WO2007134207A2 - Use of a thermococcales-derived alpha-amylase for starch liquefaction or saccharification - Google Patents

Abstract

The present invention relates to a process of producing a fermentation product, especially ethanol, from starch-containing material using an alpha-amylase and a carbohydrate-source generating enzyme. The invention also relates to a composition comprising an alpha-amylase and a carbohydrate-source generating enzyme as well as the use such compositions for producing fermentation products.

Description

Process of producing a fermentation product

CROSS-REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form. The 5 computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

I O The present invention relates to processes of using an aSpha-amylase and a carbohydrate-source generating enzyme for producing a fermentation product, such as ethanol. The invention also reiates to a composition comprising combination of alpha- amyiase and carbohydrate-source generating enzyme, and the use thereof.

15 Description of the Related Art

A vast number of commercial products, including fermentation products such as alcohols (e.g., ethanol methanol, and butanoi) are produced from starch-containing material. Enzymatic processes on geiatϊnized or un-geiatinized starch-containing material are used widely in industry. ASpha-amylase is used, in conventional liquefaction processes. 0 for thinning the aqueous siurry of geiatinized starch-containing material. Aipha-amyiase converts Song starch polymers into shorter chains and Sess viscous dextrins. A carbohydrate- source generating enzyme, such as espeeiaily gSucoamylase, is then used to convert dextrins into Sow rnolecuSar sugars, e.g., DPi.._y, that can be metabolized by a fermenting organism, such as yeast, into the desired fermentation product, 5 Richardson et ai. (The Journal of BiologicaS Chemistry, Voi. 277, No. 29, pp. 267501-

26507 (2002)) discioses a chimeric alpha-amylase for starch iiquefaction.

WO 2002/38787 discloses a process of producing ethano! including secondary liquefaction step carried out in the presence of a thermostable acid alpha-amyiase.

Despite the vast number of processes used and suggested in the art of fermentation 0 product production there is stili a need for further improvements.

Summary of the invention

The present invention provides processes for producing fermentation products from gelatinized or un-gelatinized (i.e., uncooked) starch-containing material. 5 In the first aspect the invention relates to a process for producing a fermentation product from starch-containing material comprising the steps of: (a) liquefying starch-containing material with an alpha-amySase;

(b) saccharifying the liquefied materia! using a carbohydrate-source generating enzyme;

(C) fermenting using a fermenting organism. wherein the aipha-amylase used in iiquefaction step (a) is selected from the group consisting of:

(v) the aipha-arnylase shown in SEQ iD NO: 2, or i) an alieiic variant thereof having alpha-arnyiase activity; or ii) a fragment thereof having alpha-amylase activity; (X) an alpha-amyiase having an amino acid sequence which has at feast

60% identity with amino acids 1 to 435 of SEQ !D NO: 2;

(y) an aipha-amylase which is encoded by a nucieotSde sequence (i) which hybridizes under at ieast Sow stringency conditions with nucleotides 4 to 1308 of SEQ ID NO: 1 , or (ii) a complementary strand of (i); (z) a variant comprising a conservative substitution, deletion, and/or insertion of one or more amino acids in positions 1 to 435 of SEQ iD NO: 2, In the second aspect the invention relates to processes for producing fermentation products from starch-containing materia! comprising:

(a) saccharifying starch-containing material with an alpha-amyiase at a temperature below the initial gβlatinization temperature of said starch-containing material,

(b) fermenting using a fermenting organism, wherein the alpha-amyiase used in saccharification step (a) or simultaneous sacchariftcation and fermentation in combined steps (a) and (b) is selected from the group consisting of:

(v) the aipha-amylase shown in SEQ iD NO: 2, or i) an allelic vaήant thereof having alpha-amyiase activity; or ii) a fragment thereof having alpha-amylase activity; (x) an alpha-amyiase having an amino acid sequence which has at ieast 60% identity with amino acids 1 to 435 of SEQ iD NO: 2;

(y) an aipha-amylase which is encoded by a nucieotide sequence (i) which hybridizes under at ieast low stringency conditions with nucleotides 4 to 1308 of SEQ ID NO: 1 , or (ii) a complementary strand of (i); or

(z) a variant comprising a conservative substitution, deletion, and/or insertion of one or more amino acids in positions 1 to 435 of SEQ SD NO: 2. The invention also relates to compositions comprising a carbohydrate-source generating enzyme and an aipha-amylase as described in the "Alpha-Amy!ase°-section below. Finally the invention relates to the use of a composition of the invention in processes of the invention.

Definitions Alpha-Amylase activity; The term aipha-amySase (Alpha- 1 ,4-giucan 4 glucanαhydroiases, EC 3.2, 1.1) is defined as an enzyme which catalyzes hydroSysis of starch and other linear and branched 1 ,4 glucosidic oligo- and polysaccharides. For purposes of the present invention, alpha-amyiase activity is determined according to the procedure described in the 'Materials & Methods'-section below, Glucøamylase activity; The term glucoamylase (1 ,4-alpha-D-giucan glucohydroiase, EC 3.2.1.3} is defined as an enzyme, which catalyzes the release of D- glucose from the non-reducing ends of starch or related oiigo- and polysaccharide molecules. For purposes of the present invention, giucoamyiase activity is determined according to the procedure described in the 'Materials & Methods -section below. Identity: The reiatedness between two amino acid sequences or between two nucleotide sequences Ss described by the parameter "identity".

