Method for producing a fermentation product

Abstract

The present invention relates to a process for producing a fermentation product from starch-containing material, wherein a bacterial alpha-amylase, a raw starch hydrolyzing alpha-amylase and a carbohydrate-source generating enzyme are present and / or added during liquefaction. The present invention also relates to compositions suitable for use in the process of the present invention.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201280061023.2, filed on October 9, 2012, entitled "Method for producing a fermentation product". TECHNICAL FIELD

[0002] The present invention relates to a method for producing a fermentation product from starch-containing material. The present invention also relates to compositions suitable for use in the method of the present invention.

[0003] Reference to Sequence Listing

[0004] The present invention comprises a sequence listing in computer readable form, which is incorporated herein by reference. BACKGROUND

[0005] The production of fermentation products, such as ethanol, from starch-containing material is well known in the art. Today, two different types of processes are used in industry. The most commonly used process, often referred to as the "conventional process", involves liquefaction of gelatinized starch at high temperature (typically at 80-90°C, pH 5-6) using bacterial alpha-amylase, followed by simultaneous saccharification and fermentation (SSF) in the presence of glucoamylase and a fermenting organism. Another well-known process, often referred to as the "raw starch hydrolysis" process (RSH process), involves simultaneous saccharification and fermentation of granular starch at a temperature below the initial gelatinization temperature, typically in the presence of acidic fungal alpha-amylase and glucoamylase.

[0006] Although fermentation product production processes have improved significantly over the past decade, significant amounts of residual starch material are not converted into the desired fermentation product, such as ethanol. Thus, there is a desire and need for a process for producing a fermentation product, such as ethanol, from starch-containing material that is capable of providing a higher fermentation product yield compared to conventional processes. SUMMARY

[0007] The present invention relates to a method for producing a fermentation product, such as in particular ethanol, from starch-containing material using a fermenting organism.

[0008] In a first aspect, the present invention relates to a method for producing a fermentation product, such as ethanol, comprising the steps of:

[0009] i) using

[0010] - a bacterial alpha-amylase;

[0011] - a raw starch hydrolyzing alpha-amylase;

[0012] - a carbohydrate source generating enzyme having a thermostability of at least 70% at 70°C, pH 5.3,

[0013] liquefying the starch-containing material at a temperature of 60-80°C;

[0014] ii) saccharification using a saccharogenic enzyme;

[0015] iii) fermentation using a fermenting organism.

[0016] In embodiments, a protease, such as a metalloprotease, is also present and / or added during liquefaction in step i).

[0017] In embodiments, a pullulanase is also present and / or added during liquefaction in step i).

[0018] In embodiments, the bacterial alpha-amylase is derived from a strain of Bacillus, preferably a Bacillus stearothermophilus strain. The Bacillus stearothermophilus alpha-amylase can be truncated. In embodiments, the bacterial alpha-amylase is derived from a Bacillus stearothermophilus (SEQ ID NO: 3 in WO 99 / 019467 or SEQ ID NO: 1 herein) which is truncated to have about 491 amino acids. In embodiments, the Bacillus stearothermophilus alpha-amylase is truncated as described above and further has the I181*+G182* deletion (relative to SEQ ID NO: 3 in WO 99 / 019467 or SEQ ID NO: 1 herein) or the I181*+G182* deletion and the N193F substitution. In preferred embodiments, the bacterial alpha-amylase is derived from a Bacillus stearothermophilus alpha-amylase (SEQ ID NO: 3 in WO 99 / 019467 or SEQ ID NO: 1 herein) which is truncated to have, for example, about 491 amino acids. The truncated Bacillus stearothermophilus alpha-amylase can also have mutations selected from the group consisting of:

[0019] - V59A+Q89R+E129V+K177L+R179E+I181*+G182*+N193F+H208Y+K220P+N224L+Q254S;

[0020] - E129V+K177L+R179E+I181*+G182*+N193F; and

[0021] - E129V+K177L+R179E+I181*+G182*+N193F+K220P+N224L+S242Q+Q254S.

[0022] In embodiments, the raw starch-hydrolyzing alpha-amylase is of fungal origin. In preferred embodiments, the raw starch-hydrolyzing alpha-amylase is derived from Rhizomucor pusillus and has an Aspergillus niger glucoamylase linker and SBD. In specific embodiments, the raw starch-hydrolyzing alpha-amylase is a variant of Rhizomucor pusillus alpha-amylase having an Aspergillus niger glucoamylase linker and SBD and further having one or more of the following substitutions: G128D, D143N, K192R, such as G128D+D143N or G128D+D143N+K192R (using SEQ ID NO: 14 herein for numbering).

[0023] In another embodiment, the raw starch-hydrolyzing alpha-amylase is derived from Aspergillus, such as Aspergillus niger alpha-amylase having an Aspergillus kawachii linker and SBD or Aspergillus kawachii alpha-amylase itself.

[0024] In embodiments, the sugar-source generating enzyme present and / or added during liquefaction is different from the sugar-source generating enzyme present and / or added during saccharification and / or fermentation.

[0025] A particularly contemplated sugar-source generating enzyme is a glucoamylase. In preferred embodiments, the glucoamylase added during liquefaction is from Penicillium, particularly a Penicillium oxalicum strain disclosed as SEQ ID NO: 2 in PCT / CN10 / 071753, published as WO 2011 / 127802, which is incorporated herein by reference, or SEQ ID NO: 9 or SEQ ID NO: 15 herein, or a protease-stable protein engineered variant of the Penicillium oxalicum glucoamylase having a K79V substitution disclosed in co-pending US Application No. 61 / 531,189 or US Application No. 61 / 566,046 or PCT / US12 / 053779.

[0026] In preferred embodiments, the saccharogenic enzyme is a variant of Penicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in PCT / CN10 / 071753 published as WO 2011 / 127802 and shown herein as SEQ ID NO: 9 and SEQ ID NO: 15 having a K79V substitution (numbering using the mature sequence shown in SEQ ID NO: 15). The K79V glucoamylase variant has reduced sensitivity to protease degradation relative to the parent disclosed in co-pending US Application No. 61 / 531,189 and US Application No. 61 / 566,046 or PCT / US12 / 053779, which are incorporated herein by reference.

[0027] In preferred embodiments, the saccharogenic enzyme present and / or added during saccharification and / or fermentation, e.g., SSF, is a fungal-derived glucoamylase, preferably from a strain of Aspergillus, preferably Aspergillus niger, Aspergillus awamori, or Aspergillus oryzae; or a strain of Trichoderma, preferably T. reesei; or a strain of Talaromyces, preferably T. emersonii. In embodiments, the glucoamylase present and / or added during saccharification and / or fermentation can be obtained from a strain of Pycnoporus, particularly a Pycnoporus strain described in WO 2011 / 066576 (Novozymes), or from a strain of Gloephyllum, particularly a Gloephyllum strain described in WO 2011 / 068803 (Novozymes), or a strain of Nigrofomes, particularly a Nigrofomes strain disclosed in PCT / US10 / 058375 published as WO 2012 / 064351 (Novozymes).

[0028] In a second aspect, the present application relates to a composition comprising:

[0029] - a bacterial alpha-amylase;

[0030] - a raw starch-hydrolyzing alpha-amylase;

[0031] - a saccharogenic enzyme having at least 70% thermostability at 70°C, pH 5.3.

[0032] In embodiments of the present application, a protease, e.g., a metalloprotease, and / or a pullulanase is included in the composition. Other enzymes can also be included.

[0033] Examples of suitable bacterial α-amylases, starch-hydrolyzing α-amylases, and glycogen-producing enzymes, particularly glucosylamylases, can be found below in the “Enzymes” section.

[0034] This invention also relates to the following items:

[0035] 1. A method for producing fermentation products, comprising the following steps:

[0036] a) Use

[0037] - Bacterial α-amylase;

[0038] -α-amylase hydrolyzes raw starch;

[0039] - A glycogen-producing enzyme with at least 70% thermal stability at 70°C and pH 5.3.

[0040] The starch-containing material is liquefied at a temperature of 60–80°C.

[0041] b) Saccharification using glycogen-producing enzymes;

[0042] c) Fermentation using fermentation organisms.

[0043] 2. The method according to Project 1, wherein the fermentation product is an alcohol, preferably ethanol, particularly fuel ethanol, potable ethanol and / or industrial ethanol.

[0044] 3. The method according to Project 1 or 2, wherein the bacterial α-amylase is derived from a strain of Bacillus (also known as Geobacillus).

[0045] 4. The method according to any one of items 1 to 3, wherein the raw starch hydrolytic α-amylase is of fungal origin, preferably a variant of Rhizomucor pusillus α-amylase having an Aspergillus niger glucosylase linker with SBD and one or more of the following mutations: G128D, D143N, K192R, such as G128D+D143N or G128D+D143N+K192R (numbered using SEQ ID NO:14 herein).

[0046] 5. The method according to any one of items 1 to 4, wherein the glycogen-generating enzyme is a glucosylamylase having a thermostability of at least 75%, preferably at least 80%, and more preferably at least 85% at 70°C and pH 5.3.

[0047] 6. The method according to any of items 1-5, wherein the saccharogenic enzyme is a glucoamylase having a relative activity of at least 80%, preferably at least 85%, preferably at least 90% at pH 4.5.

[0048] 7. The method according to any of items 1-6, wherein the saccharogenic enzyme is a glucoamylase having a pH stability of at least 80%, at least 85%, at least 90%, at least 95%, at least 100% at pH 4.5.

[0049] 8. The method according to any of items 1-7, wherein the saccharogenic enzyme is a glucoamylase, preferably from a strain of Penicillium, in particular a strain of Penicillium oxalicum disclosed as SEQ ID NO: 9 and 15 herein, or a variant thereof having a substitution of K79V (numbering using SEQ ID NO: 15 herein).

[0050] 9. The method according to item 8, wherein the glucoamylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the mature polypeptide shown in SEQ ID NO: 9 herein.

[0051] 10. The method according to any of items 1-9, further wherein a protease is present or added during liquefaction.

[0052] 11. The method according to any of items 1-10, further wherein a pullulanase is present during liquefaction and / or saccharification.

[0053] 12. A composition comprising:

[0054] - a bacterial alpha-amylase;

[0055] - a raw starch-hydrolyzing alpha-amylase;

[0056] - a saccharogenic enzyme having a thermal stability of at least 70% at 70°C, pH 5.3.

[0057] 13. The composition according to item 12, wherein the alpha-amylase is derived from a strain of the genus Bacillus, such as a strain of Bacillus stearothermophilus or Geobacillus stearothermophilus, in particular a variant of Bacillus stearothermophilus or Bacillus stearothermophilus alpha-amylase, such as the one shown herein as SEQ ID NO: 1.

[0058] 14. The composition according to item 12 or 13, wherein the raw starch hydrolyzing alpha-amylase is of fungal origin, preferably a variant of Rhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylase linker with SBD and additionally one or more of the following mutations: G128D, D143N, K192R, such as

[0059] G128D+D143N or G128D+D143N+K192R (numbering using SEQ ID NO: 14 herein).

[0060] 15. The composition according to any of items 12-14, wherein the carbohydrate source generating enzyme is a glucoamylase, preferably a glucoamylase having a thermal stability of at least 75%, preferably at least 80%, preferably at least 85% at 70°C, pH 5.3.

[0061] 16. The composition according to any of items 12-15, wherein the carbohydrate generating enzyme is a glucoamylase having a relative activity of at least 80%, preferably at least 85%, preferably at least 90% at pH 4.5.

[0062] 17. The composition according to any of items 12-16, wherein the carbohydrate generating enzyme is a glucoamylase having a pH stability of at least 80%, at least 85%, at least 90%, at least 95%, at least 100% at pH 4.5.

[0063] 18. The composition according to any of items 12-17, wherein the carbohydrate source generating enzyme is a glucoamylase, preferably derived from a strain of the genus Penicillium, in particular a strain of Penicillium oxalicum disclosed as SEQ ID NO: 9 and 15 herein, or a variant thereof having a K79V substitution (numbering using SEQ ID NO: 15 herein).

[0064] 19. The composition according to any of items 12-18, wherein the glucoamylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the mature polypeptide shown in SEQ ID NO: 9 herein.

[0065] 20. The composition according to any of items 12-19, further comprising a protease and / or a pullulanase. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1 Ethanol concentration after liquefaction using B. stearothermophilus alpha-amylase variant, raw starch hydrolyzing alpha-amylase variant from Rhizomucor pusillus and glucoamylase variant of Penicillium oxalicum followed by SSF 54 hours using E. emersonii glucoamylase and Saccharomyces cerevisae at 75°C and pH 4.8 is shown. DETAILED DESCRIPTION

[0067] The present invention relates to a method for producing a fermentation product, such as in particular ethanol, from starch-containing material using a fermenting organism.

[0068] The inventors of the present invention have shown that the method of the present invention has several advantages. Example 5 shows an increased yield in an ethanol process, wherein in the ethanol process a combination of bacterial alpha-amylase, raw starch degrading alpha-amylase and glucoamylase is present during liquefaction at 75°C and pH 4.8, followed by 54 hours of simultaneous saccharification and fermentation (SSF) using Saccharomyces cerevisae.

[0069] In a first aspect, the present invention relates to a method for producing a fermentation product, such as ethanol, from starch-containing material, comprising the steps of:

[0070] i) liquefying the starch-containing material using

[0071] - a bacterial alpha-amylase;

[0072] - a raw starch hydrolyzing alpha-amylase;

[0073] - a sugar source generating enzyme having at least 70% thermostability at 70°C, pH 5.3,

[0074] liquefying the starch-containing material at a temperature between 60-80°C;

[0075] ii) saccharifying using the sugar source generating enzyme;

[0076] iii) fermenting using a fermenting organism.

[0077] In embodiments, the process of the application further comprises the following steps prior to liquefaction step i):

[0078] a) reducing the particle size of the starch-containing material, preferably by dry milling;

[0079] b) forming a slurry comprising the starch-containing material and water.

[0080] The slurry can comprise 10 to 55 w / w-% dry solids (DS), preferably 25 to 45 w / w-% dry solids (DS), more preferably 30 to 40 w / w-% dry solids (DS) of the starch- containing material.

[0081] The starch-containing material used as starting material is typically reduced in particle size, e.g. by milling, to open up the structure and allow further processing.

[0082] Generally, there are two types of milling: wet milling and dry milling. In dry milling, the whole kernel is ground and used. Wet milling gives a good separation of germ and meal (starch granules and proteins) and is typically used for the production of e.g. syrups using starch hydrolysates. Both dry and wet milling are well known in the art. According to the present application, dry milling is preferred. In embodiments, the particle size is reduced to 0.05 to 3.0 mm, preferably 0.1 to 0.5 mm, or such that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material can pass through a sieve having a mesh size of 0.05 to 3.0 mm, preferably 0.1 to 0.5 mm. In another embodiment, at least 50%, preferably at least 70%, more preferably at least 80%, in particular at least 90% of the starch-containing material can pass through a sieve having a mesh size of #6.

[0083] The pH during liquefaction step i) is typically 4 to 6, preferably 4.5 to 5.0 or 4.5 to 4.8 or 5 to 6. The temperature during liquefaction can be 70 to 80°C, e.g. 75 to 80°C, preferably about 75°C. Typically, the starch-containing material is heated during liquefaction step (i) for 0.1 to 10 hours, e.g. 1 to 3 hours, e.g. about 1.5 hours.

[0084] The bacterial alpha-amylase, raw starch-hydrolyzing alpha-amylase and saccharogenic enzyme, in particular glucoamylase, and optionally the protease and / or the pullulanase can be added to the aqueous slurry to initiate liquefaction (thinning). In embodiments, a part of the enzyme blend is added to the aqueous slurry, while the remaining enzymes are added in the liquefaction step i).

[0085] In embodiments, the aqueous slurry can be jet-cooked, thereby further pasting the slurry prior to liquefaction in step i). The jet-cooking can be performed at a temperature of 95-145°C, such as 105-125°C, such as 110-145°C, preferably 120-140°C, such as 125-135°C, preferably about 130°C, for about 1-15 minutes, preferably about 3-10 minutes, in particular about 5 minutes.

