 1-5 published in 1994 (Eur. J. Biochem., 223: 1-5), 1995 (Eur. J. Biochem. 232: 1-6); 1996 (Eur. J. Biochem. 237: 1-5), 1997 (Eur. J. Biochem. 250: 1-6) and 1999 (Eur. J. Biochem. 264: 610-650), respectively. sequence list <110> Danisco America <120> Methods and compositions for improving ethanol production <130> NB41362-WO-PCT <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 1571 <212> DNA <213> Hypocrea jecorina <400> 1 atgcactcac gactcctcat cacggccctc ttcctgggcc tcatcgccct ggtctcggcc 60 tcggccatcc ccatccacca gaagcgcggc tccttcaagg tcgagcggag ggccaacccc 120 aacttcaccg gcaatgacgg cctcaaggcc atggccaagg cgtaccgcaa gttcggctgg 180 accatgcccc aggacctcaa ggacgcgctg gcggtgcggg aggcggccgt caaggcccgc 240 cgggctgcgg cggctgtggc tgcgcaggag gctgctgcca agaagaagag cagacgatcg ctgttggatc tcctgggtga gcttggcctg ctcgggggga acggaggcaa tggatcaaac 360 gatgatgcca atgccaatcc taacggtggc agcggaagac atcaccacgg caatggaat ggcaacggca acggcaacgg aaatggtcaa ggacaaggcc aaggccaggc tggaggaac cagactcagc ctgccccggc agcccagccc gcctctggcc aggtcggag cgtcacaaac 540 acgcccgagg gcaacgacgt cgagttcctg tcccccgtca agatcggcgg ccagacgctc 600. aacctcgact tcgacaccgg ctcctctgac ctctgggtct tcaaccggc catggatccc 660 tcgctgacgg ccggccacac cctgtacgat cccaccaaga gcaagacctt caagcagatc 720 cagggcgcgc agttcctcgt ccagtacggc gacggctcag gcgccgaggg cgtcgtcggc 780 acggacgttg tcgacgttgg cggcgccgtc ttcgacgccc aggccgtcga gattgccacc 840 gccgtcacgc agcagtttgt cgacgaccag cagaacgacg ggctcatggg cctcgccttt 900 tccaagctca acacggtcca gccgcagcag caaaagacgt tcctcgacaa cgtccagagc tccctcgccg agcccgtctt caccgccgac ctcaagaagg gccagcccgg cacgtacacc 1020 tttggcgccg tcgacgcctc cgccttccag ggcgacctga cctgggtcga cgtcgacaac 1080 tcgcagggct tctggcagtt cagcagcgag tcctttgccg tcgacggcgg cgcgacccag 1140 caggccacgg ccggcggcca ggccattgcc gacaccggca ccaccctgct gctggccgac 1200 cccatcatcg tccagggcta ctacgcaaag gtccagggcg cccagaacga tgcccaggcc 1260 ggtggcttta ccgtgccgtg cgatgcccag ctgcccgact tggacctgga tgttggcgga 1320 aagtatgtgg cccgcatcag cggttcggac ctcaactttg cgccggttca gggaaacagt 1380 aagttgctac cgtatcatct ttttactgag agacgacttc acaggactga catttttcga 1440 cagcttgctt tggtggtctt caggcaacga cgcagggcgg cctgggtgtc tatggcgaca 1500 tcttcttcaa gtcgcagttt gtggcgttca acattggcaa caacacgctg ggcctggctc 1560 ctcatgctta g 1571 <210> 2 <211> 430 <212> PRT <213> Hypocrea jecorina <400> 2 Met