For purposes of the present invention, the degree of identity between two amino acid sequences is determined by the Ciustal method (Higgins, 1989, CAS/OS 5: 151-153) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wl) with an identity table and the following multiple aiignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwisβ alignment parameters are Ktupie=1 _: gap penalty=3, windows=5_r and diagona!s=5.

For purposes of the present invention, the degree of identity between two nucieotide sequences is determined by the Wilbur- Lipman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA 80: 726-730) using the LASERGENE™ MEGAUGN™ software (DNASTAR, Inc., Madison, Wl) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters are Ktuple=3, gap penalty=3, and windows=20.

Subsequence: The term "subsequence" is defined herein as a nucleotide sequence having one or more nucleotides deleted from the 5' and/or 3" enά of SEQ ID NO: 1 , or homoiogous sequences thereof, wherein the subsequence encodes an aipha-amyiase.

Fragment: The term "fragment^* is defined herein as a polypeptide having one or more amino acids deleted from the amino and/or carboxyi terminus of SEQ ID NO: 2, or homologous sequences thereof, wherein the fragment has giucoamyiase activity. Allelic variant: The term "aSleliε variant¹¹ denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus. Alielic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be siSent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An alieSic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene. ArtϊfSciaS variant: When used herein, the term "artificiai variant" means a poiypeptide having aipha-amylase activity produced by an organism expressing a modified nucleotide sequence of SEQ iD NO; 1. The modified nucleotide sequence is obtained through human intervention by modification of the nucleotide sequence disclosed in SEQ ID NO: 1.

Detaited Description of the Invention Production of fermentation products

In this aspect the present invention relates to a process for producing a fermentation product, especially ethanol, from starch-containing material, which process includes a liquefaction step and sequentially or simultaneously performed saccharification and fermentation steps.

According to the present invention fermentation products, such as ethanoS, may advantageously be produced using an aipha-amylase referred to below in combination with a carbohydrate-source generating enzyme. Due to the thermostability of the alpha-amySase the enzyme action time during liquefaction would be prolonged at a pH around 5.6 and below. Further, when combining said aipha-amylase with a carbohydrate-source generating enzyme, especially glucoamylase, a more robust production process is obtained.

Therefore, in the first aspect the invention relates to a process for producing a fermentation product from starch-containing materia! comprising the steps of: (a) liquefying starch-containing material with an alpha-amyjase;

(b) saccharifying the liquefied material using a carbohydrate-source generating enzyme;

(G) fermenting using a fermenting organism. wherein the aSpha-amylase used in liquefaction step (a) is selected from the group consisting of:

(V) the aipha-amylase shown in SEQ SD NO: 2, or i) an allelic variant thereof having alpha-amylase activity, or ii) a fragment thereof having alpha-amylase activity, (x) an alpha-amyiase having an amino acid sequence which has at least 60% identity with amino acids 1 to 435 of SEQ ID NO: 2; (y) an alpha-amylase which is encoded by a nucleotide sequence (i) which hybridizes under at least Sow stringency conditions with nucleotides 4 to 1308 of SEQ ID NO: 1 , or (ii) a complementary strand of (i); or

(z) a variant comprising a conservative substitution, deletion, and/or insertion of one or more amino acids in positions 1 to 435 of SEQ SD NO: 2.

In a preferred embodiment one or more carbohydrases, especialiy a second alpha- amyiase or a pullulanase, or a combination thereof, may be introduced at step (a). According to the invention a second alpha-amyiase may be present during liquefaction in step (a). The second alpha-amylase may be of bacteήa! or fungal origin, preferabSy an acid alpha- amylase, especially acid fungal aipha-amylase; of plant origin, such as of corn, wheat or barley origin. Examples of contempiated second alpha-amyiases and puiiulanases are described below in the section "Additional Enzymes".

The fermentation product, especially ethanol, may optionally be recovered after fermentation, e.g., by distillation. Suitable starch-containing starting materials are iisted in the section "Starch-Containing Materials"-section foeSow, Contempiated enzymes are listed in the "Enzymes^*-section beiow. The liquefaction step is carried out in the presence of an alpha-amylase as defined in above and in the "Alpha-Amylasβs"-sβction beiow. In a preferred embodiment the carbohyd rate-source generating enzyme used for sacchariftcation step (b), or combined steps (b) and (c) (i.e. , SSF), is a glucoamylase. The fermentation step (C), or combined/simultaneous steps (b) anύ (c), are preferably carried out in the presence of yeast, preferably a strain of Saccharomyces, such as Saccharomyces cerevisae. Suitable fermenting organisms are listed in the "Fermenting Organisrns"-section below. In a preferred embodiment step (b) anά (c) are carried out simultaneously (i.e,, as SSF). Because of the properties of the aipha-amylase used no calcium ions need to be added during liquefaction. In a particuiar embodiment, the process of the invention further comprises, prior to the step (a), the steps of:

1) reducing the particle size of the starch-containing material, preferably by milling;

2) forming a slurry comprising the starch-containing materia! and water. The aqueous slurry may contain from 10-55 wt-%, preferably 25-40 wt-%, more preferably 30-35 wt-% starch-containing material. The slurry may be heated to above the gelatinization temperature and aipha-amylase may be added to initiate liquefaction (thinning). The slurry may in one embodiment be jet-cooked to further gelatinize the slurry before being subjected to an alpha-amylase in step (a) of the invention, More specifically liquefaction may in one embodiment be carried out as a three- step hot slurry process. The slurry is heated to between 60-1 Q5"C, preferably 80-95"C, and alpha-amylase may be added to initiate liquefaction (thinning). In one embodiment the slurry is then jet-cooked at a temperature between 95-14CTC, preferably 105-125⁰C, for 1-15 minutes, preferably for 3-10 minute, especially around 5 minutes. The slurry is cooied to 60- 105⁰C and more alpha-amySase is added to finalize hydrolysis (secondary liquefaction). The liquefaction process may be carried out at a pH from 3-7, in particuiar at a pH between 4-6. especially at a pH between 4-5. It is to be understood that aiS aSpha-amySase may be added as a single dose, e.g., before jet-cooking.