[0086] In embodiments, saccharification step ii) and fermentation step iii) are performed sequentially or simultaneously. In preferred embodiments, steps ii) and iii) are performed simultaneously (SSF process). In preferred embodiments, a saccharogenic enzyme, preferably a glucoamylase, is added. The saccharogenic enzyme, such as a glucoamylase, can be different from the enzyme added in liquefaction step i).

[0087] In embodiments, saccharification step ii) is performed at a temperature of 20-75°C, preferably 40-70°C, such as about 60°C, at a pH of 4-5, such as about pH 4.5.

[0088] Furthermore, fermentation step iii) or simultaneous saccharification and fermentation (SSF) can be performed at a temperature of 25-40°C, such as 28-35°C, such as 30-34°C, preferably about 32°C, wherein the fermentation is performed for 6-120 hours, in particular 24-96 hours, such as about 54 hours.

[0089] Examples of suitable bacterial alpha-amylases can be found in the section "Bacterial alpha-amylases" below. In preferred embodiments, the bacterial alpha-amylase is a Bacillus alpha-amylase, preferably from a Bacillus stearothermophilus strain, in particular the one shown in SEQ ID NO: 3 in WO99 / 019467 or herein SEQ ID NO: 1, such as truncated, e.g. having about 491 amino acids, such as 485-495 amino acids. The Bacillus stearothermophilus alpha-amylase can also be a variant, such as one of the ones listed below and / or disclosed in WO 201 1 / 082425 (incorporated by reference).

[0090] Examples of raw starch-hydrolyzing alpha-amylases can be found in the section "Raw starch-hydrolyzing alpha-amylases" below. In embodiments, the raw starch-hydrolyzing alpha-amylase is of fungal origin. In preferred embodiments, the raw starch-hydrolyzing alpha-amylase is derived from a Rhizomucor pusillus alpha-amylase and has an Aspergillus niger glucoamylase linker and SBD. In preferred embodiments, the raw starch-hydrolyzing alpha-amylase is a variant of the above having one of the following substitutions: G128D + D143N or G128D + D143N + K192R (using SEQ ID NO: 14 herein for numbering).

[0091] Examples of saccharogenic enzymes, including in particular glucoamylases, can be found in the section "Saccharogenic enzymes" below. The saccharogenic enzymes have a thermostability of at least 70%, such as at least 75%, preferably at least 80%, preferably at least 85% at 70°C and pH 5.3.

[0092] In a preferred embodiment, the glucoamylase is from Penicillium, in particular from the Penicillium oxalicum strain disclosed as SEQ ID NO: 2 in PCT / CN10 / 071753 published as WO 2011 / 127802 (incorporated herein by reference) and shown herein as SEQ ID NO: 9 and SEQ ID NO: 15, or a protease-stable protein engineered variant of the Penicillium oxalicum glucoamylase disclosed in co-pending US application 61 / 531,189 or US application 61 / 566,046 or PCT / US12 / 053779 and having the substitution K79V.

[0093] In an embodiment, a protease is also present during the liquefaction step i). Examples of proteases can be found in the section "Proteases" below. In an embodiment, the protease is a metalloprotease. In a preferred embodiment, the protease is derived from Thermoascus, preferably from a strain of Thermoascus aurantiacus, in particular Thermoascus aurantiacus CGMCC No. 0670 (classified as EC 3.4.24.39) disclosed in SEQ ID NO: 3 herein or amino acids 1-177 (mature polypeptide) of SEQ ID NO: 1 of WO 2010 / 008841.

[0094] Starch-containing material

[0095] According to the present application, any suitable starch-containing material can be used. The starting material is typically selected based on the desired fermentation product. Examples of starch-containing materials suitable for use in the process of the present application include whole grains, corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, beans or sweet potatoes, or mixtures thereof, or starches derived therefrom, or cereals. Both waxy and non-waxy types of corn and barley are encompassed. Preferred starch-containing materials are corn and wheat.

[0096] Saccharification and fermentation

[0097] One or more saccharogenic enzymes, in particular glucoamylases, are present and / or added during the saccharification step ii) and / or the fermentation step iii). The saccharogenic enzyme can preferably be a glucoamylase, but can also be an enzyme selected from the group consisting of beta-amylases, maltogenic amylases and alpha-glucosidases.

[0098] Examples of saccharide source generating enzymes (including glucoamylases) can be found in the section "Saccharide source generating enzymes present and / or added during saccharification and / or fermentation" below.

[0099] When saccharification and fermentation are performed sequentially, the saccharification in step ii) can be performed using conditions well known in the art. For example, the saccharification step ii) can last for about 24 to about 72 hours. However, typically a pre-saccharification of typically 40-90 minutes at a temperature of 30-65 °C, typically about 60 °C, is performed before the saccharification during fermentation in simultaneous saccharification and fermentation (SSF). The saccharification can be performed at a temperature of 20-75 °C, preferably 40-70 °C, typically about 60 °C, at a pH of 4-5, typically about pH 4.5.

[0100] Simultaneous saccharification and fermentation (SSF) is widely used in industrial scale fermentation product production processes, in particular ethanol production processes. When performing SSF, the saccharification step ii) and the fermentation step iii) are performed simultaneously. There is no hold stage for saccharification, meaning that the fermenting organism, e.g. yeast, can be added together with the enzymes. According to the present application, SSF is typically performed at a temperature of 25-40 °C, such as 28-35 °C, such as 30-34 °C, preferably about 32 °C. In embodiments, the fermentation is performed for 6-120 hours, in particular 24-96 hours. In embodiments, the pH is 3.5-5, in particular 3.8-4.3.

[0101] Fermentation medium

[0102] "Fermentation media" or "fermentation medium" refers to the environment in which fermentation is performed and includes the fermentation substrate (i.e. the saccharide source metabolized by the fermenting organism). The fermentation media can comprise nutrients and growth stimulants for the fermenting organism. Nutrients and growth stimulants are widely used in the fermentation art and include nitrogen sources such as ammonia; urea, vitamins and minerals, or combinations thereof.

[0103] Fermenting organism

[0104] The term "fermenting organism" refers to any organism suitable for fermentation in the process of the present application, including bacterial and fungal organisms. The fermenting organism is capable of producing the desired fermentation product. Particularly suitable fermenting organisms are capable of fermenting, i.e. converting, a saccharide such as glucose or maltose, directly or indirectly, into the desired fermentation product, such as ethanol. Examples of fermenting organisms include fungal organisms such as yeast. Preferred yeast, in particular for ethanol production, include Saccharomyces strains, in particular Saccharomyces cerevisiae.

[0105] In one embodiment, the fermenting organism is added to the fermentation medium such that the count of viable fermenting organism, e.g., yeast, per mL of fermentation medium is 10 5 ~ 10 12 , preferably 10 7 ~ 10 10 , and especially about 5 x 10 7 .

[0106] Commercially available yeasts include, for example, RED STAR ETHANOL RED TM Yeast (available from Fermentis / Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART, and THERMOSACC TM Fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM AFT and XR (available from NABC-North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).

[0107] Fermentation product

[0108] The term "fermentation product" refers to a product produced by a process that includes a fermentation step using a fermenting organism. Fermentation products included in accordance with the present application include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B 12and hormones. In a preferred embodiment, the fermentation product is ethanol, such as fuel ethanol; potable ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. Preferred beer types include ales, stouts, porters, lagers, bitters, malt liquor, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer, or light beer. Preferred fermentation processes used include alcohol fermentation processes. The fermentation product is preferably ethanol. The fermentation product, such as ethanol, can be used as a fuel, typically blended with gasoline. However, in the case of ethanol, it can also be used as potable ethanol. In a preferred embodiment, the fermentation product is fuel ethanol.

[0109] Recovery

[0110] Following fermentation, the fermentation product can be separated from the fermentation medium. The broth can be distilled to extract the desired fermentation product. Alternatively, the desired fermentation product can be extracted from the fermentation medium by micro- or membrane filtration techniques. The fermentation product can also be recovered by gas stripping or other methods known in the art.

[0111] Enzyme

[0112] Bacterial alpha-amylase

[0113] According to the present application, bacterial alpha-amylase is present and / or added during liquefaction, together with a raw starch hydrolyzing enzyme and a saccharogene having a thermostability of at least 70%, such as at least 75%, preferably at least 80%, preferably at least 85% at 70°C and pH 5.3.

[0114] Optionally, protease and / or a pullulanase is also present or added during liquefaction.

[0115] The term "bacterial alpha-amylase" refers to any bacterial alpha-amylase classified under EC 3.2.1.1. The bacterial alpha-amylase used according to the present application can for example be obtained from a strain of the genus Bacillus, which is sometimes also referred to as the genus Geobacillus. In a preferred embodiment, the Bacillus alpha-amylase is obtained from a strain of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis, but can also be obtained from other Bacillus.

[0116] Specific examples of bacterial alpha-amylases include the Bacillus amyloliquefaciens alpha-amylase of SEQ ID NO: 5 in WO 99 / 19467, the Bacillus licheniformis alpha-amylase of SEQ ID NO: 4 in WO 99 / 19467, and the Bacillus stearothermophilus alpha-amylase of SEQ ID NO: 3 in WO 99 / 19467 (all sequences incorporated herein by reference). In embodiments, the alpha-amylase can be an enzyme having at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of the sequences shown in SEQ ID NO: 3, 4, or 5 in WO 99 / 19467, respectively.

[0117] In embodiments, the bacterial alpha-amylase can be an enzyme having at least 60%, such as at least 70%, at least 80%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or at least 100% identity to any of the sequences shown in SEQ ID NO: 3 in WO 99 / 19467 or SEQ ID NO: 1 herein.

[0118] Bacillus alpha-amylases can also be variants and / or hybrids. Examples of such variants can be found in any of WO 96 / 23873, WO 96 / 23874, WO 97 / 41213, WO 99 / 19467, WO 00 / 60059, and WO 02 / 10355 (all documents incorporated herein by reference). Specific alpha-amylase variants are disclosed in US Patents 6,093,562, 6,187,576, 6,297,038, and 7,713,723 (incorporated herein by reference) and include Bacillus stearothermophilus alpha-amylase (often referred to as BSG alpha-amylase) variants having one or two amino acid deletions at the R179, G180, 1181, and / or G182 positions, preferably a double deletion as disclosed in WO 96 / 23873 - see e.g. page 20, lines 1-10 (incorporated herein by reference), preferably a deletion corresponding to positions 181 and 182 compared to the amino acid sequence of the Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 of WO 99 / 19467 or herein SEQ ID NO: 1, or a deletion of amino acids R179 and G180 using the numbering of SEQ ID NO: 3 of WO 99 / 19467 or herein SEQ ID NO: 1 (documents incorporated herein by reference). Even more preferred are Bacillus alpha-amylases, in particular Bacillus stearothermophilus alpha-amylases, having a double deletion corresponding to the deletion of positions 181 and 182 and further comprising the N193F substitution (also denoted I181*+G182*+N193F) compared to the wild-type BSG alpha-amylase amino acid sequence shown in SEQ ID NO: 3 of WO 99 / 19467 or herein SEQ ID NO: 1. The bacterial alpha-amylase can also have a substitution at a position corresponding to S239 in the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 99 / 19467, or be an S242 variant of the Bacillus stearothermophilus alpha-amylase of SEQ ID NO: 3 of WO 99 / 19467 or herein SEQ ID NO: 1. In a preferred embodiment, the variant is an S242A, E, or Q variant of the Bacillus stearothermophilus alpha-amylase, preferably an S242Q variant (using the numbering of SEQ ID NO: 1).

[0119] In an embodiment, the variant has an E188 mutation, e.g. an E188P substitution (using the numbering of SEQ ID NO: 1) in the Bacillus stearothermophilus alpha-amylase.

[0120] In preferred embodiments, the bacterial alpha-amylase can be a truncated Bacillus licheniformis alpha-amylase. In particular, the truncation is such that the Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 in WO 99 / 19467 or herein SEQ ID NO: 1 is about 491 amino acids in length.

[0121] Bacterial hybrid alpha-amylase

[0122] The bacterial alpha-amylase can also be a hybrid bacterial alpha-amylase, such as an alpha-amylase comprising the 445 C-terminal amino acid residues of a Bacillus licheniformis alpha-amylase (shown in SEQ ID NO: 4 in WO 99 / 19467) and the 37 N-terminal amino acid residues of an alpha-amylase from Bacillus amyloliquefaciens (shown in SEQ ID NO: 5 in WO 99 / 19467). In preferred embodiments, the hybrid has one or more, in particular all, of the following substitutions: G48A + T49I + G107A + H156Y + A181T + N190F + I201F + A209V + Q264S (using Bacillus licheniformis numbering in SEQ ID NO: 4 of WO 99 / 19467). Also preferred are variants having one or more of the following mutations (or corresponding mutations in other Bacillus alpha-amylases): H154Y, A181T, N190F, A209V, and Q264S and / or a deletion of two residues between positions 176 and 179, preferably a deletion of E178 and G179 (using position numbering in SEQ ID NO: 5 of WO 99 / 19467).

[0123] In preferred embodiments, the bacterial alpha-amylase is the mature part of the chimeric alpha-amylase disclosed in Richardson et al., 2002, The Journal of Biological Chemistry 277(29):26501-26507 (referred to as BD5088) or a variant thereof. This alpha-amylase is identical to the one shown in SEQ ID NO: 2 in WO 2007 / 134207. The mature enzyme sequence starts after the initial "Met" amino acid at position 1.

[0124] In embodiments, the bacterial alpha-amylase is a thermostable bacterial alpha-amylase. In embodiments, the thermostable bacterial alpha-amylase is of the kind disclosed in WO 2011 / 082425 (incorporated herein by reference). In embodiments, the thermostable bacterial alpha-amylase is obtained from a strain of Bacillus (or Geobacillus), in particular a strain of Bacillus stearothermophilus, especially Bacillus stearothermophilus disclosed as SEQ ID NO: 3 in WO 99 / 019467 or herein SEQ ID NO: 1, with the double deletions I181 + G182 and the substitution N193F, and further comprising the mutations:

[0125] - V59A + Q89R + G108A + E129V + K177L + R179E + H208Y + K220P + N224L + Q254S + M284V;

[0126] - V59A + Q89R + G112D + E129V + K177L + R179E + K220P + N224L + Q254S;

[0127] - V59A + Q89R + E129V + K177L + R179E + H208Y + K220P + N224L + Q254S;

[0128] - V59A + Q89R + E129V + K177L + R179E + H208Y + K220P + N224L + Q254S + M284V;

[0129] - V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + D269E + D281N;

[0130] - V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + I270L;

[0131] - V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + H274K;

[0132] - V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + Y276F;

[0133] - V59A + G108A;

[0134] - V59A+G108A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S+M284V;

[0135] - V59A+G108A+S242Q+M284V;

[0136] - V59A+G108A+M284V;

[0137] - V59A+E129V+R157Y+K177L+R179E+K220P+N224L+S242Q+Q254S;

[0138] - V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S;

[0139] - V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S+M284V;

[0140] - V59A+E129V+K177L+R179E+H208Y+M284V;

[0141] - V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;

[0142] - V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+H274K;

[0143] - V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F;

[0144] - V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+D281N;

[0145] - V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;

[0146] - V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+G416V;

[0147] V59A+ E129V+ K177L+ R179E+ K220P+ N224L+ Q254S+ M284T;

[0148] V59A+ E129V+ K177L+ R179E+ K220P+ N224L+ Q254S+ M284T;

[0149] V59A+ H208Y+ K220P+ N224L+ Q254S+ M284V;

[0150] V59A+ M284V;

[0151] A91L+ M96I+ E129V+ K177L+ R179E+ K220P+ N224L+ S242Q+ Q254S;

[0152] G108A+ M284V;

[0153] E129V+ K177L+ R179E;

[0154] E129V+ K177L+ R179E+ K220P+ N224L+ S242Q+ Q254S;

[0155] E129V+ K177L+ R179E+ K220P+ N224L+ S242Q+ Q254S+ Y276F+ L427M;

[0156] E129V+ K177L+ R179E+ K220P+ N224L+ S242Q+ Q254S+ M284T;

[0157] E129V+ K177L+ R179E+ K220P+ N224L+ S242Q+ Q254S+ N376*+ I377*;

[0158] E129V+ K177L+ R179E+ K220P+ N224L+ Q254S;

[0159] E129V+ K177L+ R179E+ K220P+ N224L+ Q254S+ M284T;

[0160] E129V+ K177L+ R179E+ S242Q;

[0161] E129V+ K177L+ R179E+ M284V;

[0162] E129V+ K177L+ R179V+ K220P+ N224L+ S242Q+ Q254S;

[0163] - K220P + N224L + S242Q + Q254S;

[0164] - K220P + N224L + Q254S;

[0165] - S242Q + M284V;

[0166] - M284V.