His Ser Arg Leu Leu Ile Thr Ala Leu Phe Leu Gly Leu Ile Ala 1 5 10 15 Leu Val Ser Ala Ser Ala Ile Pro Ile His Gln Lys Arg Gly Ser Phe 20 25 30 Lys Val Glu Arg Arg Ala Asn Pro Asn Phe Thr Gly Asn Asp Gly Leu 35 40 45 Lys Ala Met Ala Lys Ala Tyr Arg Lys Phe Gly Trp Thr Met Pro Gln 50 55 60 Asp Leu Lys Asp Ala Leu Ala Val Arg Glu Ala Ala Val Lys Ala Arg 65 70 75 80 Arg Ala Ala Ala Ala Ala Gly Gly Asn Gln Thr Gln Pro Ala Pro Ala 85 90 95 Ala Gln Pro Ala Ser Gly Gln Val Gly Ser Val Thr Asn Thr Pro Glu 100 105 110 Gly Asn Asp Val Glu Phe Leu Ser Pro Val Lys Ile Gly Gly Gln Thr 115 120 125 Leu Asn Leu Asp Phe Asp Thr Gly Ser Ser Asp Leu Trp Val Phe Asn 130 135 140 Thr Ala Met Asp Pro Ser Leu Thr Ala Gly His Thr Leu Tyr Asp Pro 145 150 155 160 Thr Lys Ser Lys Thr Phe Lys Gln Ile Gln Gly Ala Gln Phe Leu Val 165 170 175 Gln Tyr Gly Asp Gly Ser Gly Ala Glu Gly Val Val Gly Thr Asp Val 180 185 190 Val Asp Val Gly Gly Ala Val Phe Asp Ala Gln Ala Val Glu Ile Ala 195 200 205 Thr Ala Val Thr Gln Gln Phe Val Asp Asp Gln Gln Asn Asp Gly Leu 210 215 220 Met Gly Leu Ala Phe Ser Lys Leu Asn Thr Val Gln Pro Gln Gln Gln 225 230 235 240 Lys Thr Phe Leu Asp Asn Val Gln Ser Ser Leu Ala Glu Pro Val Phe 245 250 255 Thr Ala Asp Leu Lys Lys Gly Gln Pro Gly Thr Tyr Thr Phe Gly Ala 260 265 270 Val Asp Ala Ser Ala Phe Gln Gly Asp Leu Thr Trp Val Asp Val Asp 275 280 285 Asn Ser Gln Gly Phe Trp Gln Phe Ser Ser Glu Ser Phe Ala Val Asp 290 295 300 Gly Gly Ala Thr Gln Gln Ala Thr Ala Gly Gly Gln Ala Ile Ala Asp 305 310 315 320 Thr Gly Thr Thr Leu Leu Leu Ala Asp Pro Ile Ile Val Gln Gly Tyr 325 330 335 Tyr Ala Lys Val Gln Gly Ala Gln Asn Asp Ala Gln Ala Gly Gly Phe 340 345 350 Thr Val Pro Cys Asp Ala Gln Leu Pro Asp Leu Asp Leu Asp Val Gly 355 360 365 Gly Lys Tyr Val Ala Arg Ile Ser Gly Ser Asp Leu Asn Phe Ala Pro 370 375 380 Val Gln Gly Asn Thr Cys Phe Gly Gly Leu Gln Ala Thr Thr Gln Gly 385 390 395 400 Gly Leu Gly Val Tyr Gly Asp Ile Phe Phe Lys Ser Gln Phe Val Ala 405 410 415 Phe Asn Ile Gly Asn Asn Thr Leu Gly Leu Ala Pro His Ala 420 425 430 <210> 3 <211> 2388 <212> DNA <213> Aspergillus niger <400> 3 atgaagttct tctcgacaat ctgcagtttg acccttgcgg tctctgcgtt ggccttgcca 60 acttctgacc atgttattca tgagaagcga tctggtaccc catctcgatg ggagaagatc 120 agtcgggtca atggtactga gcatgtcttg gtgcgcatcg gtctgacgca gaacaatctt 180 gatcgagcct