The saccharification in step (b) may be carried out using conditions we!! known in the art. For instance, a full saccharification process may last up to from about 24 to about 72 hours. In one embodiment a pre-saccharification of typically 40-90 minutes at a temperature between 30-85⁰C, typicaily about 60⁰C, is carried out, followed by compiete saccharification during fermentation in a simuStaneous sacchanfication and fermentation process (Le,, SSF), Saccharification is typicaiSy carried out at temperatures from 30-85⁰C, typicalSy around 6O⁰C, and at a pH between 4 and 5, normaiiy at about pH 4,5. The most wideiy used process in fermentation product, especially eihanoi, production is a simultaneous saccharification anύ fermentation (SSF) process, in which there is no holding stage for the saccharification, meaning that the fermenting organism, such as yeast, and enzyme(s) may be added together. SSF may typically be carried out at a temperature between 25''C and 4O⁰C, such as between 29^C1C and 35"C, such as between 30"C and 34⁰C, such as around 32°C. According to the invention the temperature may be adjusted up or down duήng fermentation.

PlQ.PAssg.s fpf.pr^^^

In this aspect the invention relates to processes for producing a fermentation product from starch-containing material without cooking (i.e., no geiaiinization) of the starch- containing materia!. According to the invention a desired fermentation product, such as ethanol, may be produced without Siquefying the aqueous slurry containing the starch- containing materia!, in one embodiment a process of the invention includes saccharifying (e.g., mϋled) starch-containing materia!, e.g., granular starch, beiow the initia! geiatinization temperature in the presence of an alpha-amyiase as defined in the "Alpha-Amyiase^M-seetion below; and further a carbohydrate-source generating enzyme, preferably a giucoamyiase, disclosed in the "Carbohydrate-Source Generating Enzymes"-section beiow, to produce sugars that can be fermented and/or converted into a desired fermentation product by a suitabie fermenting organism. Accordingly, in this aspect the invention reiatβs to a process for producing a fermentation product from starch-containing materia! comprising: (a) saccharifying starch-containing material with an aipha-arnyiase at a temperature below the initial gelatinizatiαn temperature of said starch-containing material,

(b) fermenting using a fermenting organism, wherein the alpha-amyiase used in sacchariftcation step (a) or simultaneous saccharification and fermentation in combined step (a) and (b) is seiected from the group consisting of. (v) the aipha-arnylase shown in SEQ iD NO: 2, or f) an alieiic variant thereof having alpha-amyiase activrty; or ii) a fragment thereof having alpha-amyiase activity; (x) an alpha-amyiase having an amino acid sequence which has at Sβast 60% identity with amino acids 1 to 435 of SEQ iD NO: 2;

(y) an aipha-amylase which is encoded by a nucleotide sequence (i) which hybridizes under at least low stringency conditions with nucleotides 4 to 1308 of SEQ ID NO: 1 , or (ti) a complementary strand of (i): or

(z) a variant comprising a conservative substitution, deletion, and/or insertion of one or more amino acids in positions 1 to 435 of SEQ !D NO: 2.

In one embodiment an acid alpha-amyiase, such as an acid fungal aipha-amylase, or a plant aipha-amylase is also added during saccharification or fermentation or simuitaneous steps (a) &nά (b). The additional aipha-amylase may be derived from a bacteria or fungal cell, such as a filamentous fungus. Examples of additional alpha-amySases, preferably acid alpha-amyiases, are described in the "Additional Enzymβs'-sβction below.

Steps (a) and (b) of the process of the invention may be earned out sequentially or simultaneously. The fermentation product may be recovered after fermentation.

The term "... below the initial geiatinization temperature..." means the lowest temperature at which geiatinization of the starch in question commences. Starch heated in water begins to gelatinize between about 5CTC and 75"C; the exact temperature of geiatinization depends on the specific starch and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species. to the particular variety of the piant species as wei! as with the growth conditions, In the context of this invention the initial geiatinization temperature of a given starch-containing materia! can be defined as the temperature at which birefringence is iost in 5% of the starch granules using the method described by Gorinstein. S. and Lii. C, Starch/StaYke, Vo! 44

(12) pp. 461-466 (1992),

Before step (a) a slurry of starch-containing material, such as granular starch, having between 10-55 wt-% dry solids (DS), preferably between 25-40 wt-% dry solids, more preferably 30-35 wt-% dry solids of starch-containing material, may be prepared. The slurry may include water and/or process water, such as thin stiilage (backset), scrubber water, evaporator condensate or distillate, side stripper water from distiiiatioπ, or other fermentation product plant process water. Because the process is carried out below the initial gelatinization temperature and thus no significant viscosity increase takes piace, high leveis of stillage may be used if desired, in an embodiment the aqueous slurry contains from about 1 to about 70 vøl,-% stillage, preferably 15-60 vol.-% stiiiage. especially from about 30 to 50 vo!,-% stiilage.