[0167] In an embodiment, the bacterial alpha-amylase variant can be an enzyme having at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identity to any of the sequences shown in SEQ ID NO: 3 in WO 99 / 19467 or SEQ ID NO: 1 herein.

[0168] In an embodiment, the bacterial alpha-amylase has a T1 / 2 (min) at pH 4.5, 75°C and 0.12 mM CaCI2 of at least 20, e.g., at least 25, e.g., at least 30, e.g., at least 40, e.g., at least 50, e.g., at least 60, e.g., at least 70, e.g., at least 80, e.g., at least 90, e.g., at least 100, e.g., at least 1 10, e.g., at least 120, e.g., at least 130, e.g., at least 140, e.g., at least 150, e.g., at least 160, e.g., at least 170, e.g., at least 180, e.g., 20-300, e.g., 50-300, e.g., 60-300, e.g., 70-300, e.g., 80-300, e.g., 90-300, e.g., 100-300, e.g., 120-300, e.g., 140-300, e.g., 160-300, e.g., 180-300.

[0169] The bacterial alpha-amylase is typically added in an amount of 0.0005-5 KNU / g DS, preferably 0.001-1 KNU / g DS, e.g., about 0.06 KNU / g DS or 0.0005-5 KNU(S) / g DS, preferably 0.001-1 KNU(S) / g DS, e.g., about 0.060 KNU(S) / g DS, if it is a B. stearothermophilus alpha-amylase.

[0170] Examples of commercially available compositions comprising a bacterial alpha-amylase include BAN TM , TERMAMYL TM SC, LIQUOZYME TM X, LIQUOZYME TM SC, (Novozymes), SPEZYME TMFRED, SPEZYME TM AA, SPEZYME TM DELTA AA, GC358, GC980 and SPEZYME TM RSL (Danisco A / S), and FUELZYME by Verenium, USA TM .

[0171] Raw starch hydrolyzing alpha-amylase

[0172] In the process of the application, a raw starch hydrolyzing alpha-amylase is present during the liquefaction step i) together with a bacterial alpha-amylase and a saccharogenic enzyme having a thermostability of at least 70%, such as at least 75%, preferably at least 80%, preferably at least 85% at 70°C and pH 5.3.

[0173] Optionally, a protease and / or a pullulanase is also present or added during liquefaction.

[0174] As used herein, "raw starch hydrolyzing alpha-amylase" means an alpha-amylase capable of degrading raw starch granules directly at temperatures below the gelatinization temperature of starch. The gelatinization temperature of starch can be between 51 °C and 78 °C, and the initial gelatinization temperature can vary from about 51 °C to 68 °C.

[0175] The raw starch hydrolyzing alpha-amylase can be of any origin. In a preferred embodiment, the raw starch hydrolyzing alpha-amylase is derived from a fungal organism, such as a filamentous fungus.

[0176] In an embodiment, the raw starch hydrolyzing alpha-amylase is derived from an Aspergillus strain, such as A. niger or A. candidus.

[0177] In a preferred embodiment, the fungal acid raw starch hydrolyzing alpha-amylase is a hybrid alpha-amylase.

[0178] In an embodiment, the raw starch hydrolyzing enzyme is a hybrid enzyme comprising an amino acid sequence of a catalytic module having alpha-amylase activity and an amino acid sequence of a carbohydrate binding module, and optionally a linker, wherein the catalytic module is of fungal origin. Particular examples of such enzymes are the enzymes specifically disclosed in Table 3 and Table 4 of WO 2005 / 003311. Raw starch hydrolyzing enzymes include those in the following table:

[0179] Variants Catalytic module Linker SBD JA001 Aspergillus niger AA (SP288) Aspergillus candidus AA Aspergillus candidus AA JA002 SP288 Aspergillus candidus AA Aspergillus niger AMG JA003 SP288 Aspergillus candidus AA Aspergillus emersonii AMG JA004 SP288 Aspergillus candidus AA Athelia rolfsii AMG JA005 SP288 Aspergillus candidus AA Bacillus sp. MA JA007 Aspergillus candidus AA Aspergillus candidus AA Aspergillus candidus AA JA008 SP288 Aspergillus niger AMG Aspergillus niger AMG JA009 SP288 Athelia rolfsii AMG Aspergillus niger AMG JA010 SP288 PEPT Aspergillus niger AMG JA011 SP288 Athelia rolfsii AMG Athelia rolfsii AMG JA012 SP288 Aspergillus candidus AA Aspergillus niger AMG + Athelia rolfsii AMG

[0180] In a preferred embodiment, the raw starch hydrolyzing enzyme has an A. niger catalytic domain and an A. candidus alpha-amylase (AA) or a Roerich A. tuba glucoamylase (AMG) SBD.

[0181] In another preferred embodiment, the raw starch-degrading enzyme is an A. candidus alpha-amylase.

[0182] Other specific examples of raw starch-hydrolyzing hybrid alpha-amylases of interest include those disclosed in WO 2006 / 069290, particularly the M. minutissimus alpha-amylase with the A. niger glucoamylase linker and SBD (SEQ ID NO: 101 in US Application No. 60 / 638,614).

[0183] In a preferred embodiment, the raw starch-hydrolyzing alpha-amylase is a hybrid alpha-amylase consisting of the M. minutissimus alpha-amylase with the A. niger glucoamylase linker and SBD disclosed as V039 in Table 5 of WO 2006 / 069290 (Novozymes A / S).

[0184] Also included is the M. giganteus alpha-amylase with the A. roxburghii glucoamylase linker and SBD (SEQ ID NO: 102 in US Application No. 60 / 638,614).

[0185] In another embodiment, the raw starch-hydrolyzing alpha-amylase is a hybrid alpha-amylase consisting of the M. giganteus alpha-amylase disclosed in WO 2006 / 069290 (Novozymes A / S).

[0186] The raw starch-hydrolyzing alpha-amylase used in Example 5 herein is a variant of the M. minutissimus alpha-amylase disclosed in co-pending US Provisional Application No. 61 / 505,192, which is incorporated herein by reference.

[0187] RSH AA 96 is a M. minutissimus alpha-amylase variant with the A. niger glucoamylase linker and SBD and the following substitutions: G128D + D143N (using the numbering of SEQ ID NO: 14 herein).

[0188] RSH AA 101 is a M. minutissimus alpha-amylase variant with the A. niger glucoamylase linker and SBD and the following substitutions: G128D + D143N + K192R (using the numbering of SEQ ID NO: 14 herein).

[0189] In one embodiment, α-amylase hydrolyzing raw starch is defined as an enzyme having a raw starch degradation index of at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, or at least 2, wherein the raw starch degradation index is the ratio of the activity degrading raw starch to the activity degrading gelatinized starch (Ra / Ga). Preferably, α-amylase hydrolyzing raw starch is defined as an enzyme having a raw starch degradation index greater than 1. The activity against gelatinized starch is determined by measuring the release of glucose produced by the enzyme in a 2% gelatinized (e.g., corn) starch reaction mixture. The activity is determined by the release of reducing sugars produced per hour at 4 mol per mg of pure active enzyme. Subsequently, the same assay can be used to measure the enzyme activity against raw starch, but instead of 2% gelatinized (e.g., corn) starch, 2% raw (e.g., corn) starch is used. In both assays, the temperature is 40°C, the same pH and buffer are used, and the incubation time is 6 hours, as described in the “Materials and Methods” section below.

[0190] The starch-hydrolyzing α-amylase used in this invention also includes α-amylases with high sequence identity to the starch-hydrolyzing α-amylases described herein. In embodiments, the starch-hydrolyzing α-amylase has an amino acid sequence identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the starch-hydrolyzing α-amylases disclosed herein. For example, raw starch hydrolytic α-amylases include hybrid α-amylases that have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the α-amylase disclosed in Table 5 of WO 2006 / 069290 or SEQ ID NO:13 or 14 herein.

[0191] In one embodiment, α-amylase for hydrolyzing raw starch can be added in the liquefaction step i) at an amount of 0.01 to 1000 micrograms of enzyme protein (EP) per g DS, for example, 0.1 to 500 micrograms of EP per g DS, for example, 1 to 200 micrograms of EP per g DS, for example, 1 to 100 micrograms of EP per g DS.

[0192] Saccharogenic enzyme

[0193] According to the present application, a saccharogenic enzyme (preferably a glucoamylase) having a thermostability of at least 70%, such as at least 75%, preferably at least 80%, preferably at least 85% at 70°C and pH 5.3 is present and / or added during liquefaction, together with bacterial alpha-amylase and raw starch hydrolyzing alpha-amylase. Protease and / or pullulanase can also be present and / or added during liquefaction step i).

[0194] The term "saccharogenic enzyme" includes any enzyme that generates a fermentable sugar. The saccharogenic enzyme is capable of producing a sugar that can be used as an energy source for the fermenting organism in question, for example in a process for the production of a fermentation product, such as ethanol, according to the present application. The sugar produced can be converted, directly or indirectly, into the desired fermentation product, preferably ethanol. According to the present application, mixtures of saccharogenic enzymes can be used. Specific examples include glucoamylases (as glucose generators), beta-amylases and maltogenic amylases (as maltose generators).

[0195] In embodiments, the saccharogenic enzyme, preferably a glucoamylase, has a thermostability of at least 70%, such as at least 75%, preferably at least 80%, preferably at least 85% at 70°C, pH 5.3.

[0196] In embodiments, the saccharogenic enzyme, preferably a glucoamylase, has a relative activity of at least 80%, preferably at least 85%, preferably at least 90% at pH 4.5, as determined according to Example 4 (pH optimum conditions).

[0197] In embodiments, the saccharogenic enzyme, preferably a glucoamylase, has a pH stability of at least 80%, at least 85%, at least 90%, at least 95%, at least 100% at pH 4.5, as determined according to Example 4 (pH stability).

[0198] In specific and preferred embodiments, the saccharogenic enzyme is a glucoamylase, preferably of fungal origin, preferably of filamentous fungi, such as from a strain of Penicillium, in particular a Penicillium oxalicum strain disclosed as SEQ ID NO: 2 and shown as SEQ ID NO: 9 herein in WO2011 / 127802 (which is incorporated herein by reference) or a Penicillium oxalicum variant with a K79V substitution (numbering using SEQ ID NO: 15 herein) disclosed in co-pending US Application No. 61 / 531,189 or US Application No. 61 / 566,046 or PCT / US12 / 053779. The K79V glucoamylase variant has reduced sensitivity to protease degradation compared to the parent.

[0199] In an embodiment, the glucoamylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the mature polypeptide shown as SEQ ID NO: 2 in PCT / CN10 / 071753 published as WO 201 1 / 127802 or shown as mature polypeptide in SEQ ID NO: 9 herein.

[0200] Protease

[0201] According to the present application, a protease can be present and / or added during liquefaction in step i) together with the bacterial alpha-amylase, raw starch-hydrolyzing alpha-amylase, and the saccharide source generating enzyme (preferably a glucoamylase) having at least 70% thermostability at 70°C and pH 5.3.

[0202] The protease can be any protease. In a preferred embodiment, the protease is an acid protease of microbial origin, preferably of fungal or bacterial origin. Preferred are acid fungal proteases, other proteases can also be used.

[0203] Suitable proteases include microbial proteases, such as fungal and bacterial proteases. Preferred proteases are acid proteases, i.e. proteases characterized by the ability to hydrolyze proteins under acidic conditions at pH 7 or below.

[0204] Acid fungal proteases can be obtained from Aspergillus, Candida, Coriolus, Endothia, Enthomophtra, Irpex, Mucor, Penicillium, Rhizopus, Sclerotium and Torulopsis. In particular, the protease can be obtained from Aspergillus aculeatus (WO 95 / 02044), Aspergillus awamori (Hayashida et al., 1977, Agric. Biol. Chem. 42(5), 927-933), Aspergillus niger (see, e.g., Koaze et al., 1964, Agr. Biol. Chem. Japan 28:216), Aspergillus saitoi (see, e.g., Yoshida, 1954, J. Agr. Chem. Soc. Japan 28:66), or Aspergillus oryzae, e.g., pepA protease; and from Mucor miehei or Mucor pusillus.

[0205] The protease can be a neutral or alkaline protease, e.g., obtained from a Bacillus strain. A specific protease is obtained from Bacillus amyloliquefaciens and has a sequence available in Swissprot under accession number P06832. The protease can have at least 90% sequence identity, e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% identity, to the amino acid sequence disclosed in Swissprot database under accession number P06832.

[0206] The protease can have at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% identity, to the amino acid sequence disclosed as SEQ ID NO: 1 in WO 2003 / 048353 or SEQ ID NO: 3 herein.

[0207] The protease can be a papain-like protease selected from the group of EC 3.4.22. *(Cysteine proteases) proteases, such as EC 3.4.22.2 (Papain), EC 3.4.22.6 (Chymopapain), EC 3.4.22.7 (Ficain), EC 3.4.22.14 (Actinidin), EC 3.4.22.15 (Cathepsin L), EC 3.4.22.25 (Glycyl endopeptidase), and EC 3.4.22.30 (Caricain).

[0208] In an embodiment, the protease is a protease preparation from a strain of Aspergillus, such as Aspergillus oryzae. In another embodiment, the protease is from a strain of Rhizomucor, preferably Rhizomucor miehei. In another embodiment, the protease is a protease preparation, preferably a mixture of a proteolytic preparation from a strain of Aspergillus, such as Aspergillus oryzae, and a protease from a strain of Rhizomucor, preferably Rhizomucor miehei.

[0209] Aspartic proteases are described, for example, in the Handbook of Proteolytic Enzymes, Edited by A.J. Barrett, N.D. Rawlings and J.F. Woessner, Academic Press, San Diego, 1998, Chapter 270. Examples of aspartic proteases include those disclosed, for example, in Berka et al., 1990, Gene 96:313; Berka et al., 1993, Gene 125:195-198; and Gomi et al., 1993, Biosci. Biotech. Biochem. 57:1095-1100, which are incorporated herein by reference.

[0210] The protease can also be a metalloprotease, defined as a protease selected from the group of:

[0211] (a) a protease belonging to EC 3.4.24 (Metalloendopeptidases); preferably EC 3.4.24.39 (Acid protease);

[0212] (b) a metalloprotease belonging to Group M in the above-mentioned handbook;

[0213] (c) a metalloprotease not assigned to a family (name: MX family) or belonging to any of the families MA, MB, MC, MD, ME, MF, MG, MH (defined on pages 989-991 in the above-mentioned handbook);

[0214] (d) other families of metalloproteases (as defined on pages 1448-1452 in the above-mentioned handbook);

[0215] (e) metalloproteases having the HEXXH motif;

[0216] (f) metalloproteases having the HEFTH motif;

[0217] (g) metalloproteases belonging to any of the M3, M26, M27, M32, M34, M35, M36, M41, M43 or M47 family (as defined on pages 1448-1452 of the above Handbook);

[0218] (h) metalloproteases belonging to the M28E family; and

[0219] (i) metalloproteases belonging to the M35 family (as defined on pages 1492-1495 of the above Handbook).