atgagtatct catgagcgtg tatgttgctt cctcccccct atattgttcc 240 gactgactag tcaggtctga ctccgtgtcg cctaactacg gcaaattctg gactccagaa 300 gaggtgcaca gtaccttcgc gccgtcgaat gagactgtca acgctgtgcg caactggctt 360 attgaatccg gtgtggatga gtctcgcttg gttcacacca aaaaccaagg ctggattgtg 420 tttgatgcaa ccaccaaaga agcggaaaat ctcctccaca ccaagtatta ccactacaca 480 gaccggattt ctggcttcaa gacactcgca gcagaagagt atgtctccca tttattctca 540 gagatatagt atactaacaa gacgaccagg taccgcgtcc cccagaagat ccaacaacac 600 atcgacttca ttaaacccgg cgttcttctc ccattgacct ccaagggacc atccgccaag 660 cacaccaaga aatacaaacc cttgaagcag acatcagtca acgccacgtc tctcaccacg 720 tgcgacgagg tcatcactcc agcttgtgtc gccgctctgt acaagatccc tcacgcaagc 780 ggcaacgtca gtgcaagcaa ctcgcttggg atcttcgaag aaggagacta ctacgcccag 840 gaagatctgg atctcttttt ccgcaacttc accccgtaca ttccgaaggg aacacaccct 900 aagccggcat ttatcgatgg agcctcagcg cccgtgagcg ttgctgatgc tggtgcggag 960 tcggatttgg acttccagct tgcgtatcca attgtttacc ctcagaccat cacgctgtat 1020 cagacagatg actatgacta tgcgagtggg gaggttgaga ctgatggatt ctttaacacc 1080 tttcttgatg ctgttgatgg ggtaagtagc ctctggaatt ttgggattta gctaatgtct 1140 cagtcatact gcacctactg tgcctatggg gaatgcggag acagcccgac tctcgatccc 1200 acttacccag ataactccac cggaggttac aagggacagt tgatgtgcgg tgtttataag 1260 cccacgaatg tcatctccgt ttcttatggt ggccaggagg cagacctccc ggcttactac 1320 cagcagcgac aatgcaatga gtatgccatc cccacctcta tttatacagc ccgaattaac 1380 aacaggaata gattcctcaa gctcggtctc cagggcattt ccatcctctt cgcctctggc 1440 gatgacggcg tcgcagggcc cccaggcgac gactcgacta acgggtgtct gggaaatgga 1500 accatcttca gtcctgcgtt ccccaattcg tatgttttct tctcttgcaa tattcttgca 1560 acaaccacat ctaacttccc tagttgcccc tgggtgacta atgtcggagc caccaaactc 1620 taccccggaa agaccatcgc agacggggaa agcgccgtcg tcgacccggc cggccacccc 1680 tactcggtcg cattctcctc tggtggtggc ttcagcaaca tctatactat tccagactat 1740 caagccgaag cagtagcaga gtaggtgctt ttctatcatc ctatacgcgc aatctaacag 1800 cccccattca gatacttcaa aaagcacaac ccaccctatc cttactacga aggcaacgcc 1860 agcttcggca aaaacggcgg tgtctacaac cgtcttggac gcgggtaccc cgacgtggca 1920 gcgaacggcg acaacatcgc cgagtacaac gcgggagaat tcatacttga gggtggaact 1980 agtgctagta cgttactacc ctactacatc cacccaactc cgttatattt caattactaa 2040 tgacaatgat taaaggtacc ccgatcttct cctccgtgat taaccgcatc atcgagaagc 2100 gaatcgcggc aggaaagggc ccactaggct tcctgaaccc ggttctgtat cggaatgcgt 2160 gggcgttgaa