The starch-containing materia! may be prepared by reducing the particle size, preferably by dry or wet miiiSng, to between 0.05 to 3,0 mm, preferabiy between 0.1-0,5 mm. After being subjected to a process of the invention at least 60%. at ieast 70%, at Seast 80%. at ieast 90%, at least 90%, at ieast 91%, at ieast 92%, at least 93%, at least 94%, at ieast 95%, at least 96%, at ieast 97%, at least 98%, or preferably at ieast 99% of the dry solids of the starch-containing material is converted into a soiuble starch hydroiysate.

The process of the invention is conducted at a temperature beiow the initial gelatinization temperature. Preferably the temperature at which step (a) is carried out sequentiaily is between 30-75¹⁵C, preferably between 45-60⁰C. in a preferred embodiment step (a) anύ step (b) are carried out as a simultaneous saccharification and fermentation process (i.e., one-step fermentation), in such preferred embodiment the process may typically be carried out at temperatures between 25'C and 4OX, such as between 29'C and 35⁰C, such as between 3O^C1C and 34"C, such as around 32*C. According to the invention the temperature may be adjusted up or down during fermentation,

In an embodiment steps (a) and (b) are carried out simuitaneously (i.e., one-step fermentation) so that the sugar ievel, such as glucose Sevel, is kept at a low levei such as below 6 wt,-%, preferably beiow about 3 wt,-%, preferably beiow about 2 wt,-%, more preferred beiow about 1 wt-%,, even more preferred below about 0.5%, or even more preferred 0.25% wt.-%, such as beiow about 0.1 wt.-%. Such iow leveis of sugar can be accompiished by simply employing adjusted quantities of enzyme and fermenting organism, A skiiled person in the art can easiiy determine which quantities of enzyme anά fermenting organism to use. The employed quantities of enzyme and fermenting organism may aSso be seiected to maintain low concentrations of maltose in the fermentation broth. For instance, the maitose ievel may be kept beiow about 0.5 wt.-% or below about 0,2 wt.-%.

The process of the invention may be carried out at a pH in the range between 3-7, preferably from pH 3-6, or more preferabiy from pH 4-5. _■^g_,r_,^_,h-contaiπj_.og_..matejiajs

Any suitable starch-containing starting material, including granuiar starch, may be used according to the present invention, As indicated above the starch-containing material may either be gelatinized or un-gelatinized (i.e., uncooked). The actual starting material is generally seiected based on the desired fermentation product. Examples of starch-containing starting materials, suitable for use in a process of present invention, include tubers, roots, stems, whole grains, corns, cobs, wheat, barley, rye, miio, sago, cassava, tapioca, sorghum, rice peas, beans, or sweet potatoes, or mixtures thereof, or cereals, sugar-containing raw materials, such as moiasses, fruit materials, sugar cane or sugar beet potatoes, and ceiiulose-confaining materials, such as wood or plant residues, or mixtures thereof. Contemplated are both waxy and non-waxy types of corn and barley.

The term "granular starch" means raw uncooked starch, i.e., starch in its natural form found in cereal, tubers or grains. Starch is formed within plant cells as tiny granules insoluble in water. When put in cold water, the starch granules may absorb a small amount of the liquid and swell. At temperatures up to 50⁰C to 75°C the swelling may be reversible. However, with higher temperatures an irreversible sweiiing cailed "gelatinization" begins. Granular starch to be processed may be a highly refined starch quality, preferably at least 90%, at least 95%, at least 97% or at ieast 99.5% pure or it may be a more crude starch containing materia! comprising milled whole grain including non-starch fractions such as germ residues and fibers.

In an embodiment the starch-containing material is fractionated into one or more components, including fiber, germ, and a mixture of starch and protein (endosperm).

Fractionation may according to the invention be clone using any suitable technology or apparatus. For instance, Satake Corporation (Japan), has manufactured a system suitable for fractionation of plant material such as corn.

The germ and fiber components may be fractionated from the remaining potion of the endosperm, In an embodiment of the invention the starch-containing material Ss plant endosperm, preferably corn endosperm. Further, the endosperm may be reduced in particle size &nά combined with the larger pieces of the fractionated germ and ftber components for fermentation.

Fractionation can be accomplished, e.g., using the apparatus disclosed in US application publication no. 2004/0043117 (hereby incorporated by reference). Suitable methods and apparatus for fractionation include a sieve, sieving and eiutriation. Suitable apparatus also include friction mis, such as rice or grain polishing mills (e.g., those manufactured by Satake Corporation (Japan), Kett, or Rapsco, TX, USA).

Reducing the particle size of starch-containing, plant maferiai The starch-containing materia!, such as whole grain, used in a process of the invention, may preferably be reduced in particle size in order to open up the structure and expose more surface area. This may be done by miing. Two miiiing processes are preferred according to the invention: wet and dry miing. In dry miing whole kernels are milled and used. Wet milling gives a good separation of germ and meal (starch granules and protein) and is often applied at locations where the starch hydrolysate is used in production of syrups. Both dry and wet miiiing is weii known in the art of starch processing and is equaiiy contempSated for the process of the invention. Examples of other contempiated technologies for reducing the particle size of the starch-containing plant material include emulsifying technology and rotary pulsation. The starch-containing material may be reduced in particle size to between 0.05 to 3.0 mm, or so that at ieast 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material fit through a sieve with a 0.05 to 3.0 mm screen, preferably 0.1-0.5 mm screen.