[0220] In other embodiments, the metalloprotease is a hydrolase whose nucleophilic attack on the peptide bond is mediated by a water molecule, which is activated by a divalent metal cation. Examples of divalent cations are zinc, cobalt or manganese. The metal ion can be held in situ by amino acid ligands. The number of ligands can be five, four, three, two, one or zero. In embodiments, the number is two or three, preferably three.

[0221] There is no limitation on the source of the metalloprotease used in the method of the present application. In embodiments, the metalloprotease is classified as EC 3.4.24, preferably EC 3.4.24.39. In one embodiment, the metalloprotease is an acid-stable metalloprotease, such as a fungal acid-stable metalloprotease, such as a metalloprotease from a Thermoascus strain, preferably a T. luteus strain, in particular T. luteus CGMCC No. 0670 (classified as EC 3.4.24.39). In another embodiment, the metalloprotease is from an Aspergillus strain, preferably an A. oryzae strain.

[0222] In one embodiment, the metalloprotease has at least 80%, at least 82%, at least 85%, at least 90%, at least 95%, or at least 97%, such as at least 98%, such as at least 99% sequence identity to amino acids -178 to -177, -159 to -177, or preferably to amino acids 1 to 177 (mature polypeptide) of WO 2010 / 008841 SEQ ID NO: 1 or herein SEQ ID NO: 3 (T. luteus metalloprotease); and has metalloprotease activity. In specific embodiments, the metalloprotease consists of an amino acid sequence identical to the above-mentioned SEQ ID NO: 1 or herein shown as SEQ ID NO: 3.

[0223] The metalloprotease from H. flavescens is a preferred example of a metalloprotease suitable for use in the methods of the present application. Another metalloprotease is derived from A. oryzae and comprises the sequence of SEQ ID NO: 11 or amino acids -23 to 353; -23 to 374; -23 to 397; 1 to 353; 1 to 374; 1 to 397; 177 to 353; 177 to 374; or 177 to 397 of SEQ ID NO: 11 disclosed in WO 2003 / 048353; or the sequence of SEQ ID NO: 10 disclosed in WO 2003 / 048353.

[0224] Another metalloprotease suitable for use in the methods of the present application is the A. oryzae metalloprotease comprising SEQ ID NO: 5 of WO 2010 / 008841, or the metalloprotease is an isolated polypeptide having at least about 80%, at least 82%, at least 85%, at least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 5; and has metalloprotease activity. In a specific embodiment, the metalloprotease consists of the amino acid sequence of SEQ ID NO: 5 of WO 2010 / 008841, incorporated herein by reference.

[0225] In a specific embodiment, the amino acid sequence of the metalloprotease differs from the amino acid sequence of the H. flavescens or A. oryzae metalloprotease by forty, thirty-five, thirty, twenty-five, twenty, or fifteen amino acids in amino acids -178 to 177, -159 to 177, or +1 to 177.

[0226] In another embodiment, the amino acid sequence of the metalloprotease differs from the amino acid sequence of these metalloproteases by ten, or nine, or eight, or seven, or six, or five amino acids in amino acids -178 to 177, -159 to 177, or +1 to 177, such as by four, three, two, or one amino acid.

[0227] In a specific embodiment, the metalloprotease a) comprises, or b) consists of, the amino acid sequence of:

[0228] i) amino acids -178 to 177, -159 to 177, or +1 to 177 of the amino acid sequence of SEQ ID NO: 1 of WO 2010 / 008841;

[0229] ii) amino acids -23 to 353, -23 to 374, -23 to 397, 1 to 353, 1 to 374, 1 to 397, 177 to 353, 177 to 374, or 177 to 397 of the amino acid sequence of SEQ ID NO: 3 of WO 2010 / 008841;

[0230] iii) the amino acid sequence of SEQ ID NO: 5 of WO 2010 / 008841 ; or

[0231] i), ii) and iii) sequences having protease activity or fragments thereof.

[0232] A fragment of amino acids -178 to -177, -159 to -177, or +1 to -177 of SEQ ID NO: 1 of WO 2010 / 008841 or -23 to 353, -23 to 374, -23 to 397, 1 to 353, 1 to 374, 1 to 397, 177 to 353, 177 to 374, or 177 to 397 of SEQ ID NO: 3 of WO 2010 / 008841 is a polypeptide having one or more amino acids deleted from the amino and / or carboxyl terminus of these amino acid sequences. In one embodiment, the fragment comprises at least 75 amino acid residues, or at least 100 amino acid residues, or at least 125 amino acid residues, or at least 150 amino acid residues, or at least 160 amino acid residues, or at least 165 amino acid residues, or at least 170 amino acid residues, or at least 175 amino acid residues.

[0233] In another embodiment, the metalloprotease is combined with another protease, such as a fungal protease, preferably an acid fungal protease.

[0234] Commercially available products include ESPERASE TM , FLAVOURZYME TM , NOVOZYM TM FM 2.0L and iZyme BA (available from Novozymes A / S, Denmark) and GC106 from Genencor International, Inc., USA TM and SPEZYME TM FAN.

[0235] The protease can be present in an amount of 0.0001 to 1 mg enzyme protein per g DS, preferably 0.001 to 0.1 mg enzyme protein per g DS. Alternatively, the protease can be present in an amount of 0.0001 to 1 LAPU / g DS, preferably 0.001 to 0.1 LAPU / g DS and / or 0.0001 to 1 mAU-RH / g DS, preferably 0.001 to 0.1 mAU-RH / g DS.

[0236] In embodiments, the protease used in the method of the application is a thermostable protease, preferably the one disclosed in WO 2011 / 072191 (incorporated herein by reference), which can have

[0237] i) a thermostability value higher than 20% determined as relative activity at 80°C / 70°C; and / or

[0238] ii) a thermostability value higher than 10% determined as relative activity at 85°C / 70°C.

[0239] In embodiments, the protease has:

[0240] - a thermostability value higher than 30%, higher than 40%, higher than 50%, higher than 60%, higher than 70%, higher than 80%, higher than 90% determined as relative activity at 80°C / 70°C, and / or

[0241] - a thermostability value higher than 12%, higher than 14%, higher than 16%, higher than 18%, higher than 20% determined as relative activity at 85°C / 70°C; and / or

[0242] - a thermostability value higher than 20%, higher than 30%, higher than 40%, higher than 50%, higher than 60%, higher than 70%, higher than 80%, higher than 90% determined as residual activity at 80°C; and / or

[0243] a thermostability value higher than 20%, higher than 30%, higher than 40%, higher than 50%, higher than 60%, higher than 70%, higher than 80%, higher than 90% determined as residual activity at 84°C.

[0244] The purified variants can have a thermostability higher than 90, higher than 100 at 85°C determined using the Zein-BCA assay as disclosed in Example 3.

[0245] The determination of "relative activity" and "residual activity" is determined as described in Example 2.

[0246] In preferred embodiments, the thermostable protease used in the method of the application is a "metalloprotease" defined as a protease belonging to EC 3.4.24 (metallopeptidases), preferably EC 3.4.24.39 (acidic metalloprotease).

[0247] To determine whether a given protease is a metalloprotease, reference is made to the "Handbook of Proteolytic Enzymes" and the principles indicated therein. This determination method can be performed on all types of proteases, naturally occurring or wild-type proteases; or genetically engineered or synthetic proteases.

[0248] Protease activity can be determined using any suitable assay, wherein a substrate is employed, including a peptide bond relevant for the specificity of the protease in question. Likewise, the assay pH and the assay temperature will be suitable for the protease in question. Examples of assay pH values are pH 6, 7, 8, 9, 10 or 11. Examples of assay temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70 or 80 °C.

[0249] Examples of protease substrates are caseins, such as Azurine-cross-linked casein (AZCL-casein). Two protease assays are described in the "Materials & Methods" section below, wherein the so-called "AZCL-casein assay" is the preferred assay.

[0250] In embodiments, the protease has at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 100% of the activity of the JTP196 protease variant or the protease Pfu as determined by the AZCL-casein assay.

[0251] There is no limitation to the origin of the thermostable protease used in the method of the present application, as long as it fulfils the thermostable characteristics defined above. The protease can be, for example, a variant of a wild-type protease, as long as the protease has the thermostable characteristics defined above. In preferred embodiments, the protease is a variant of a metalloprotease as defined above. In embodiments, the protease used in the method of the present application is of fungal origin, such as a fungal metalloprotease, such as a fungal metalloprotease obtained from a Thermoascus strain, preferably a Thermoascus flavus strain, in particular a Thermoascus flavus CGMCC No. 0670 (classified as EC 3.4.24.39).

[0252] In embodiments, the protease is a variant of the mature part of the metalloprotease of SEQ ID NO: 2 disclosed in WO 2003 / 048353 or the mature part of SEQ ID NO: 1 disclosed in WO 2010 / 008841 and SEQ ID NO: 3 disclosed herein, having the following mutations:

[0253] - S5*+N26R+D79L+S87P+A112P+D142L;

[0254] - S5*+D79L+S87P+A112P+D142L;

[0255] - N26R+T46R+D79L+S87P+A112P+D142L;

[0256] -A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;

[0257] -A27K+D79L+Y82F+D104P+A112P+A126V+D142L;

[0258] -A27K+Y82F+S87G+D104P+A112P+A126V+D142L;

[0259] -A27K+D79L+S87P+A112P+T124V+D142L;

[0260] -A27K+D79L+S87P+A112P+A126V+D142L;

[0261] -A27K+D79L+S87P+A112P+D142L.

[0262] -A27K+Y82F+D104P+A112P+A126V+D142L;

[0263] -S36P+D79L+S87P+A112P+D142L;

[0264] -A37P+D79L+S87P+A112P+D142L;

[0265] -S38T+D79L+S87P+A112P+A126V+D142L;

[0266] -T46R+D79L+S87P+T116V+D142L;

[0267] -S49P+D79L+S87P+A112P+D142L;

[0268] -S50P+D79L+S87P+A112P+D142L;

[0269] -S70V+D79L+Y82F+S87G+Y97W+A112P+D142L;

[0270] -S70V+D79L+Y82F+S87G+A112P+D142L;

[0271] -D79L+P81R+S87P+A112P+D142L;

[0272] -D79L+Y82F+S87G+Y97W+D104P+A112P+D142L;

[0273] - D79L+Y82F+S87G+D104P+A112P+D142L;

[0274] - D79L+Y82F+S87G+A112P+A126V+D142L;

[0275] - D79L+Y82F+S87G+A112P+D142L;

[0276] - D79L+Y82F+S87P+A112P+T124V+D142L;

[0277] - D79L+Y82F+S87P+A112P+A126V+D142L;

[0278] - D79L+Y82F+S87P+A112P+D142L;

[0279] - D79L+S87P+N98C+A112P+G135C+D142L;

[0280] - D79L+S87P+D104P+A112P+D142L;

[0281] - D79L+S87P+A112P+T124V+A126V+D142L;

[0282] - D79L+S87P+A112P+T124V+D142L;

[0283] - D79L+S87P+A112P+D142L;

[0284] - D79L+S87P+A112P+D142L+T141C+M161C;

[0285] - Y82F+S87G+S70V+D79L+D104P+A112P+D142L;

[0286] - Y82F+S87G+D79L+D104P+A112P+A126V+D142L.

[0287] In an embodiment, the thermally stable protease variant has at least 75% identity to the mature part of the polypeptide of SEQ ID NO: 2 disclosed in WO 2003 / 048353 or the mature part of SEQ ID NO: 1 in WO 2010 / 008841 or SEQ ID NO: 3 herein, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% but less than 100% identity.

[0288] In an embodiment, the protease is derived from a strain of the genus Pyrococcus, such as a strain of Pyrococcus furiosus. In an embodiment, the protease is the one shown in SEQ ID NO: 1 in US patent No. 6,358,726-B1 (Takara Shuzo Company). In another embodiment, the protease is the one disclosed in SEQ ID NO: 16 herein, or a protease having at least 80% identity to SEQ ID NO: 1 in US patent No. 6,358,726-B1 or SEQ ID NO: 16 herein, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity. Pyrococcus furiosus protease can be purchased from Takara Bio, Japan.

[0289] Pyrococcus furiosus protease is a thermally stable protease. The commercial Pyrococcus furiosus protease product (Pfu S) from Takara Bio, Japan was found to have a thermal stability of 110% (80°C / 70°C) and 103% (90°C / 70°C) at pH 4.5 as determined in Example 2 herein.

[0290] Pullulanase

[0291] According to the present application, a pullulanase can further be present during liquefaction, saccharification and / or fermentation.

[0292] In an embodiment, a pullulanase is present and / or added during the liquefaction step i).

[0293] In another embodiment, a pullulanase is present and / or added during saccharification or simultaneous saccharification and fermentation (SSF).

[0294] Purinases (EC 3.2.1.41, pullulan 6-glucanohydrolase) are debranching enzymes characterized by their ability to hydrolyze alpha-1,6-glucosidic bonds in, for example, amylopectin and pullulan.

[0295] The pullulanase can be any pullulanase. In embodiments, the pullulanase is a bacterial pullulanase, in particular a pullulanase from a Bacillus strain, in particular from a Bacillus deramificans strain. EP 605,040 discloses such pullulanases from Bacillus deramificans.

[0296] In embodiments, the pullulanase is a variant disclosed in WO 00 / 01796. Pullulanases of interest include the pullulanase from Bacillus amyloderamificans disclosed in U.S. Patent No. 4,560,651 (incorporated herein by reference), the pullulanase disclosed as SEQ ID NO: 2 in WO 01 / 151620 (incorporated herein by reference), Bacillus deramificans disclosed as SEQ ID NO: 4 in WO 01 / 151620 (incorporated herein by reference), and the pullulanase from Bacillus acidopullulyticus disclosed as SEQ ID NO: 6 in WO 01 / 151620 (incorporated herein by reference) and described in FEMS Mic. Let. 115:97-106 (1994).

[0297] Other pullulanases of interest according to the present application include pullulanases from Pyrococcus woesei, in particular Pyrococcus woesei DSM No. 3773 disclosed in WO 92 / 02614.

[0298] In embodiments, the pullulanase is a GH57 family pullulanase. In embodiments, the pullulanase comprises an X47 domain as disclosed in WO 201 1 / 087836 (incorporated herein by reference). More specifically, the pullulanase can be derived from a strain of the genus Thermococcus, including Thermococcus litoralis and Thermococcus hydrothermalis, such as the Thermococcus hydrothermalis pullulanase truncated at the X4 site after the X47 domain shown in SEQ ID NO: 1 1 (i.e., amino acids 1 to 782 of SEQ ID NOS: 1 1 and 12). The pullulanase can also be a hybrid of Thermococcus litoralis and Thermococcus hydrothermalis pullulanases or a hot T. hydrothermalis / T. litoralis hybrid enzyme with a truncated site X4 as disclosed in WO 201 1 / 087836 (incorporated herein by reference) or disclosed herein in SEQ ID NO: 12.

[0299] In another embodiment, the pullulanase is derived from a strain of the genus Thermococcus, such as in particular Thermococcus hydrothermalis. In embodiments, the pullulanase is a variant of Thermococcus hydrothermalis. In embodiments, the pullulanase comprises an X47 domain. In embodiments, the pullulanase is truncated, such as that disclosed in WO 201 1 / 087836. In embodiments, the pullulanase comprises an X46 domain such as that disclosed in WO 201 1 / 076123.

[0300] The pullulanases added during liquefaction and saccharification and / or fermentation can be different. For example, in embodiments, the pullulanase present and / or added during the liquefaction step i) is derived from Thermococcus hydrothermalis, while the pullulanase optionally added during saccharification and / or fermentation is derived from Bacillus deramificans. Pullulanase activity can be determined as NPUN. The assay for determining NPUN is described in the "Materials & Methods" section below.

[0301] According to the present application, the pullulanase can be added in an effective amount, which includes a preferred amount of about 0.0001 to 10 mg of enzyme protein per gram of DS, preferably 0.0001 to 0.10 mg of enzyme protein per gram of DS, more preferably 0.0001 to 0.010 mg of enzyme protein per gram of DS. Pullulanase activity can be determined as NPUN. The assay for determining NPUN is described in the "Materials & Methods" section below.