tgatattacg aatgggtcga atccgggttg tggaacggag gggttctata 2220 ctgctcctgg gtatgtattt ccctctcgtt tttttatttc tttattatcg tacttcgata 2280 tatggagcca tgctaacaat cggtgaacaa aatagatggg atcccgtcac cggtctcgga 2340 acgcctaact tcccgaaatt gctagacgtg ttcctgaacc tcccgtaa 2388 <210> 4 <211> 646 <212> PRT <213> Aspergillus niger <400> 4 Met Lys Phe Phe Ser Thr Ile Cys Ser Leu Thr Leu Ala Val Ser Ala 1 5 10 15 Leu Ala Leu Pro Thr Ser Asp His Val Ile His Glu Lys Arg Ser Gly 20 25 30 Thr Pro Ser Arg Trp Glu Lys Ile Ser Arg Val Asn Gly Thr Glu His 35 40 45 Val Leu Val Arg Ile Gly Leu Thr Gln Asn Asn Leu Asp Arg Ala Tyr 50 55 60 Glu Tyr Leu Met Ser Val Ser Asp Ser Val Ser Pro Asn Tyr Gly Lys 65 70 75 80 Phe Trp Thr Pro Glu Glu Val His Ser Thr Phe Ala Pro Ser Asn Glu 85 90 95 Thr Val Asn Ala Val Arg Asn Trp Leu Ile Glu Ser Gly Val Asp Glu 100 105 110 Ser Arg Leu Val His Thr Lys Asn Gln Gly Trp Ile Val Phe Asp Ala 115 120 125 Thr Thr Lys Glu Ala Glu Asn Leu Leu His Thr Lys Tyr Tyr His Tyr 130 135 140 Thr Asp Arg Ile Ser Gly Phe Lys Thr Leu Ala Ala Glu Glu Tyr Arg 145 150 155 160 Val Pro Gln Lys Ile Gln Gln His Ile Asp Phe Ile Lys Pro Gly Val 165 170 175 Leu Leu Pro Leu Thr Ser Lys Gly Pro Ser Ala Lys His Thr Lys Lys 180 185 190 Tyr Lys Pro Leu Lys Gln Thr Ser Val Asn Ala Thr Ser Leu Thr Thr 195 200 205 Cys Asp Glu Val Ile Thr Pro Ala Cys Val Ala Ala Leu Tyr Lys Ile 210 215 220 Pro His Ala Ser Gly Asn Val Ser Ala Ser Asn Ser Leu Gly Ile Phe 225 230 235 240 Glu Glu Gly Asp Tyr Tyr Ala Gln Glu Asp Leu Asp Leu Phe Phe Arg 245 250 255 Asn Phe Thr Pro Tyr Ile Pro Lys Gly Thr His Pro Lys Pro Ala Phe 260 265 270 Ile Asp Gly Ala Ser Ala Pro Val Ser Val Ala Asp Ala Gly Ala Glu 275 280 285 Ser Asp Leu Asp Phe Gln Leu Ala Tyr Pro Ile Val Tyr Pro Gln Thr 290 295 300 Ile Thr Leu Tyr Gln Thr Asp Asp Tyr Asp Tyr Ala Ser Gly Glu Val 305 310 315 320 Glu Thr Asp Gly Phe Phe Asn Thr Phe Leu Asp Ala Val Asp Gly Ser 325 330 335 Tyr Cys Thr Tyr Cys Ala Tyr Gly Glu Cys Gly Asp Ser Pro Thr Leu 340 345 350 Asp Pro Thr Tyr Pro Asp Asn Ser Thr Gly Gly Tyr Lys Gly Gln Leu 355 360 365 Met Cys Gly Val Tyr Lys Pro Thr Asn Val Ile Ser Val Ser Tyr Gly 370 375 380 Gly Gln Glu Ala Asp Leu Pro Ala Tyr Tyr Gln Gln Arg Gln Cys Asn 385 390 395 400 Glu Phe Leu Lys Leu Gly Leu Gln Gly Ile Ser Ile Leu Phe Ala Ser 405 410 415 Gly Asp Asp Gly Val Ala Gly