Fermentation Products

The term "fermentation product" means a product produced by a process including a fermentation step using a fermenting organism. Fermentation products contemplated according to the invention include alcohols (e.g., ethanoi, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid): ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H_? and CO-); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B<?, beta-carotene): and hormones, In a preferred embodiment the fermentation product is ethanoi, e.g., fuel ethanol; dnnking ethanoi, i.e., potable neutral spirits; or industrial ethanoi or products used in the consumable alcohol industry (e.g. _r beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Preferred beer types comprise aies, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohoS beer, low-alcohol beer, Sow-calorie beer or Sight beer. Preferred fermentation processes used include alcohol fermentation processes, as are well known in the art. Preferred fermentation processes are anaerobic fermentation processes, as are well known in the art, FgriTientinQ Organisms

"Fermenting organism" refers to any organism, including bacterial and fungal organisms, suitable for use in a fermentation process and capable of producing desired a fermentation product. Especially suitable fermenting organisms are able to ferment, i.e., convert, sugars, such as glucose or maltose, directly or indirectly into the desired fermentation product. Examples of fermenting organisms include fungal organisms, such as yeast. Preferred yeast includes strains of Saccharomycβs spp., in particular, Saccharomycβs cerevϊsiae. Cornmerciaily available yeast include, e.g., RED STAR™/Lesaffrβ, ETHANOL RED™ (available from Red Star/Lesaffre, USA) FAL! (available from Fleischrnann's Yeast, a division of Burns Philp Food inc., USA), SUPERSTAR! (available from Ailtech), GERT STRAND (available from Gert Strand AB, Sweden) and FERMIOL™ (avaiiable from DSM Specialties).

ENZYMES Alpha- Amylase

The alpha-amySase used in a process of the invention may be an alpha-amyiase selected from the group consisting of:

(v) the alpba-amyiase shown in SEQ !D NO: 2, or i) an allelic variant thereof having aSpha- amylase activity, or ii) a fragment thereof having alpha-amyiase activity,

(x) the alpha-amySase having an amino acid sequence which has at least 60% identity with amino acids 1 to 435 of SEQ ID NO: 2;

(y) the alpna-amyiase which is encoded by a nucleotide sequence (i) which hybridizes under at least Sow stringency conditions with nucleotides 4 to 1308 of SEQ ID NO: 1 _« or (ii) a compiementary strand of (i);

(z) a variant comprising a conservative substitution, deietion, and/or insertion of one or more amino acids in positions 1 to 435 of SEQ ID NO: 2,

In a preferred embodiment the alpha-amyiase is the mature part of the alpha- amySase disclosed in Richardson et ai. (The Journal of Biological Chemistry, Vol. 277, No 29, pp. 267501-26507 (2002)), referred to as BD5088. This alpha-amyiase is the same as the one shown in SEQ ID NO: 2, The mature enzyme sequence starts after the initial "Met^*1 amino acid in position 1.

In a preferred embodiment the aipha-amyiase used in a process of the invention is derived from a microorganism, preferably a bacterium, of the order ThermococcaSes. The alpha-amylase may be a hybrid alpha-amylase such as the BD5088 alpha-amySase made from alpha-amyiases from three microorganisms within the order Thwmococcales. The alpha-amyiases may according to the process of the invention be added in an amount of 0.1 to 10 AFAUZg DS, preferably 0.10 to 5 AFAU/g DS₁ especially 0.3 to 2 AFAU/g

DS. When measured in KNU units the aipha-amySase activity is preferably present in an amount of 0.0005-5 KNU per g DS₁ preferably 0.001-1 KNU per g DS, such as around 0.050 KNU per g DS.

In a preferred embodiment the alpha-amyiase is the commercially available product sold as ULTRA THIN™ (Valley Research, USA),

_■Hybridization The alpha-amylase used in a process of the invention may in one embodiment be encoded by polynucleotides (i) which hybridizes under at ieast low stringency conditions, preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with a nucleotide sequence with nucleotides 4 to 1308 of SEQ SD NO: 1 , or (ii) a subsequence of (i), or (iii) a complementary strand of (i) or (ii) {J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989« Molecular Cloning, A Laboratory Manual, 2ύ edition, Cold Spring Harbor, New York). A subsequence of SEQ ID NO: 1 contains at ieast 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides.

The nucleotide sequence of SEQ ID NO: 1 , or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO: 2, or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having aipha-amyiasβ activity from strains of different genera or species of especially the order Thermococcaies according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at ieast 14, preferably at least 25, more preferably at ieast 35, and most preferably at least 70 nucleotides in length, it is however, preferred that the nucleic acid probe is Bt ieast 100 nucleotides in length. For example, the nucleic acid probe may be at ieast 200 nucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides in length. Even longer probes may be used, e.g. , nucleic acid probes which are at least 600 nucleotides, at least preferably at ieast 700 nucleotides, more preferably at least 800 nucleotides, or most preferably at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for exampie, with ³²P, ³H, ^3;S, biotin, or avidin). Such probes are encompassed by the present invention. A genomic DNA or cDNA library prepared from such other organisms may, therefore, be screened for DNA which hybridizes with the probes described above and which encodes a poϊypeptide having alpha-amyiasβ activity. Genomic or other DNA from such other organisms may be separated by agarose or poiyacrylamide gei electrophoresis, or other 5 separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitroceiiuiose or other suitable carrier material, in order to identify a cione or DNA which is homologous with SEQ ID NO: 1 , or a subsequence thereof, the carrier material is used in a Southern blot.

For iong probes of at least 100 nucleotides in length, low to very high stringency

H⁾ conditions are defined as prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micro g/mi sheared and denatured salmon sperm DNA. and either 25% formamide for low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, foilowing standard Southern blotting procedures for 12 to 24 hours optimaily.