[0302] Commercially available pullulanase products include PROMOZYME D, PROMOZYME TM D2 (Novozymes A / S, Denmark), OPTIMAX L-300 (Danisco, USA) and AMANO 8 (Amano, Japan).

[0303] Saccharogenic enzyme present and / or added during saccharification and / or fermentation

[0304] According to the present application, a saccharogenic enzyme, preferably a glucoamylase, can be present and / or added during saccharification and / or fermentation. The saccharogenic enzyme can be different from the saccharogenic enzyme, preferably a glucoamylase, present and / or added in the liquefaction step i).

[0305] Glucoamylase

[0306] According to the present application, the glucoamylase present and / or added during saccharification and / or fermentation can be derived from any suitable source, for example from a microorganism or a plant. Preferred glucoamylases are of fungal or bacterial origin, selected from Aspergillus glucoamylases, in particular Aspergillus niger G1 or G2 glucoamylase (Boel et al., 1984, EMBO J. 3(5): 1097-1102) or variants thereof, such as those disclosed in WO 92 / 00381, WO 00 / 04136 and WO 01 / 04273 (from Novozymes, Denmark); Aspergillus awamori glucoamylase disclosed in WO 84 / 02921, Aspergillus oryzae glucoamylase (Agric. Biol. Chem. 55(4): 941-949 (1991)), or variants or fragments thereof. Other Aspergillus glucoamylase variants include variants with enhanced thermal stability: G137A and G139A (Chen et al., 1996, Prot. Eng. 9: 499-505); D257E and D293E / Q (Chen et al., 1995, Prot. Eng. 8: 575-582); N182 (Chen et al., 1994, Biochem. J. 301: 275-281); disulfide bonds, A246C (Fierobe et al., 1996, Biochemistry 35: 8698-8704); and introduction of Pro residues at positions A435 and S436 (Li et al., 1997, Protein Eng. 10: 1199-1204).

[0307] Other glucoamylases include those from Ro's Acremonium (formerly known as Corticium rolfsii) (see US Patent No. 4,727,026 and Nagasaka et al., 1998, "Purification and properties of the raw-starch-degrading glucoamylases from Corticium rolfsii, Appl. Microbiol. Biotechnol. 50:323-330), Talaromyces, particularly from Talaromyces emersonii (WO 99 / 28448), Talaromyces leycettanus (US Patent No. RE32,153), Talaromyces duponti, Talaromyces thermophilus (US Patent No. 4,587,215). In a preferred embodiment, the glucoamylase used in the saccharification and / or fermentation process is the Talaromyces emersonii glucoamylase disclosed in WO 99 / 28448.

[0308] In embodiments, the glucoamylase is a glucoamylase exhibiting at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to the mature enzyme sequence shown as SEQ ID NO: 7 in WO 99 / 28448.

[0309] Bacterial glucoamylases of interest include those from Clostridium, particularly C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO 86 / 01831) and Trametes cingulata, Pachykytospora papyracea; and Leucopaxillus giganteus as disclosed in WO 2006 / 069289; or Peniophora rufomarginata as disclosed in WO 2007 / 124285; or mixtures thereof. According to the present application, also hybrid glucoamylases are included. An example is the hybrid glucoamylase disclosed in WO 2005 / 045018. Specific examples include the hybrid glucoamylases disclosed in Tables 1 and 4 of Example 1 (hybrids incorporated herein by reference).

[0310] In embodiments, the glucoamylase is derived from a strain of the genus Pycnoporus, in particular a strain of the genus Pycnoporus as described in WO 2011 / 066576 (Novozymes), or from a strain of the genus Gloeophyllum, in particular a strain of the genus Gloeophyllum as described in WO 2011 / 068803 (Novozymes), or from a strain of the genus Physisporinus, in particular a strain of the genus Physisporinus as disclosed in PCT / US10 / 058375 published as WO 2012 / 064351 (Novozymes).

[0311] Also included are glucoamylases exhibiting a high identity to any of the above-mentioned glucoamylases, i.e. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the above-mentioned mature enzyme sequences.

[0312] In embodiments, the glucoamylase is a glucoamylase exhibiting at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature enzyme sequence shown as any one of SEQ ID NO: 2, 4 or 6 in WO 2011 / 066576.

[0313] In embodiments, the glucoamylase is a glucoamylase exhibiting at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity to the mature enzyme sequence shown as any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18 in WO 2011 / 068803.

[0314] In embodiments, the carbohydrate-source generating enzyme (e.g. preferably a glucoamylase) can be added in an amount of 0.0001-20 AGU / g DS, preferably 0.001-10 AGU / g DS, in particular 0.01-5 AGU / g DS, such as 0.1-2 AGU / g DS.

[0315] Commercially available compositions comprising glucoamylases include AMG 200L; AMG 300L; SAN TM SUPER, SAN TM EXTRA L, SPIRIZYME TM PLUS, SPIRIZYME TM FUEL, SPIRIZYME TM B4U, SPIRIZYME TMULTRA and AMG TM E (from Novozymes A / S); OPTIDEX TM 300, GC480, GC417 (from Genencor Int.); AMIGASE TM and AMIGASE TM PLUS (from DSM); G-ZYME TM G900, G-ZYME TM and G990 ZR (from Genencor Int.).

[0316] Maltogenic amylase

[0317] The saccharogenic enzyme present and / or added during saccharification and / or fermentation can also be a maltogenic alpha-amylase. A "maltogenic alpha-amylase" (pullulan 1,4-alpha-maltohydrolase, E.C. 3.2.1.133) is capable of hydrolyzing amylose and amylopectin to maltose in the alpha-configuration. A maltogenic amylase from Bacillus stearothermophilus strain NCIB 11837 is commercially available from Novozymes A / S. Maltogenic alpha-amylases are described in US Patents Nos. 4,598,048, 4,604,355 and 6,162,628, which are incorporated herein by reference.

[0318] In a preferred embodiment, the maltogenic amylase can be added in an amount of 0.05-5 mg total protein per gram DS or 0.05-5 MANU / g DS.

[0319] Composition comprising a bacterial alpha-amylase, a raw starch hydrolyzing alpha-amylase and a saccharogenic enzyme

[0320] The composition of the application can be added in the process of the application during the liquefaction step i). The composition of the application comprises a bacterial alpha-amylase, a raw starch-hydrolyzing alpha-amylase and a saccharogenic enzyme. The composition can also comprise a protease and / or a pullulanase and other enzymes.

[0321] Thus, in this aspect, the application relates to a composition comprising

[0322] - a bacterial alpha-amylase;

[0323] - a raw starch-hydrolyzing alpha-amylase;

[0324] - a saccharogenic enzyme having a thermal stability of at least 70% at 70°C and pH 5.3.

[0325] In an embodiment, the bacterial alpha-amylase is derived from a Bacillus sp. starch. Suitable bacterial alpha-amylases are described in the section "Bacterial alpha-amylases" above.

[0326] In embodiments, the bacterial alpha-amylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identity to the polypeptide mature part of SEQ ID NO: 3 disclosed in WO 99 / 019467 or SEQ ID NO: 1 herein.

[0327] In preferred embodiments, the bacterial alpha-amylase is derived from a Bacillus stearothermophilus alpha-amylase, in particular a variant comprising a double deletion corresponding to the deletion of positions 181 and 182 and further comprising a N193F substitution (also denoted I181*+G182*+N193F). The Bacillus stearothermophilus alpha-amylase can be a variant disclosed in WO 201 1 / 082425 or specifically disclosed below.

[0328] A preferred bacterial alpha-amylase is derived from a Bacillus stearothermophilus alpha-amylase as set forth in SEQ ID NO: 1 herein, truncated to have about 491 amino acids and having mutations selected from the group consisting of:

[0329] - V59A+Q89R+E129V+K177L+R179E+I181*+G182*+N193F+H208Y+K220P

[0330] + N224L+Q254S;

[0331] - E129V+K177L+R179E+I181*+G182*+N193F; and

[0332] - E129V+K177L+R179E+I181*+G182*+N193F+K220P+N224L+S242Q+Q254S.

[0333] In embodiments, the bacterial alpha-amylase variant has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% but less than 100% identity to the polypeptide mature part of SEQ ID NO: 3 disclosed in WO 99 / 019467 or SEQ ID NO: 1 herein.

[0334] Suitable raw starch hydrolyzing alpha-amylases are described in the section "Raw starch hydrolyzing alpha-amylases" above.

[0335] In embodiments, the raw starch-hydrolyzing alpha-amylase is of fungal origin. In preferred embodiments, the raw starch-hydrolyzing alpha-amylase is a variant of Rhizomucor pusillus alpha-amylase having an Aspergillus niger glucoamylase linker with SBD and having one or more of the following substitutions: G128D, D143N, K192R, such as, in particular, G128D+D143N or G128D+D143N+K192R (using SEQ ID NO: 14 herein for numbering).

[0336] Suitable carbohydrate-source generating enzymes, preferably glucoamylases, are described in the above "Carbohydrate-source generating enzymes" section.

[0337] In embodiments, the carbohydrate-source generating enzyme is a glucoamylase having a thermal stability of at least 70%, such as at least 75%, preferably at least 80%, preferably at least 85% at 70°C and pH 5.3, is a glucoamylase. In preferred embodiments, the glucoamylase is from Penicillium, in particular a Penicillium oxalicum strain disclosed as SEQ ID NO: 2 in PCT / CN10 / 071753 published as WO 2011 / 127802, which is incorporated herein by reference, or a protease-stable protein engineered variant of a Penicillium oxalicum glucoamylase having a K79V substitution disclosed in co-pending US Application No. 61 / 531,189 or US Application No. 61 / 566,046 or PCT / US12 / 053779 (Novozymes) (using SEQ ID NO: 15 herein for numbering), shown in SEQ ID NO: 9 herein.

[0338] In another embodiment, the carbohydrate-source generating enzyme is a glucoamylase shown in SEQ ID NO: 9 or a glucoamylase having at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 9 herein.

[0339] In embodiments, the composition further comprises a protease. The protease can be of fungal or bacterial origin. Suitable proteases are described in the above "Proteases" section.

[0340] In embodiments, the protease is a metalloprotease. In embodiments, the protease is derived from a strain of the genus Thermoascus, preferably Thermoascus aurantiacus, in particular a Thermoascus aurantiacus strain disclosed in SEQ ID NO: 3 herein or

[0341] Thermoascus aurantiacus CGMCC No. 0670 (classified as EC 3.4.24.39) disclosed in amino acids 1-177 (mature polypeptide) of SEQ ID NO: 1 in WO 2010 / 008841.

[0342] In embodiments, the protease is of the kind shown as SEQ ID NO: 1 in US 6,258,726 or of the kind shown herein as SEQ ID NO: 16.

[0343] In embodiments, the protease is of the kind having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 1 in US Patent No. 6,258,726 or to SEQ ID NO: 16 herein.

[0344] In embodiments, the composition of the application comprises:

[0345] - a bacterial alpha-amylase derived from Bacillus stearothermophilus;

[0346] - a raw starch-hydrolyzing alpha-amylase derived from Rhizomucor pusillus;

[0347] - a saccharogenase having at least 70% thermostability at 70°C and pH 5.3 derived from Penicillium oxalicum.

[0348] In embodiments, the composition further comprises a protease derived from Thermoascus flavus or Pyrococcus furiosus.

[0349] In embodiments, the composition further comprises a pullulanase. Suitable pullulanases are described in the section "Pullulanases" above.

[0350] In embodiments, the pullulanase is derived from the genus Thermococcus, such as the Thermococcus hydrothermalis strain pullulanase shown in SEQ ID NO: 11 and truncated after the X47 domain (i.e. amino acids 1-782 in SEQ ID NO: 11).

[0351] Materials & Methods

[0352] Materials:

[0353] Reference alpha-amylase A: Bacillus stearothermophilus alpha-amylase having mutations I181*+G182*+N193F and truncated to 491 amino acids (numbering using SEQ ID NO: 1 herein).

[0354] Alpha-amylase (BAA 1407)： Bacillus stearothermophilus alpha-amylase having mutations I181*+G182*+N193F and truncated to 491 amino acids (numbering using SEQ ID NO: 1 herein).

[0355] Bacillus stearothermophilus alpha-amylase of SEQ ID NO: 1 having the following substitutions: V59A + Q89R + E129V + K177L + R179E + I181* + G182* + N193F + H208Y + K220P + N224L + Q254S and truncated to 491 amino acids (numbering using SEQ ID NO: 1 herein).

[0356] Alpha-amylase (RSH AA 96): The hybrid alpha-amylase consisting of a M. minutum alpha-amylase with an A. niger glucoamylase linker and SBD, disclosed as V039 in Table 5 of WO 2006 / 069290 (Novozymes A / S) or as SEQ ID NO: 14 herein, and having the following substitutions: G128D + D143N.

[0357] Alpha-amylase (RSH AA 101): The hybrid alpha-amylase consisting of a M. minutum alpha-amylase with an A. niger glucoamylase linker and SBD, disclosed as V039 in Table 5 of WO 2006 / 069290 (Novozymes A / S) or as SEQ ID NO: 14 herein, and having the following substitutions: G128D + D143N + K192R.

[0358] Glucoamylase (AMG 001): Penicillium oxalicum glucoamylase mature portion having a K79V substitution (numbering using SEQ ID NO: 15 herein) disclosed as SEQ ID NO: 2 in PCT / CN10 / 071753 published as WO2011 / 127802 and shown as SEQ ID NO: 9 and 15 herein as disclosed in co-pending US application 61 / 531,189.

[0359] Glucoamylase (AMG SPU): The Talaromyces emersonii glucoamylase disclosed in WO 99 / 28448, having about 20% glucoamylase activity from the O. placenta disclosed in WO 06 / 069289, and side activity from the hybrid alpha-amylase consisting of a M. minutum alpha-amylase with an A. niger glucoamylase linker and SBD, disclosed as V039 in Table 5 of WO 2006 / 069290 (Novozymes A / S) or as SEQ ID NO: 14 herein.

[0360] Corn: The ground corn and backset used in Example 5 were obtained from Corn LP in November 2010. The dry solids (% DS) content of the ground corn and backset were determined to be 86.78% and 7.93%, respectively, by oven drying at 105°C for 3 hours.

[0361] Yeast: RED STAR ETHANOL RED TM Available from Red Star / Lesaffre, USA. Methods

[0362] Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".

[0363] For the purposes of the present invention, the identity between two amino acid sequences and the identity between two nucleotide sequences can be determined by the "align" program, which is a Needleman-Wunsch alignment (i.e. global alignment). This program is used for the alignment of polypeptide as well as nucleotide sequences. The default scoring matrix BLOSUM50 is used for polypeptide alignment and the default identity matrix for nucleotide alignment. The penalty for the first residue gap is -12 for polypeptides and -16 for nucleotides. The penalty for other residue gaps is -2 for polypeptides and -4 for nucleotides.

[0364] "Align" is part of the FASTA package version v20u6 (see Pearson and Lipman, 1988, "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448, and Pearson, 1990, "Rapid and Sensitive Sequence Comparison with FASTP and FASTA," Methods in Enzymology 183:63-98). FASTA protein alignment uses the Smith-Waterman algorithm without restriction on gap size (see "Smith-Waterman algorithm", Smith and Waterman, 1981, J. Mol. Biol. 147:195-197).

[0365] Protease assay

[0366] AZCL-casein assay

[0367] A solution of 0.2% blue substrate AZCL-casein was suspended in Borax / NaH2PO4 buffer (pH 9) while stirring. The solution was dispensed into a microtiter plate (100 microliters per well) while stirring, 30 microliters of enzyme sample were added and the plate was incubated in an Eppendorf Thermomixer at 45°C and 600 rpm for 30 minutes. Denatured enzyme sample (boiled at 100°C for 20 min) was used as a blank. After incubation, the reaction was stopped by transferring the microtiter plate to ice and the colored solution was separated from the solids by centrifugation at 3000 rpm for 5 minutes at 4°C. 60 microliters of supernatant were transferred to a microtiter plate and the absorbance was measured at 595 nm using a BioRad microplate reader.