Pro Pro Gly Asp Asp Ser Thr Asn Gly 420 425 430 Cys Leu Gly Asn Gly Thr Ile Phe Ser Pro Ala Phe Pro Asn Ser Cys 435 440 445 Pro Trp Val Thr Asn Val Gly Ala Thr Lys Leu Tyr Pro Gly Lys Thr 450 455 460 Ile Ala Asp Gly Glu Ser Ala Val Val Asp Pro Ala Gly His Pro Tyr 465 470 475 480 Ser Val Ala Phe Ser Ser Gly Gly Gly Phe Ser Asn Ile Tyr Thr Ile 485 490 495 Pro Asp Tyr Gln Ala Glu Ala Val Ala Glu Tyr Phe Lys Lys His Asn 500 505 510 Pro Pro Tyr Pro Tyr Tyr Glu Gly Asn Ala Ser Phe Gly Lys Asn Gly 515 520 525 Gly Val Tyr Asn Arg Leu Gly Arg Gly Tyr Pro Asp Val Ala Ala Asn 530 535 540 Gly Asp Asn Ile Ala Glu Tyr Asn Ala Gly Glu Phe Ile Leu Glu Gly 545 550 555 560 Gly Thr Ser Ala Ser Thr Pro Ile Phe Ser Ser Val Ile Asn Arg Ile 565 570 575 Ile Glu Lys Arg Ile Ala Ala Gly Lys Gly Pro Leu Gly Phe Leu Asn 580 585 590 Pro Val Leu Tyr Arg Asn Ala Trp Ala Leu Asn Asp Ile Thr Asn Gly 595 600 605 Ser Asn Pro Gly Cys Gly Thr Glu Gly Phe Tyr Thr Ala Pro Gly Trp 610 615 620 Asp Pro Val Thr Gly Leu Gly Thr Pro Asn Phe Pro Lys Leu Leu Asp 625 630 635 640 Val Phe Leu Asn Leu Pro 645 <210> 5 <211> 1302 <212> DNA <213> Hypocrea jecorina <400> 5 atgcagacct ttggagcttt tctcgtttcc ttcctcgccg ccagcggcct ggccgcggcc 60 ctccccaccg agggtcagaa gacggcttcc gtcgaggtcc agtacaacaa gaactacgtc 120 ccccacggcc ctactgctct cttcaaggcc aagagaaagt atggcgctcc catcagcgac 180 aacctgaagt ctctcgtggc tgccaggcag gccaagcagg ctctcgccaa gcgccagacc 240 ggctcggcgc ccaaccaccc cagtgacagc gccgattcgg agtacatcac ctccgtctcc 300 atcggcactc cggctcaggt cctccccctg gactttgaca ccggctcctc cgacctgtgg 360 gtctttagct ccgagacgcc caagtcttcg gccaccggcc acgccatcta cacgccctcc 420 aagtcgtcca cctccaagaa ggtgtctggc gccagctggt ccatcagcta cggcgacggc 480 agcagctcca gcggcgatgt ctacaccgac aaggtcacca tcggaggctt cagcgtcaac 540 acccagggcg tcgagtctgc cacccgcgtg tccaccgagt tcgtccagga cacggtcatc 600 tctggcctcg tcggccttgc ctttgacagc ggcaaccagg tcaggccgca cccgcagaag 660 acgtggttct ccaacgccgc cagcagcctg gctgagcccc ttttcactgc cgacctgagg 720 cacggacaga gtaagtagac actcactgga attcgttcct ttcccgatca tcatgaaagc 780 aagtagactg actgaaccaa acaactagac ggcagctaca actttggcta catcgacacc 840 agcgtcgcca agggccccgt tgcctacacc cccgttgaca acagccaggg cttctgggag 900 ttcactgcct cgggctactc tgtcggcggc ggcaagctca accgcaactc catcgacggc 960 attgccgaca ccggcaccac cctgctcctc ctcgacgaca acgtcgtcga tgcctactac 1020 