15 For long probes of at least 100 nucleotides in iength. the carrier materia! is finaiiy washed three times each for 15 minutes using 2X SSC, 0.2% SOS preferably at least at 5O⁰C (low stringency), more preferably at least at 55"C (medium stringency), more preferably at least at 6CTC (medium-high stringency), even more preferably at ieast at 65"C (high stringency), and most preferably at Seast at 70"C (very high stringency).

3d For short probes which are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization. hybridization, and washing post- hybridization at about 5^"C to about 1O⁰C below the calculated T.-_κ using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0,9 M NaCI, 0.09 M TrIs-HCI pH 7.6. 6 mM EDTA, 0.5% NP-40. 1X

25 Denhardt's solution. 1 mM sodium pyrophosphate, 1 mlv5 sodium monobasic phosphate, 0.1 mM ATP, &nύ 0.2 mg of yeast RNA per ml foiiowing standard Southern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6X SCC plus 0.1 % SDS for 15 minutes and twice each for 15 minutes using 6X SSC at 5"C to 10^JC below the calculated T_m.

30 Under salt-containing hybridization conditions, the effective T- is what controls the degree of identity required between the probe anά the filter bound DMA for successful hybridization. The effective T-,, may be determined using the formula below to determine the degree of identity required for two DNAs to hybridize under various stringency conditions Effective T,,, = 81.5 + 16.6(log !VUNa^*]) + 0.41(%G+C) - 0.72{% formamide)

35 (See .wwwMsu.nødM_^ VgrjgMg_.or_..Fragfnents

As mentioned above, the alpha-amyiase used in a process of the invention may be a variant of the alpha-amyiase shown in SEQ ID NO: 2. A variant may be an aSSelic or an artificial variant, including a fragment having alpha-amyiase activity, in an embodiment of the invention the variant is an artificial variant comprising a conservative substitution, deletion, and/or insertion in positions 1-435 of SEQ ID NO: 2. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyi-termina! extensions, such as an amino- terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a smail extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain,

Exampies of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (giutamine and asparagine), hydrophobic amino acids {leucine, isoieucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (giycine, alanine, serine, threonine and methionine). Amino acid substitutions which do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. HiIi, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Aia/Ser, Val/ile, Asp/Giu, Thr/Ser, Aia/Gly, Ala/Thr, Ser/Asn, Aia/Vai, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lie, Leu/Val, Aia/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids (such as 4- hydroxyproiine, β-Λ/-methyS lysine, 2-aminoisobutyrie acid, isovaiine, anύ aipha-methyl serine) may be substituted for amino acid residues of a wild-type polypeptide. A iimited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnaturai amino acids may be substituted for amino acid residues. "Unnatural amino acids" have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids. Unnatural amino acids can be chemically synthesized, and preferably, are commercially available, and include pipecoiic acid, fhiazolidine carboxyiic acid, dehydroproiine, 3- and 4-methySpraSine, and 3,3- dimethy I proline.

Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, aiter the substrate specificity, change the pH optimum, and the like. Essential amino acids in the parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanniπg mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant moiecules are tested for biological activity (i.e., glucoamylase activity) to identity amino acid residues that are critica! to the activity of the molecule. See also, Hilton ei a/., 1996, J. Biol. Chem, 271 : 4699-4708. The active site of the enzymes or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or phαtoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et at, 1992_r Science 255: 306-312; Smith et ai. , 1992, J, MoL BbL 224: 8ΘΘ-9Q4; Wlociaver et ai... 1ΘΘ2, FEBS Lett, 309:59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides which are related to a polypeptide according to the invention. Single or multiple amino acid substitutions can be made anύ tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Oison and Sauer, 1988, Science 241 : 53- 57; Bowie and Sauer, 1989, Proc. Natl. Acad Set. USA 86: 2152-2156; WO 95/17413: or WO 95/22625. Other methods that can be used include error-prone PCR, phage display {e.g., Lowman et a/., 1991 , Biochem. 30:10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region- directed mutagenesis (Derbyshire et ai., 1986, Gene 46:145; Ner et ai. 1988. DΛM 7:127). iviutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host ceils. Mutagenized DNA molecules that encode active polypeptides can be recovered from the host ceils and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

The total number of amino acid substitutions, deletions and/or insertions of amino acids in position 1 to 558 of SEQ IO NO: 2, is 10, preferably 9, more preferabiy 8, more preferably 7, more preferably at most 6, more preferably at most 5, more preferably 4, even more preferably 3, most preferabiy 2, and even most preferably 1.

CaφohydraterSouTce.fleMratiη.jj.enzyiriβ The term "carbohydrate-source generating enzyme" includes glucoamylase (being glucose generators), beta-amyiase and maltogenic amylase (being maltose generators). A carbohydrate-source generating enzyme is capable of producing a carbohydrate that can be used as an energy-source by the fermenting organism(s) in question, for instance, when used in a process of the invention for producing a fermentation product, such as ethanol. The generated carbohydrate may be converted directly or indirectly to the desired fermentation product, preferably ethanol.

According to the invention a combination or mixture of carbohydrate-source generating enzyme and alpha-amySase may be used in a process of the invention. EspeciaSSy contempSated combinations or mixtures are incSudβ one or more giucoamyiases as disclosed below in the "Glucoamyiases'-sectioπ and an aipha-amylase as defined in the Alpha- Amylase"-section below. The ratio between alpha-amyiase activity (AFAU) per glucoamyiase activity (AGU) (AFAU per AGU) may in an embodiment of the invention be at least 0.1 , in particular at Seast 0,16, such as in the range from 0.12 to 0.50 or more.