[0368] pNA-assay

[0369] Fifty microliters of sample containing protease were added to a microtiter plate and the assay was initiated by adding 100 microliters of 1 mM pNA substrate (5 mg dissolved in 100 microliters DMSO and further diluted to 10 mL with Borax / NaH2PO4 buffer pH 9.0). The increase in OD 405 at room temperature was monitored as a measure of protease activity.

[0370] Glucoamylase activity (AGU)

[0371] Glucoamylase activity can be measured in Glucoamylase Units (AGU).

[0372] A Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme that hydrolyzes 1 micromole of maltose per minute under standard conditions of 37°C and pH 4.3, where the substrate is 23.2 mM maltose and the buffer is 0.1 M acetate, with a 5 minute reaction time.

[0373] An automated analyzer system can be used. Mutarotase is added to the glucose dehydrogenase reagent, which causes any alpha-D-glucose present to be converted to beta-D-glucose. The glucose dehydrogenase specifically reacts with beta-D-glucose in the reaction described above to form NADH, which is determined using a photometer at 340 nm as a measure of the original glucose concentration.

[0374] AMG incubation: ]]> ​ Substrate: Maltose 23.2 mM Buffer: Acetate 0.1 M pH: 4.30±0.05 Incubation temperature: 37℃±1 Reaction time: 5 minutes Enzyme working range: 0.5-4.0 AGU / mL

[0375]

[0376]

[0377] A folder describing the assay in more detail (EB-SM-0131.02 / 01) can be obtained from Novozymes A / S, Denmark upon request, which folder is incorporated herein by reference.

[0378] Alpha-amylase activity (KNU)

[0379] Alpha-amylase activity can be determined using potato starch as substrate. The method is based on the breakdown of modified potato starch by the enzyme, which is followed by mixing a sample of the starch / enzyme solution with iodine solution. Initially, a blue-black colour is formed, but during breakdown of the starch, the blue colour fades and gradually turns into a red-brown colour, which is compared with a colour glass standard.

[0380] One Kilo Novo alpha-amylase unit (KNU) is defined as the amount of enzyme which dextrinizes 5260 mg dry substrate Merck Amylum solubile starch under standard conditions (i.e. 37°C + / - 0.05; 0.0003 M Ca 2+ ; and pH 5.6).

[0381] A folder describing the assay in more detail (EB-SM-0131.02 / 01) can be obtained from Novozymes A / S, Denmark upon request, which folder is incorporated herein by reference. EB-SM-0009.02 / 01

[0382] KNU(S) alpha-amylase activity

[0383] KNU(S) is used to determine the activity of Bacillus stearothermophilus alpha-amylase and is described in pages 35-41 of WO 99 / 19467 (incorporated herein by reference).

[0384] Determination of pullulanase activity (NPUN)

[0385] Endo-pullulanase activity expressed in NPUN is measured relative to a Novozymes pullulanase standard. One pullulanase unit (NPUN) is defined as the amount of enzyme which releases 1 micromole of glucose per minute under standard conditions (0.7% red pullulan (Megazyme), pH 5, 40°C, 20 minutes). Activity is determined using red pullulan in NPUN / ml.

[0386] ​Diluted sample or standard, 1 mL, was incubated at 40°C for 2 minutes. 0.5 mL of 2% red prurolan, 0.5 M KCl, 50 mM citric acid pH 5 was added and mixed. The test tube was incubated at 40°C for 20 minutes and the reaction was stopped by adding 2.5 mL of 80% ethanol. The test tube was left at room temperature for 10-60 minutes after which the supernatant was centrifuged at 4000 rpm for 10 minutes. The OD of the supernatant was then measured at 510 nm and the activity was calculated using the standard curve.

[0387] The application is described in more detail in the following examples, which are provided to illustrate the application, but are not intended to limit the scope of the claimed application in any way. All references cited herein are specifically incorporated by reference herein for the portions described therein.

[0388] Raw starch degrading enzyme (Ra / Ga) assay

[0389] The procedure for obtaining raw starch degrading enzyme index (Ra / Ga) values is as follows:

[0390] 1) The assay is performed at a temperature of 40°C.

[0391] 2) First, the pH profile of the enzyme is obtained based on raw starch. The profile is obtained from a plot of % activity vs. pH. The pH optimum is used in the assay.

[0392] 3) Any type of starch can be used, e.g. wheat, corn, barley, rice, etc. In the examples, the raw starch used was corn starch. A 2% solution of raw starch was used. Alternatively, to obtain a gelatinized starch solution, the solution of raw starch is heated above the gelatinization temperature for at least 60 minutes. In the case of corn, the solution of raw starch is heated to 70°C for at least 60 minutes.

[0393] 4) The reaction solution contains gelatinized starch (or raw starch) and a buffer. The composition of the buffer used in the assay differs based on the pH optimum of the enzyme. The buffer composition and concentration must be the same for raw starch activity measurement and gelatinized starch activity measurement.

[0394] 5) The enzyme concentration used in the assay must be the same for both raw starch activity measurements and paste starch activity measurements. 6) Enzyme activity is measured by determining the reducing sugars in the solution. Suitable methods are as follows: Bernfield's method using dinitrosalicylic acid to determine reducing sugars is described in Bernfield, 1955, Methods Enzymology 1 : 149-158, and methods using copper-bicinchoninate to determine reducing sugars are described in Fox et al., 1991, Analytical Biochemistry 195:93-96 or Waffenschmidt et al., 1987, Anal. Biochem. 165:337-340. Prior to determining the reducing sugars, the solution is boiled for 3 minutes and centrifuged to inactivate the enzyme.

[0395] 7) The incubation time for measuring enzyme activity is 6 hours.

[0396] 8) Enzyme activity is expressed as the number of reducing sugars produced per hour per mg of pure active enzyme.

[0397] 9) Activity on paste starch is determined by measuring the release of glucose produced by the enzyme in a 2% paste (e.g., corn) starch reaction mixture, and activity on raw starch is determined by measuring the release of glucose produced by the enzyme in a 2% raw (e.g., corn) starch reaction mixture. Activity is determined by the release of reducing sugars produced at 4 mol per hour per mg of pure active enzyme.

[0398] Examples

[0399] Example 1

[0400] Stability of alpha-amylase variants

[0401] The stability of a reference alpha-amylase (B. stearothermophilus alpha-amylase with mutations 1181*+G182*+N193F, truncated to 491 amino acids (numbering using SEQ ID NO: 1 herein)) and alpha-amylase variants thereof was determined by incubating the reference alpha-amylase and variants at pH 4.5 and 5.5 and temperatures of 75°C and 85°C with 0.12 mM CaCI2, followed by determining the remaining activity using the substrate Ultra Amylase Assay Kit, E33651, Molecular Probes.

[0402] Purified enzyme samples were diluted in enzyme dilution buffer (10 mM acetate, 0.01 % Triton X100, 0.12 mM CaCI2, pH 5.0) to working concentrations of 0.5 and 1 or 5 and 10 ppm (microgram / ml). Twenty microliters of enzyme sample were transferred to a 48-well PCR MTP and 180 microliters of stability buffer (150 mM acetate, 150 mM MES, 0.01 % Triton X100, 0.12 mM CaCI2, pH 4.5 or 5.5) was added to each well and mixed. Assays were performed in duplicate using two concentrations of enzyme. Twenty microliters were removed before incubation at 75°C or 85°C and stored on ice as control samples. Incubation was performed in a PCR machine at 75°C and 85°C. After incubation, samples were diluted to 15 ng / mL in remaining activity buffer (100 mM acetate, 0.01 % Triton X100, 0.12 mM CaCI2, pH 5.5) and 25 microliters of diluted enzyme was transferred to a black 384-MTP. Residual activity was determined using EnzChek substrate by adding 25 microliters of substrate solution (100 microgram / ml) to each well. Fluorescence was determined every minute for 15 minutes using an excitation filter at 485-nm and an emission filter at 555 nm (fluorescence plate reader is Polarstar, BMG). Residual activity was normalized to control samples for each setting.

[0403] Half-life (T1 / 2(min)) was calculated using the equation T1 / 2(min) = T(min)*LN(0.5) / LN(%RA / 100), assuming logarithmic decay, where T is the assay incubation time in minutes and %RA is the % residual activity determined in the assay.

[0404] Using this assay setting, half-life was determined for the reference alpha-amylase and its variants, shown in Table 1.

[0405] Table 1

[0406]

[0407]

[0408]

[0409]

[0410] ND: not determined

[0411] The results show that the alpha-amylase variants have significantly higher half-life and stability than the reference alpha-amylase.

[0412] Example 2

[0413] Preparation of protease variants and testing of thermostability

[0414] The chemicals used are at least reagent grade commercially available products.

[0415] Strains and plasmids:

[0416] E. coli DH12S (available from Gibco BRL) was used for yeast plasmid rescue. pJTP000 is a S. cerevisiae and E. coli shuttle vector under the control of the TPI promoter, constructed from pJC039 described in WO 01 / 92502, in which the Thermoascus flavus M35 protease gene (WO 03 / 048353) has been inserted.

[0417] S. cerevisiae YNG318 competent cells: MATa Dpep4[cir+] ura3-52, leu2-D2, his4-539 for protease variant expression. It is described in J. Biol. Chem. 272(15):9720-9727 (1997).

[0418] Media and substrates

[0419] 10X base solution: Yeast nitrogen base (DIFCO) without amino acids 66.8 g / L, succinate 100 g / l, NaOH 60 g / l.

[0420] SC-glucose : 20% glucose (i.e. final concentration 2% = 2 g / 100 mL) 100 mL / L, 5% threonine 4 mL / L, 1% tryptophan 10 ml / l, 20% casamino acids 25 ml / l, 10X base solution 100 ml / l. The solution was sterilized using a filter with a pore size of 0.20 microns. Agar (2%) and H2O (about 761 mL) were separately autoclaved and sterilized. The SC-glucose solution was added to the agar solution.

[0421] YPD: Bacto peptone 20 g / l, yeast extract 10 g / L, 20% glucose 100 mL / L.

[0422] YPD + Zn : YPD + 0.25 mM ZnSO4.

[0423] PEG / LiAc solution: 40% PEG4000 50 ml, 5 M lithium acetate 1 mL.

[0424] 96-well corn protein microtiter plates:

[0425] Each well contained 200 microliters of 0.05-0.1% Zea mays protein (Sigma), 0.25 mM ZnSO4, and 1% agarose in 20 mM sodium acetate buffer, pH 4.5.

[0426] DNA manipulation

[0427] DNA manipulations and transformations were performed using standard methods of molecular biology unless otherwise indicated, as described in Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab. Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds) "Current protocols in Molecular Biology", John Wiley and Sons, 1995; Harwood, C. R. and Cutting, S. M. (eds).

[0428] Yeast transformation

[0429] Yeast transformations were performed using the lithium acetate method. 0.5 microliters of vector (digested by restriction enzymes) and 1 microliter of PCR fragment were mixed. This DNA mixture, 100 microliters of YNG318 competent cells and 10 microliters of YEAST MAKER carrier DNA (Clontech) were added to a 12 mL polypropylene tube (Falcon 2059). 0.6 mL of PEG / LiAc solution was added and mixed gently. Incubated at 30°C and 200 rpm for 30 minutes, then 42°C for 30 minutes (heat shock). Transferred to eppendorf tube and centrifuged for 5 seconds. Supernatant was removed and dissolved in 3 mL of YPD. The cell suspension was incubated at 200 rpm and 30°C for 45 minutes. The suspension was poured into SC-glucose plates and incubated at 30°C for 3 days to grow colonies. Total yeast DNA was extracted by Zymoprep yeast plasmid miniprep kit (ZYMO research).

[0430] DNA sequencing

[0431] E. coli transformations for DNA sequencing were performed by electroporation (BIO-RAD Gene Pulser). DNA was prepared by alkaline method (Molecular Cloning, Cold Spring Harbor) or using QIAquick PCR purification kit (QIAGEN) according to the manufacturer's instructions. DNA plasmids were prepared using a plasmid kit. DNA fragments were recovered from agarose gels by a Qiagen gel extraction kit. PCR was performed using a PTC-200 DNA Engine. An ABI PRISMTM 310 Genetic Analyzer was used to determine all DNA sequences.

[0432] Construction of protease expression vectors

[0433] The thermophilic ascomycete M35 protease gene was amplified using the primer pair Prot F (SEQ ID NO: 4) and Prot R (SEQ ID NO: 5). The resulting PCR fragment was introduced into S. cerevisiae YNG318 together with a pJC039 vector (described in WO 2001 / 92502) that had been restriction enzyme digested to remove the specific Humicola insolens cutinase gene.

[0434] The plasmid in the yeast clone on SC-glucose plates was recovered to confirm the internal sequence and named pJTP001.

[0435] Construction of yeast library and site-directed variants

[0436] The yeast library and site-directed variants were constructed by SOE PCR (splicing by overlap extension, see "PCR: A practical approach", p. 207-209, Oxford University press, eds. McPherson, Quirke, Taylor) followed by in vivo recombination in yeast.

[0437] Universal primers for amplification and sequencing

[0438] By SOE, the primers AM34 (SEQ ID NO: 6) and AM35 (SEQ ID NO: 7) were used together with degenerate primers (AM34 + reverse primer and AM35 + forward primer) to make DNA fragments containing any mutated fragment, or just to amplify the whole protease gene (AM34 + AM35).

[0439]

[0440] DNA fragments were recovered from agarose gels by a Qiagen gel extraction kit. The resulting purified fragments were mixed with vector digests. The mixed solution was introduced into S. cerevisiae to construct the library or to construct site-directed variants by in vivo recombination.

[0441] Relative activity assay

[0442] Yeast clones on SC-glucose were inoculated into wells of a 96-well microtiter plate containing YPD+Zn media and incubated at 28°C for 3 days. Culture supernatant was applied to a 96-well Zea protein microtiter plate and incubated at at least two temperatures (e.g., 70°C and 80°C) for more than 4 hours or overnight. The turbidity of the Zea protein in the plate was measured as A630 and the relative activity (higher / lower temperature) was determined as an indicator of improved thermal activity. Clones with higher relative activity than the parent variant were selected and the sequence determined.

[0443] Residual activity assay

[0444] Yeast clones on SC-glucose were inoculated into wells of a 96-well microtiter plate and incubated at 28°C for 3 days. Culture supernatant was incubated in 20 mM sodium acetate buffer pH 4.5 at a specific temperature (80°C or 84°C, with 4°C as a reference) for 10 minutes before measuring protease activity using azocasein (Megazyme) at 65°C to determine residual activity. Clones with higher residual activity than the parent variant were selected and the sequence determined.

[0445] Azocasein assay

[0446] Twenty microliters of sample were mixed with 150 microliters of substrate solution (4 mL of 12.5% azocasein in ethanol in 96 mL of 20 mM sodium acetate pH 4.5 containing 0.01% triton-100 and 0.25 mM ZnSO4) and incubated for 4 hours or more.

[0447] After addition of 20 microliters / well of 100% trichloroacetic acid (TCA) solution, the plate was centrifuged and 100 microliters of supernatant were removed to measure A440.

[0448] Expression of protease variants in A. oryzae

[0449] Constructs containing protease variant genes were used to build Aspergillus expression vectors. The Aspergillus expression vectors were composed of an expression cassette based on the Aspergillus niger neutral amylase II promoter fused to the Aspergillus nidulans phosphotriose isomerase untranslated leader sequence (Pna2 / tpi) and the A. niger amyloglucosidase terminator (Tamg). Also present on the plasmid was the Aspergillus selective marker amdS from A. nidulans, which enables growth on acetamide as the sole nitrogen source. The expression plasmids for the protease variants were transformed into Aspergillus as described in Lassen et al., 2001, Appl. Environ. Microbiol. 67:4701-4707. For each construct, 10-20 strains were isolated, purified, and grown in shake flasks.

[0450] Purification of expressed variants

[0451] The pH of the 0.22 pm filtered fermentation sample was adjusted to 4.0.