gccaacgtcc agtcggccca gtacgacaac cagcaggagg gtgtcgtctt cgactgcgac 1080 gaggacctcc cttcgttcag cttcggtgtt ggaagctcca ccatcaccat ccctggcgat 1140 ctgctgaacc tgactccct cgaggaggc agctccacct gctcggtgg cctccagagc 1200 agctccggca ttggcatca catctttggt gacgttgccc tcaggctgc cctggttgtc 1260 tttgacctcg gcaacgagcg cctggggctgg gctcagaaat aa 1302 <210> 6 <211> 407 <212> PRT <213> Hypocrea jecorina <400> 6 Met Gln Thr Phe Gly Ala Phe Leu Val Ser Phe Leu Ala Ala Ser Gly 1 5 10 15 Leu Ala Ala Ala Leu Pro Thr Glu Gly Gln Lys Thr Ala Ser Val Glu 20 25 30 Val Gln Tyr Asn Lys Asn Tyr Val Pro His Gly Pro Thr Ala Leu Phe 35 40 45 Lys Ala Lys Arg Lys Tyr Gly Ala Pro Ile Ser Asp Asn Leu Lys Ser 50 55 60 Leu Val Only Arg Gln Only Lys Gln Only Leu Only Lys Arg Gln Thr 65 70 75 80 Gly Ser Ala Pro Asn His Pro Ser Asp Ser Ala Asp Ser Glu Tyr Ile 85 90 95 Thr Ser Val Ser Ile Gly Thr Pro Ala Gln Val Leu Pro Leu Asp Phe 100 105 110 Asp Thr Gly Ser Ser Asp Leu Trp Val Phe Ser Ser Glu Thr Pro Lys 115 120 125 Ser Ser Ala Thr Gly His Ala Ile Tyr Thr Pro Ser Lys Ser Ser Thr 130 135 140 Ser Lys Lys Val Ser Gly Ala Ser Trp Ser Ile Ser Tyr Gly Asp Gly 145 150 155 160 Ser Ser Ser Ser Gly Asp Val Tyr Thr Asp Lys Val Thr Ile Gly Gly 165 170 175 Phe Ser Val Asn Thr Gln Gly Val Glu Ser Ala Thr Arg Val Ser Thr 180 185 190 Glu Phe Val Gln Asp Thr Val Ile Ser Gly Leu Val Gly Leu Ala Phe 195 200 205 Asp Ser Gly Asn Gln Val Arg Pro His Pro Gln Lys Thr Trp Phe Ser 210 215 220 Asn Ala Ala Ser Ser Leu Ala Glu Pro Leu Phe Thr Ala Asp Leu Arg 225 230 235 240 His Gly Gln Asn Gly Ser Tyr Asn Phe Gly Tyr Ile Asp Thr Ser Val 245 250 255 Ala Lys Gly Pro Val Ala Tyr Thr Pro Val Asp Asn Ser Gln Gly Phe 260 265 270 Trp Glu Phe Thr Ala Ser Gly Tyr Ser Val Gly Gly Gly Lys Leu Asn 275 280 285 Arg Asn Ser Ile Asp Gly Ile Ala Asp Thr Gly Thr Thr Leu Leu Leu 290 295 300 Leu Asp Asp Asn Val Val Asp Ala Tyr Tyr Ala Asn Val Gln Ser Ala 305 310 315 320 Gln Tyr Asp Asn Gln Gln Glu Gly Val Val Phe Asp Cys Asp Glu Asp 325 330 335 Leu Pro Ser Phe Ser Phe Gly Val Gly Ser Ser Thr Ile Thr Ile Pro 340 345 350 Gly Asp Leu Leu Asn Leu Thr Pro Leu Glu Glu Gly Ser Ser Thr Cys 355 360 365 Phe Gly Gly Leu Gln Ser Ser Ser Gly Ile Gly Ile Asn Ile Phe Gly 370 375 380 Asp Val Ala Leu Lys Ala Ala Leu Val Val Phe Asp Leu Gly Asn Glu 385 390 395 400 Arg Leu Gly Trp Ala Gln Lys 405