Giucoamyiases A giucoamylase used according to the invention may be derived from any suitable source, e.g., derived from a microorganism or a plant. Preferred giucoamyiases are of fungal or bacterial origin, selected from the group consisting of Aspergillus giucoamyiases, in particular A, niger GI or G2 giucoamylase (Boei et ai., 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 Movozymes, Denmark); the A awamoή gSucoamySase disclosed in WO 84/02921 , A, oryzae giucoamylase (Agric. Biol, Chem., 1991 , 55(4): 941-949), or variants or fragments thereof. Other Aspergillus gSucoamySase variants incSude variants with enhanced thermal stabiiity: G137A and G139A (Chen et a!,, 1996, Prot. Eng. 9: 499-505); D257E and D293E/Q (Chen et a!., 1995, Prot. Eng. 8: 575-582); N 182 (Chen et a!. (1994). Biochem. J. 301 : 275-281); disulphide bonds, A248C (Fierobe et ai., 1996, Biochemistry, 35: 8698-8704; and Introduction of Pro residues In position A435 and S436 (Li et al., 1997, Protein Eng. 10; 1199-1204.

Other giucoamyiases include Athelia roifsii (previously denoted Cortiάum rotfsli) giucoamylase (see U.S. Patent No. 4,727,026 and Nagasaka et al., 1998, "Purification and properties of the raw- starch- degrading gSucoamylases from Corticium roifsii, AppS Microbiol Biotechnoi 50:323-330), Talaromyαes giucoamyiases, in particuiar derived from Talaromyces emersonil (WO 99/28448), Tataromyces leycettanus (U.S. Patent No. Re. 32,153), Talaromyces duponti. Talaromyces thermophilus (U.S. Patent No. 4,587,215).

Bacterial giucoamyiases contempSated include gSucoamySases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135, 138), and C. thermohydrosuSfuncum (WO 86/01831) and Trametes cingulata disclosed in WO 2006/069289, and disclosed in SEQ ID NO: 4 herein (which reference is hereby incorporated by reference).

Aiso hybrid gSucoarnySase are contemplated according to the invention. Examples the hybrid glucoamylases disclosed in WO 2005/045018. Specific examples include the hybrid glucoamylase disclosed in Table 1 and 4 of Example 1 (which hybrids are hereby incorporated by reference.).

Preferred glucoamylases are the glucoamySase is selected from the group consisting of gSucoarnySases derived from the genus Aspergillus, preferably a strain of Aspergillus niger, Aspergillus oryzae, Aspergillus awatnoή, or the genus Athella, preferably a strain of Aihelia rolfsii, the genus Talarornyces, preferabSy a strain the Talarornyces emersonii, or the genus Rhizopus, such as a strain of Rhizopus nivius, or of the genus Humicola, preferably a strain of Humicola gήsea var. thenvoiύea, or a strain of the genus Trametes, preferably a strain of Trametβs clngulata disclosed in co-pending application PCT/US05/48724 which is hereby incorporated by reference. Glucoamyiases may in an embodiment be added in an amount of 0.001 to 10 AGU/g

DS, preferably from 0.01 to 5 AGU/g DS₁ especially 0.1 to 0.5 AGU/g DS.

Beta-amylase

At least according to the invention the a beta-amylase (E C 3,2.1 ,2) is the name traditionary given to exo-actsng maitogenic amylases, which cataiyze the hydrolysis of 1 ,4- alpha-glucosidic linkages in amyiosβ, amylopectin and related glucose polymers. Maltose units are successively removed from the non-reducing chain ends in a step-wise manner until the molecule is degraded or, in the case of amylopectin, until a branch point is reached. The maltose re-leased has the beta anorneric configuration, hence the name beta-amylase. Beta-amyiases have been isolated from various plants and microorganisms (W. M.

Fogarty and CT. Keily, Progress in Industrial Microbiology, vol. 15, pp. 112-1 15, 1979). These beta-amylases are characterized by having optimum temperatures in the range from 40⁰C to 65⁰C and optimum pH in the range from 4,6 to 7. A commercially available beta- amylase from barley is NOVOZYM™ WBA from Novozymes A/S, Denmark and SPEZYME™ BBA 1500 from Genencor Int. , USA.

Maltopenic amylase

The amylase may also be a maitogenic alpha-amyiase. A "maitogenic alpha- amyiase^* (giucan 1 ,4-aSpha-maitohydrolasβ, E.C. 3.2,1.133) is able to hydrolyzβ amyiose and amylopectin to maitose in the alpha-configuration. A maitogenic amylase from Bacillus stearothermophiius strain NCIB 11837 is commercially available from Novozymes A/S. Maltøgenic alpha-amyiases are described in US Patent nos. 4.598.048. 4.604.355 and 6,162,628, which are hereby incorporated by reference.

The maitogenic amylase may in a preferred embodiment be added in an amount of 0.05- 5 mg total protein/gram DS or 0.05- 5 MANU/g DS.

Additional Enzymes

As mentioned above, processes of the invention, both non-cook processes {i.e., un- gelatinizβd starch processes) and gelatinized starch processes (i.e.. including a liquefaction step) may in preferred embodiments include introduction of one or more additional carbohydrases, especiaSSy alpha-amyiases and/or puliufanases.

Atpha-Amyϊase

Contemplated additional aipha-arnylases may be any aipha-amylase. Preferred are alpha-amyiases of fungal or bacterial origin. The alpha-amyiase may also be of plant origin, preferably corn, wheat or bariey origin.

In a preferred embodiment the additional alpha-amyiase is an acid alpha-amyiase.