[0452] The sample was placed on an ice bath with magnetic stirring. A small aliquot of (NH4)2SO4 was added (corresponding to approximately 2.0-2.2 M (NH4)2SO4, not accounting for volume increase upon addition of compound).

[0453] After the final addition of (NH4)2SO4, the sample was incubated on ice bath for a minimum of 45 minutes with gentle magnetic stirring.

[0454] Centrifugation: Hitachi himac CR20G high speed refrigerated centrifuge equipped with R20A2 rotor head, 5°C, 20,000 rpm, 30 min.

[0455] The formed pellet was dissolved in 200 mL 50 mM sodium acetate pH 4.0.

[0456] The sample was filtered using a 0.22 micron PES PLUS membrane (IWAKI) by vacuum suction.

[0457] The sample was desalted / buffer exchanged to 50 mM sodium acetate pH 4.0 using overnight ultrafiltration in a cold room (Vivacell 250 from Vivascience equipped with 5 kDa MWCO PES membrane). The remaining sample was diluted to 200 ml using 50 mM sodium acetate pH 4.0. The conductivity of the sample was preferably less than 5 mS / cm.

[0458] The sample was loaded onto a cation exchange column equilibrated with 50 mM sodium acetate pH 4.0. Unbound sample was washed from the column using 3 column volumes of binding buffer (50 mM sodium acetate pH 4.0) and the sample was eluted using a linear gradient 0-100% elution buffer (50 mM sodium acetate + 1 M NaCl pH 4.0) in 10 column volumes.

[0459] The collected fractions were assayed by endoprotease assay (see below) after which selected fractions were subjected to standard SDS-PAGE (reducing conditions). Fractions were pooled based on endoprotease assay and SDS-PAGE.

[0460] Endoprotease assay

[0461] Protazyme OL tablets / 5 ml of 250 mM sodium acetate pH 5.0 were dissolved by magnetic stirring (substrate: Endoprotease Protazyme AK tablets - cat. # PRAK 11 / 08 from Megazyme).

[0462] Under stirring, 250 microliter of substrate solution was transferred to a 1.5 mL Eppendorf tube.

[0463] 25 microliter of sample was added to each tube (blank was sample buffer).

[0464] The tubes were incubated on a Thermomixer at 50°C with shaking (1000 rpm) for 15 minutes.

[0465] 250 microliter of 1 M NaOH was added to each tube followed by vortexing.

[0466] Centrifugation at 16,100 x G for 3 minutes at 25°C.

[0467] 200 microliter of supernatant was transferred to a MTP and the absorbance at 590 nm was recorded.

[0468]

[0469]

[0470]

[0471]

[0472] Example 3

[0473] Temperature profile of selected protease variants using purified enzymes

[0474] Selected protease variants showing good thermal stability were purified and the purified enzymes were used in the Zein-BCA assay described below. After incubation of the enzymes for 60 minutes at the indicated elevated temperatures, the remaining protease activity was determined at 60°C.

[0475] Corn protein-BCA assay

[0476] The Zein-BCA assay was performed to detect the quantification of soluble protein released from Zein by the variant proteases at different temperatures.

[0477] Instructions:

[0478] 10 microliter of 10 microgram / mL enzyme solution and 100 microliter of 0.025% Zein solution were mixed in a microtiter plate (MTP).

[0479] Incubation for 60 minutes at different temperatures.

[0480] 10 microliter of 100% trichloroacetic acid (TCA) solution was added.

[0481] Centrifuge the MTP at 3500 rpm for 5 minutes.

[0482] Take 15 microliter to a new MTP containing 100 microliter BCA assay solution (Pierce Cat#: 23225, BCA Protein Assay Kit).

[0483] Incubate at 60°C for 30 minutes.

[0484] Measure A562.

[0485] The results are shown in Table 5. All the tested protease variants show improved thermal stability compared to the wild type (WT) protease.

[0486] Table 5 Zein-BCA assay

[0487]

[0488]

[0489] Example 4

[0490] Characterization of oxalic acid Penicillium glucoamylase

[0491] Penicillium oxalicum glucoamylase is disclosed in SEQ ID NO: 9 herein.

[0492] Substrate. Substrate: 1% soluble starch (Sigma S-9765) in deionized water

[0493] Reaction buffer: 0.1 M acetate buffer, pH 5.3

[0494] Glucose concentration determination kit: Wako Glucose Assay Kit (LabAssay glucose, WAKO, Cat# 298-65701).

[0495] Reaction conditions. Mix 20 microliter soluble starch and 50 microliter acetate buffer pH 5.3. Add 30 microliter enzyme solution (50 microgram enzyme protein / ml) to a final volume of 100 microliter, after which incubate at 37°C for 15 minutes.

[0496] Determine glucose concentration by Wako kit.

[0497] All work was performed in parallel.

[0498] Optimum temperature. To assess the optimum temperature of the Penicillium oxalicum glucoamylase, the "Reaction conditions" assay described above was performed at 20, 30, 40, 50, 60, 70, 80, 85, 90 and 95°C. The results are shown in Table 6.

[0499] Table 6 Optimum temperature

[0500] Temperature (°C) 20 30 40 50 60 70 80 85 90 95 Relative activity (%) 63.6 71.7 86.4 99.4 94.6 100.0 92.9 92.5 82.7 82.8

[0501] From the results it can be seen that the optimal temperature for the glucoamylase of Penicillium oxalicum is between 50°C and 70°C and that the glucoamylase retains more than 80% activity at 95°C.

[0502] Thermostability. To assess the thermostability of the glucoamylase of Penicillium oxalicum the reaction condition assay was modified in that the enzyme solution and acetate buffer were pre-incubated for 15 minutes at 20, 30, 40, 50, 60, 70, 75, 80, 85, 90 and 95°C. After the incubation 20 microliter of starch was added to the solution and the assay was performed as described above.

[0503] The results are shown in Table 7.

[0504] Table 7 Thermostability

[0505] Temperature (°C) 20 30 40 50 60 70 80 85 90 95 Relative activity (%) 91.0 92.9 88.1 100.0 96.9 86.0 34.8 36.0 34.2 34.8

[0506] From the results it can be seen that the glucoamylase of Penicillium oxalicum is stable up to 70°C, retaining more than 80% activity after 15 minutes of pre-incubation.

[0507] Optimal pH. To assess the optimal pH of the glucoamylase of Penicillium oxalicum the reaction condition assay described above was performed at pH 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 and 11.0. The following buffers were used to replace the acetate buffer described in the reaction condition assay: 100 mM succinate, HEPES, CHES, CAPSO, 1 mM CaCl2, 150 mM KCl, 0.01% Triton X-100, pH adjusted to 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 or 11.0 with HC1 or NaOH.

[0508] The results are shown in Table 8.

[0509] Table 8. Optimal pH

[0510]

[0511] From the results it can be seen that the glucoamylase of Penicillium oxalicum has the highest activity at pH 5.0 under the given conditions. The glucoamylase of Penicillium oxalicum is active over a broad pH range, retaining more than 50% activity from pH 2 to 7.

[0512] pH stability. To evaluate the thermal stability of the oxalate PenG, the reaction condition assay was modified to use the buffers described in the section on the optimum pH, and the enzyme solution (50 micrograms / mL) was pre-incubated at pH 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 for 20 hours. After pre-incubation, 20 microliters of soluble starch was added to the solution to a final volume of 100 microliters, and the assay was performed as described above.

[0513] The results are shown in Table 9.

[0514] Table 9 pH stability

[0515]

[0516] From the results, it can be seen that the oxalate PenG is stable from pH 3 to pH 7 after pre-incubation for 20 hours, and that it decreases activity at pH 8.

[0517] Example 5

[0518] Improved ethanol production process for liquefaction at 75 °c and ph 4.80

[0519] Ground corn and counter-current were used for this study. The dry solids (% DS) content of the ground corn and counter-current were determined to be 86.78% and 7.93%, respectively, by oven drying at 105°C for 3 hours.

[0520] Mash preparation. Nine different corn slurries were prepared for liquefaction. To 5 x 200 g lab-o-mat jars (Mathis, Inc.) were added 35.29 g ground corn, 37.71 g tap water, and 30.00 g counter-current. The counter-current ratio was set at 30% for all slurries. After the addition of these ingredients, the pH of the slurry was adjusted to 4.80 using 40% H2SO4, if necessary. Deionized water was used to prepare stock solutions of all concentrated enzymes. Aliquots of all enzymes were added to the individual lab-o-mat jars to achieve the final concentrations shown in Table 10. Finally, deionized water was added to the slurries to ensure that the starting DS% was the same for all slurries.

[0521] Table 10

[0522]

[0523] All of the jars were then closed. They were placed into the lab-o-mat and liquefied using the following program (Table 11):

[0524] Table 11.

[0525] Liquefaction: Ramp 5 deg / min rpm = 30 Lab-o-mat Ramp time 17 min Liquefaction temperature 75℃ Liquefaction time 113 min

[0526] At the end of the procedure described in Table 11, all of the cans were removed from the lab-o-mat and immediately cooled in an ice bath.

[0527] Fermentation Set-up. After complete cooling, urea and penicillin were added to all of the mashes to achieve final concentrations of 750 and 3 ppm, respectively. The mashes were adjusted to pH 5.0 using 40% H2SO4or 50% NaOH. The final dry solids % of the mashes were measured and recorded as 32.44%.

[0528] Approximately 5 g of each mash was transferred to pre-weighed 15 mL plastic Falcon centrifuge tubes for fermentation. A small hole was drilled into the lid of each tube to allow for CO2release during fermentation. Five replicate fermentations were prepared for each treatment. After the mash was transferred, all of the tubes were re-weighed to obtain the initial sample weight. Thereafter, 100 microliters of rehydrated Red Star Ethanol Red yeast (rehydrated by weighing 5.5 g of dry yeast into a 150 mL Erlenmeyer flask, adding 100 mL of tap water and stirring in a 32°C water bath for 30 minutes), an aliquot of diluted AMG SPU glucoamylase (diluted in deionized water) was added to each tube to achieve a starting concentration of 0.50 AGU / g DS. The appropriate amount of deionized water was finally added to each tube so that the total volume of liquid in each tube was the same relative to the sample weight. Thereafter, all of the tubes were re-weighed and then placed in a pre-heated water bath set at 32°C. Fermentation proceeded for a total of 54 hours. After approximately 7 hours, the tubes were vortexed vigorously and then re-weighed twice daily for the remainder of the fermentation time. The number of grams of ethanol produced per gram of dry solids in each tube was calculated from the weight loss data according to the following equation:

[0529]

[0530] After 54 hours of fermentation, three replicate tubes were removed for HPLC analysis. The removed samples were treated with 50 microliters of 40% H2SO4to stop fermentation and vortexed thoroughly. The samples were then centrifuged at 1570 x g for 10 minutes and then filtered through a 0.45 micron syringe filter into HPLC vials. The samples were finally analyzed by HPLC to quantify the amounts of DP4+, DP3, DP2, glucose, fructose, lactic and acetic acids, glycerol, and ethanol.

[0531] Results

[0532] Figure 1The average HPLC results obtained for each treatment after 54 hours of fermentation are shown. A significant increase in ethanol production was measured when RSH AA 96, RSH AA 101 and AMG 001 glucoamylases were added on top of the liquefaction BAA 1407 alpha amylase.

[0533] The invention is described in the following paragraphs:

[0534] Paragraph 1. A process for producing a fermentation product from a starch- containing material, comprising the steps of:

[0535] i) liquefying the starch-containing material using

[0536] - a bacterial alpha-amylase;

[0537] - a raw starch-hydrolyzing alpha-amylase;

[0538] - a saccharogenic enzyme having a thermostability of at least 70% at 70°C, pH 5.3,

[0539] liquefying the starch-containing material at a temperature of 60-80°C;

[0540] ii) saccharifying using the saccharogenic enzyme;

[0541] iii) fermenting using a fermenting organism.

[0542] Paragraph 2. The process according to paragraph 1, further comprising the steps of:

[0543] a) reducing the particle size of the starch-containing material, preferably by dry milling;

[0544] b) forming a slurry comprising the starch-containing material and water.

[0545] Paragraph 3. The process according to paragraph 1 or 2, wherein at least 50%, preferably at least 70%, more preferably at least 80%, in particular at least 90% of the starch- containing material can pass through a sieve having a #6 mesh.

[0546] Paragraph 4. The process according to any of paragraphs 1-3, wherein the pH during the liquefaction step i) is between 4 and 6, preferably between 4.5 and 5.0 or between 4.5 and 4.8 or between 5.0 and 6.0.

[0547] Paragraph 5. The process according to any of paragraphs 1-4, wherein the temperature during liquefaction is between 70 and 80°C, such as between 75 and 80°C, preferably about 75°C.

[0548] Paragraph 6. The process according to any of paragraphs 1-5, wherein the liquefaction is carried out for between 0.1 and 10 hours, such as between 1 and 3 hours, such as about 1.5 hours.

[0549] Paragraph 7. The method according to paragraph 6, wherein the jet-cooking step is performed after liquefaction of step i), e.g. wherein the jet-cooking is performed at a temperature of 110-145 °C, preferably 120-140 °C, such as 125-135 °C, preferably about 130 °C, for about 1-15 minutes, preferably about 3-10 minutes, in particular about 5 minutes.

[0550] Paragraph 8. The method according to any of paragraphs 1-7, wherein saccharification and fermentation are performed sequentially or simultaneously.

[0551] Paragraph 9. The method according to any of paragraphs 1-8, wherein saccharification is performed at a temperature of 20-75 °C, preferably 40-70 °C, such as about 60 °C, at a pH of 4-5, such as about pH 4.5.

[0552] Paragraph 10. The method according to any of paragraphs 1-9, wherein fermentation or simultaneous saccharification and fermentation (SSF) is performed at a temperature of 25-40 °C, such as 28-35 °C, such as 30-34 °C, preferably about 32 °C, wherein fermentation is performed for 6-120 hours, in particular 24-96 hours.

[0553] Paragraph 11. The method according to any of paragraphs 1-10, wherein the fermentation product is an alcohol, preferably ethanol, in particular fuel ethanol, potable ethanol and / or industrial ethanol.

[0554] Paragraph 12. The method according to any of paragraphs 1-11, wherein the fermentation product is recovered after fermentation, e.g. by distillation.

[0555] Paragraph 13. The method according to any of paragraphs 1-12, wherein the starch-containing raw material is whole grain.

[0556] Paragraph 14. The method according to any of paragraphs 1-13, wherein the starch-containing material is derived from corn, wheat, barley, rye, milo, sago, cassava, manioc flour, tapioca flour, sorghum, rice or potato.

[0557] Paragraph 15. The method according to any of paragraphs 1-14, wherein the fermenting organism is a yeast, preferably a strain of the genus Saccharomyces.

[0558] Paragraph 16. The method according to any of paragraphs 1-15, wherein the fermenting organism is a strain of Saccharomyces cerevisiae.

[0559] Paragraph 17. The method according to any of paragraphs 1-16, wherein the bacterial alpha-amylase is derived from a strain of the genus Bacillus (also known as Geobacillus).

[0560] Paragraph 18. The method according to paragraph 17, wherein the bacterial alpha-amylase is obtained from Bacillus or Geobacillus, such as a Bacillus stearothermophilus strain, in particular a Bacillus stearothermophilus alpha-amylase or a variant of Bacillus stearothermophilus, such as the one shown in SEQ ID NO: 3 in WO 99 / 019467 or SEQ ID NO: 1 herein, in particular the Bacillus stearothermophilus alpha-amylase is truncated, preferably has 491 amino acids.

[0561] Paragraph 19. The method according to any of paragraphs 1-18, wherein the bacterial alpha-amylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identity to the polypeptide mature part of SEQ ID NO: 3 disclosed in WO 99 / 019467 or SEQ ID NO: 1 herein.