Claims

1. A method for producing ethanol from a starch-containing material, the method comprising: (a) The starch-containing material is liquefied in the presence of α-amylase at a temperature higher than the initial gelatinization temperature of the starch-containing material. (b) Using a saccharifying enzyme, the liquefied material obtained in step (a) is saccharified, and (c) Under conditions suitable for ethanol production, use ethanol to produce the material obtained in step (b) from the host fermentation. Steps (b) and / or (c) are carried out in the presence of a mixture of aspartic protease and serine protease, and The aspartic protease is shown in SEQ ID NO:2, and the serine protease is shown in SEQ ID NO:

4.

2. A method for producing ethanol from a starch-containing material, the method comprising: (a) Using a saccharifying enzyme, the starch-containing material is saccharified at a temperature below the initial gelatinization temperature of the starch-containing material, and (b) Under conditions suitable for ethanol production, the material obtained in step (a) is produced by host fermentation using ethanol. Steps (a) and / or (b) are carried out in the presence of a mixture of aspartic protease and serine protease, and The aspartic protease is shown in SEQ ID NO:2, and the serine protease is shown in SEQ ID NO:

4.

3. The method of claim 1 or claim 2, wherein saccharification and fermentation are carried out simultaneously.

4. The method of claim 1 or claim 2, further comprising recovering the produced ethanol.

5. The method of claim 1, wherein the amount of ethanol produced is increased relative to the amount of ethanol produced using the same method except that steps (b) and / or (c) are performed in the presence of an aspartic protease or a serine protease rather than a mixture thereof.

6. The method of claim 2, wherein the amount of ethanol produced is increased relative to the amount of ethanol produced using the same method except that steps (a) and / or (b) are performed in the presence of an aspartic protease or a serine protease rather than a mixture thereof.

7. The method of claim 1, wherein the ethanol production rate is increased relative to the ethanol production rate of the same method except that steps (b) and / or (c) are performed in the presence of an aspartic protease or a serine protease rather than a mixture thereof.

8. The method of claim 2, wherein the ethanol production rate is increased relative to the ethanol production rate of the same method except that steps (a) and / or (b) are performed in the presence of an aspartic protease or a serine protease rather than a mixture thereof.

9. The method of claim 1, wherein the amount of glycerol produced is reduced relative to the amount of glycerol produced by the same method except that steps (b) and / or (c) are performed in the presence of an aspartic protease or a serine protease rather than a mixture thereof.

10. The method of claim 2, wherein the amount of glycerol produced is reduced relative to the amount of glycerol produced using the same method except that steps (a) and / or (b) are performed in the presence of an aspartic protease or a serine protease rather than a mixture thereof.

11. A method for producing ethanol from a starch-containing material, the method comprising fermenting the starch-containing material with an ethanol production host under conditions suitable for ethanol production, and recovering the produced ethanol, wherein the ethanol production host expresses and secretes a heterologous aspartic protease and a heterologous serine protease, and wherein the aspartic protease is as shown in SEQ ID NO:2, and the serine protease is as shown in SEQ ID NO:

4.

12. A method for producing ethanol from a starch-containing material, the method comprising, under conditions suitable for ethanol production, fermenting the starch-containing material with at least two types of ethanol as a host, and recovering the produced ethanol, wherein the first ethanol produces a host expressing and secreting a heterologous aspartic protease and the second ethanol produces a host expressing and secreting a heterologous serine protease, wherein the aspartic protease is as shown in SEQ ID NO:2 and the serine protease is as shown in SEQ ID NO:

4.

13. The method of claim 11 or claim 12, wherein the starch-containing material has been liquefied and saccharified.

14. A protease composition comprising a mixture of an aspartic protease and a serine protease, wherein the aspartic protease is as shown in SEQ ID NO:2 and the serine protease is as shown in SEQ ID NO:

4.

15. The protease composition of claim 14, for use in the production of ethanol from starch-containing materials.

16. The protease composition of claim 14, wherein the serine protease excludes enzymes specified as EC 3.4.

14.

17. The protease composition of claim 14, wherein the serine protease excludes enzymes specified as EC 3.4.

16.

18. The protease composition of claim 14, wherein it is mixed with a starch-containing material.

19. The protease composition of claim 14, wherein the protease composition is mixed with the starch-containing material after the starch-containing material has been liquefied.

20. The protease composition of claim 14, wherein it is mixed with a granular starch composition for obtaining starch-containing material.

21. The protease composition of claim 14, wherein it is mixed with a starch-containing material in a simultaneous saccharification and fermentation (SSF) process.

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