[0562] Paragraph 20. The method according to any of paragraphs 17-19, wherein the bacterial alpha-amylase has a T1 / 2 (min) at pH 4.5, 75°C and 0.12 mM CaCI2 of at least 20, such as at least 25, such as at least 30, such as at least 40, such as at least 50, such as at least 60, such as at least 70, such as at least 80, such as at least 90, such as at least 100, such as at least 110, such as at least 120, such as at least 130, such as at least 140, such as at least 150, such as at least 160, such as at least 170, such as at least 180, such as 20-300, such as 50-300, such as 60-300, such as 70-300, such as 80-300, such as 90-300, such as 100-300, such as 120-300, such as 140-300, such as 160-300, such as 180-300.

[0563] Paragraph 21. The method according to any of paragraphs 17-20, wherein the bacterial alpha-amylase is obtained from a Bacillus stearothermophilus alpha-amylase which is truncated to have 491 amino acids and has mutations selected from the group consisting of:

[0564] - V59A + Q89R + E129V + K177L + R179E + I181* + G182* + N193F + H208Y + K220P + N224L + Q254S;

[0565] - E129V + K177L + R179E + I181* + G182* + N193F; and

[0566] - E129V + K177L + R179E + I181*+ G182*+ N193F + K220P + N224L + S242Q + Q254S.

[0567] Paragraph 22. The process according to any of paragraphs 17-21, wherein the B. stearothermophilus or G. stearothermophilus alpha-amylase is a variant having the following mutations: I181*+ G182*, preferably I181*+ G182*+ N193F, wherein the numbering is performed using SEQ ID NO: 3 in WO 99 / 019467 or SEQ ID NO: 1 herein.

[0568] Paragraph 23. The process according to paragraph 1, wherein the bacterial alpha-amylase is a chimeric alpha-amylase disclosed in Richardson et al., 2002, The Journal of Biological Chemistry 277(29):26501-26507, preferably one designated as BD5088 or shown as amino acids 1-435 of SEQ ID NO: 2 in WO 2007 / 134207.

[0569] Paragraph 24. The process according to paragraphs 1-23, wherein the raw starch hydrolyzing alpha-amylase is of fungal origin, preferably a variant of a Rhizomucor pusillus alpha-amylase having the Aspergillus niger glucoamylase linker with the SBD and additionally one or more of the following mutations: G128D, D143N, K192R, such as G128D+D143N or G128D+D143N+K192R (numbering using SEQ ID NO: 14 herein).

[0570] Paragraph 25. The process according to any of paragraphs 1-24, wherein the saccharogenic enzyme present and / or added during the liquefaction step i) is a glucoamylase.

[0571] Paragraph 26. The process according to paragraph 25, wherein the saccharogenic enzyme is a glucoamylase having a thermal stability of at least 75%, preferably at least 80%, preferably at least 85% at 70°C, pH 5.3.

[0572] Paragraph 27. The process according to paragraph 25 or 26, wherein the saccharogenic enzyme is a glucoamylase having a relative activity of at least 80%, preferably at least 85%, preferably at least 90% at pH 4.5.

[0573] Paragraph 28. The process according to any of paragraphs 25-27, wherein the saccharogenic enzyme is a glucoamylase having a pH stability of at least 80%, at least 85%, at least 90%, at least 95%, at least 100% at pH 4.5.

[0574] Paragraph 29. The method according to any of paragraphs 25-28, wherein the saccharogenic enzyme is a glucoamylase, preferably derived from a strain of Penicillium, in particular a strain of Penicillium oxalicum disclosed as SEQ ID NO: 2 or herein SEQ ID NO: 9 and 15 in WO 2011 / 127802 published as PCT / CN10 / 071753, or a variant thereof having a substitution of K79V (numbering using SEQ ID NO: 15 herein).

[0575] Paragraph 30. The method according to any of paragraphs 25-29, wherein the glucoamylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the mature polypeptide shown as SEQ ID NO: 2 in WO 2011 / 127802 published as PCT / CN10 / 071753 or shown as SEQ ID NO: 9 herein.

[0576] Paragraph 31. The method according to any of paragraphs 1-30, wherein a glucoamylase is present and / or added during saccharification and / or fermentation.

[0577] Paragraph 32. The method according to any of paragraphs 1-31, wherein the glucoamylase present and / or added during saccharification and / or fermentation is of fungal origin, preferably from a strain of Aspergillus, preferably A. niger, A. awamori or A. oryzae; or a strain of Trichoderma, preferably T. reesei; or a strain of Talaromyces, preferably T. emersonii, or a strain of Pycnoporus, or a strain of Ischnoderma.

[0578] Paragraph 33. The method according to any of paragraphs 1-32, wherein a protease is also present or added during liquefaction.

[0579] Paragraph 34. The method according to paragraph 33, wherein the protease is of fungal or bacterial origin.

[0580] Paragraph 35. The method according to paragraph 33 or 34, wherein the protease has a thermostability value above 20% determined as relative activity at 80°C / 70°C.

[0581] Paragraph 36. The method according to any of paragraphs 33-35, wherein the protease has a thermostability value above 30%, above 40%, above 50%, above 60%, above 70%, above 80%, above 90% determined as relative activity at 80°C / 70°C.

[0582] Paragraph 37. The method according to any of paragraphs 33-36, wherein the protease is a metalloprotease.

[0583] Paragraph 39. The method according to any of paragraphs 33 to 38, wherein the protease is a variant of the metalloprotease disclosed as the mature part of SEQ ID NO. 2 in WO 2003 / 048353 or the mature part of SEQ ID NO: 1 in WO 2010 / 008841 or SEQ ID NO: 3 herein.

[0584] Paragraph 39. The method according to any of paragraphs 33 to 38, wherein the protease is a variant of the metalloprotease disclosed as the mature part of SEQ ID NO. 2 in WO 2003 / 048353 or the mature part of SEQ ID NO: 1 in WO 2010 / 008841 or SEQ ID NO: 3 herein.

[0585] Paragraph 40. The method according to any of paragraphs 33 to 39, wherein the protease variant has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%, to the mature part of the polypeptide of SEQ ID NO: 2 disclosed in WO 2003 / 048353 or the mature part of SEQ ID NO: 1 in WO 2010 / 008841 or SEQ ID NO: 3 herein.

[0586] Paragraph 41. The method according to any of paragraphs 33 to 40, wherein the protease is derived from a strain of the genus Pyrococcus.

[0587] Paragraph 42. The method according to any of paragraphs 33 to 41, wherein the protease is derived from a strain of Pyrococcus furiosus.

[0588] Paragraph 43. The method according to any of paragraphs 33 to 42, wherein the protease is of the kind shown in SEQ ID NO: 1 in US Patent No. 6,258,726 or SEQ ID NO: 13 herein.

[0589] Paragraph 44. The method according to any of paragraphs 33 to 43, wherein the protease is of the kind having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 1 in US Patent No. 6,258,726 or SEQ ID NO: 13 herein.

[0590] Paragraph 45. The method according to any of paragraphs 1 to 44, wherein a pullulanase is also present during liquefaction and / or saccharification.

[0591] Paragraph 46. A composition comprising

[0592] a bacterial alpha-amylase;

[0593] a raw starch-hydrolyzing alpha-amylase;

[0594] a saccharide source generating enzyme having at least 70% thermostability at 70°C, pH 5.3.

[0595] Paragraph 47. The composition according to paragraph 46, wherein the alpha-amylase is derived from a strain of the genus Bacillus, such as a strain of Bacillus stearothermophilus or Bacillus caldovelox, in particular a variant of a Bacillus stearothermophilus or Bacillus caldovelox alpha-amylase, such as the one shown as SEQ ID NO: 3 in WO 99 / 019467 or the one shown as SEQ ID NO: 1 herein.

[0596] Paragraph 48. The composition according to paragraph 46 or 47, wherein the Bacillus stearothermophilus or Bacillus caldovelox alpha-amylase is a variant having the mutations: I181*+G182*, preferably I181*+G182*+N193F, wherein the numbering is made using SEQ ID NO: 3 in WO 99 / 019467 or SEQ ID NO: 1 herein.

[0597] Paragraph 49. The composition according to any of paragraphs 46-48, wherein the Bacillus stearothermophilus or Bacillus caldovelox alpha-amylase is truncated to have about 491 amino acids.

[0598] Paragraph 50. The composition according to any of paragraphs 46-49, wherein the bacterial alpha-amylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identity to the polypeptide mature part of SEQ ID NO: 3 disclosed in WO 99 / 019467 or SEQ ID NO: 1 herein.

[0599] Paragraph 51. The composition according to any of paragraphs 46-50, wherein the alpha-amylase has a T1 / 2 (min) at pH 4.5, 75°C and 0.12 mM CaCI2 of at least 20, such as at least 25, such as at least 30, such as at least 40, such as at least 50, such as at least 60, such as at least 70, such as at least 80, such as at least 90, such as at least 100, such as at least 110, such as at least 120, such as at least 130, such as at least 140, such as at least 150, such as at least 160, such as at least 170, such as at least 180, such as 20-300, such as 50-300, such as 60-300, such as 70-300, such as 80-300, such as 90-300, such as 100-300, such as 120-300, such as 140-300, such as 160-300, such as 180-300.

[0600] Paragraph 52. The composition according to any of paragraphs 46-51, wherein the bacterial alpha-amylase is derived from a Bacillus stearothermophilus alpha-amylase, which is truncated to have about 491 amino acids and has mutations selected from the group consisting of: -V59A+Q89R+E129V+K177L+R179E+I181*+G182*+N193F+H208Y+K220P+N224L+Q254S;

[0601]

[0602] +K220P+N224L+S242Q+Q254S.

[0603] +K220P+N224L+S242Q+Q254S.

[0604] Paragraph 53. The composition according to any of paragraphs 46-52, wherein the raw starch-hydrolyzing alpha-amylase is of fungal origin, preferably a variant of a Rhizomucor pusillus alpha-amylase having an Aspergillus niger glucoamylase linker with a SBD and one or more of the following mutations: G128D, D143N, K192R, such as G128D+D143N or G128D+D143N+K192R (using SEQ ID NO: 14 herein for numbering).

[0605] Paragraph 54. The composition according to any of paragraphs 46-53, wherein the saccharide source generating enzyme is a glucoamylase, preferably a glucoamylase having a thermal stability of at least 75%, preferably at least 80%, preferably at least 85% at 70°C, pH 5.3.

[0606] ​Paragraph 55. The composition according to any of paragraphs 46-54, wherein the sugar generating enzyme is a glucoamylase having a relative activity of at least 80%, preferably at least 85%, preferably at least 90% at pH 4.5.

[0607] Paragraph 56. The composition according to any of paragraphs 46-55, wherein the sugar generating enzyme is a glucoamylase having a pH stability of at least 80%, at least 85%, at least 90%, at least 95%, at least 100% at pH 4.5.

[0608] Paragraph 57. The composition according to any of paragraphs 46-56, wherein the sugar source generating enzyme is a glucoamylase, preferably from a strain of Penicillium, in particular a Penicillium oxalicum strain disclosed as SEQ ID NO: 2 in PCT / CN10 / 071753 published as WO 2011 / 127802, or a variant thereof having a substitution of K79V (numbering using SEQ ID NO: 15 herein).

[0609] Paragraph 58. The composition according to any of paragraphs 46-57, wherein the glucoamylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, for example even at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the mature polypeptide shown as SEQ ID NO: 2 in PCT / CN10 / 071753 published as WO 2011 / 127802, or shown as SEQ ID NO: 9 herein.

[0610] Paragraph 59. The composition according to any of paragraphs 46-58, further comprising a protease.

[0611] Paragraph 60. The composition according to any of paragraphs 46-59, further comprising a metalloprotease.

[0612] Paragraph 61. The composition according to any of paragraphs 46-60, wherein the protease has a thermostability of higher than 30%, higher than 40%, higher than 50%, higher than 60%, higher than 70%, higher than 80%, higher than 90% determined as relative activity at 80°C / 70°C.

[0613] Paragraph 62. The composition according to any of paragraphs 46-61, wherein the protease has a thermostability of higher than 12%, higher than 14%, higher than 16%, higher than 18%, higher than 20% determined as relative activity at 85°C / 70°C.

[0614] Clause 63. The composition according to any of clauses 46-62, wherein the protease is a variant of the metalloprotease from Chryseomonas luteola CGMCC No. 0670 shown in SEQ ID NO: 3 herein.

[0615] Clause 64. The composition according to any of clauses 46-63, wherein the protease is from a Pyrococcus strain.

[0616] Clause 65. The composition according to any of clauses 46-64, wherein the protease is from a Pyrococcus furiosus strain.

[0617] Clause 66. The method according to any of clauses 46-65, wherein the protease is the one shown in SEQ ID NO: 1 in US 6,258,726 or SEQ ID NO: 13 herein.

[0618] Clause 67. The method according to any of clauses 46-66, wherein the protease is the one which is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to SEQ ID NO: 1 in US 6,258,726 or SEQ ID NO: 13 herein.

[0619] Clause 68. The composition according to any of clauses 46-67, further comprising a pullulanase.

[0620] Clause 69. The composition according to any of clauses 46-68, comprising:

[0621] - a bacterial alpha-amylase derived from Bacillus stearothermophilus;

[0622] - a raw starch-hydrolyzing alpha-amylase derived from Rhizomucor pusillus;

[0623] - a carbohydrate-source generating enzyme having at least 70% thermostability at 70°C and pH 5.3 derived from Penicillium oxalicum.

Claims

1. A method of producing a fermentation product comprising the steps of: i) liquefying a starch-containing material using - a Bacillus stearothermophilus alpha-amylase having the mutations V59A + Q89R + E129V + K177L + R179E + I181 + G182 + N193F + H208Y + K220P + N224L + Q254S and being truncated to 491 amino acids, using the numbering of SEQ ID NO: 1, - a raw starch-hydrolyzing alpha-amylase, which is a variant of Rhizomucor pusillus alpha-amylase having an Aspergillus niger glucoamylase linker to SBD, which is SEQ ID NO: 14 and having the following substitutions: G128D+D143N+K192R or G128D+D143N; liquefying the starch-containing material at a temperature in the range of 60-80 °C; ii) saccharifying using a sugar source generating enzyme; iii) fermenting using a fermenting organism.

2. The method according to claim 1, wherein the fermentation product is an alcohol.

3. The method according to claim 1, wherein, A protease is also present or added during liquefaction.

4. The method according to claim 1, wherein a pullulanase is also present during liquefaction and / or saccharification.

5. A method of producing a fermentation product comprising the steps of: i) liquefying a starch-containing material using - a Bacillus stearothermophilus alpha-amylase having the mutations V59A + Q89R + E129V + K177L + R179E + I181 + G182 + N193F + H208Y + K220P + N224L + Q254S and being truncated to 491 amino acids, using the numbering of SEQ ID NO: 1, - a raw starch-hydrolyzing alpha-amylase, which is a variant of Rhizomucor pusillus alpha-amylase having an Aspergillus niger glucoamylase linker to SBD, which is SEQ ID NO: 14 and having the following substitutions: G128D+D143N+K192R; liquefying the starch-containing material at a temperature in the range of 60-80 °C; ii) saccharifying using a sugar source generating enzyme; iii) fermenting using a fermenting organism.

6. The method according to claim 5, said temperature range is in the range of 70-80 °C.

7. The method according to claim 6, wherein, Step ii and step iii can occur simultaneously.

8. A composition comprising: - a B. lentus alpha-amylase having the mutations V59A + Q89R + E129V + K177L + R179E + I181 + G182 + N193F + H208Y + K220P + N224L + Q254S and truncated to 491 amino acids, using the numbering of SEQ ID NO: 1 ; - a raw starch-hydrolyzing alpha-amylase, which is a variant of Rhizomucor pusillus alpha-amylase having an Aspergillus niger glucoamylase linker to SBD, which is SEQ ID NO: 14 and having the following substitutions: G128D+D143N+K192R or a combination thereof, wherein said raw starch-hydrolyzing alpha-amylase has alpha-amylase activity.

9. The composition of claim 8, wherein, further comprising a protease.

10. The composition of claim 8, wherein, further comprising a pullulanase.

11. The composition of claim 8, wherein, further comprising a protease and a pullulanase.

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