The acid alpha-amySase may be of fungal or bacterial origin. The term "acid alpha-amyiase" means an aipha-amylase (E.C. 3.2,1.1) which added in an effective amount has activity optimum at a pH in the range of 2 to 7, preferably from 3 to 6, or more preferably from 3,5- 5.5.

Bacterial Alpha-Amylases

A bacteria! alpha-amyiase may preferably be derived from the genus Bacillus,

In a preferred embodiment the Bacillus aipha-amylase is derived from a strain of B, licheniformis, B. amytoliquefaciens, B. subtiiis or B. stearothenvopNlus, but may also be derived from other Bacillus sp. Specific examples of contemplated aipha-amylases include the Bacillus licheniformis aipha-amylase {BLA) shown in SEQ ID NO: 4 in WO 99/19467, the Bacillus amyloliquefaciens aipha-amylase (BAN) shown in SEQ ID NO: 5 in WO 99/19467, and the Bacillus stearothermophilus aipha-amylase (BSG) shown in SEQ ID NO: 3 in WO 99/19487, in an embodiment of the invention the aipha-amylase is an enzyme having a degree of identity of at least 60%, preferably at ieast 70%, more preferred at least 80%, even more preferred at least 90%, such as at least 95%, at least 96%, at least 97%, at ieast 98% or at least 99% identity to any of the sequences shown as SEQ ID NOS: 1 , 2, 3, 4, or 5, respectively, in WO 99/19467. The Badtlus aipha-amylase may also be a variant and/or hybrid, especiaily one described in any of WO 96/23873, WO 96/23874, WO 97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documents hereby incorporated by reference). Specifically contemplated aipha-amylase variants are disclosed in US patent nos. 6,093,562, 6.297,038 or US patent no, 6,187,576 (hereby incorporated by reference) and include Bacillus sieamthermophilus aipha-amylase (BSG aipha-amylase) variants having a deletion of one or 5 two amino acid in position 179 to 182, preferably a double deletion disclosed in WO 1996/023873 - see e.g., page 20, lines 1 -10 (hereby incorporated by reference), preferably corresponding to delta(181-182) compared to the wild-type BSG aipha-amylase amino acid sequence set forth in SEQ ID NO; 3 disclosed in WO 99/19467 or deletion of amino acids 179 and 180 using SEQ ID NO. 3 in WO 99/19467 for numbering (which reference is hereby

H⁾ incorporated by reference). Even more preferred are Bacillus alpha-amySases, especially Baαilus stearothermophsius alpha-amylase, which have a double deletion corresponding to delta(181-182) and further comprise a N193F substitution {also denoted 1181^* + G182^* + N193F) compared to the wild-type BSG alpha-arnyiase amino acid sequence set forth in SEQ ID NO: 3 disclosed in WO 99/19467.

15 The alpha-amyiase may also be a maiiogenic aipha-amyiase. A "maltogenic alpha- amylase" (glucan 1 _;4-a!pha-matohydrolase, E,C, 3,2.1 ,133} is able to hydrolyze amyiose and amyiopectin to maStosβ in the aipha-configu ration. A maltogenic aipha-amylase from Bacillus stearothermophilus strain NCIB 11837 is commercially available from Novozymes A/S, Denmark. The maltogenic alpha-amyiase is described in U.S. Patent Nos. 4.598,048.

2(1 4,604,355 anύ 6,182,628, which are hereby incorporated by reference.

Other bacterial alpha-amyiases contemplated may be derived from Pyrococcus sp., such as Pyrococcus fuήosus, such as the ones disclosed in VVO 94/19454 which is hereby incorporated by reference.

25 Bacterial Hybrid Aipha-Amylases

A hybrid alpha-amylase specificaily contemplated comprises 445 C-terminai amino acid residues of the Bacillus licheniformis alpha-amylase (shown as SEQ ID NO: 4 in WO 99/19467) and the 37 N-termtnai amino acid residues of the alpha-amylase derived from Bacillus amyloliquefaciens (shown as SEQ ID NO. 3 in WO 99/194676), with one or more,

30 especially ail, of the following substitution:

G48A+T49i+G 107A+H156Y+A181T+N190F+i201 F+A209V+Q264S (using the Bacillus licheniformis numbering). Also preferred are variants having one or more of the foliowing mutations (or corresponding mutations in other Bacillus alpha-amylase backbones): H154Y, A181T, N190F, A209V and Q264S and/or deletion of two residues

35 between positions 176 and 179, preferably deletion of E178 and G 179 (using the SEQ ID NO: 5 numbering of WO 99/19467). The bacterial alpha-amyiase may be added in amounts well-known in the art

Fungal Alpha-Amylases

Acid fungai aipha-amyiases include acid alpha-amyiases derived from a strain of the genus Aspergillus, such as Aspergillus otyzaβ, Aspergillus niger. and Aspergillus kawachii .

A preferred acid fungal aSpba-amylase is a FungamyS-Sike alpha-amyiase which is preferably derived from a strain of Aspergillus oryzae. in the present disclosure, the term Tungamyl-like aipha-amyiase" indicates an alpha-amyiase which exhibits a high identity, i.e. more than 70%, more than 75%, more than 80%. more than 85% more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or even 100% identity to the mature part of the amino acid sequence shown in SEQ SD NO: 10 in WO 96/23874,

Another preferred acid alpha-amyiase is derived from a strain Aspergillus niger. In a preferred embodiment the acid fungai aipha-amylase is the one from A, niger disciosed as ^* AMYA-ASPNG" in the Swiss-prot/TeEMBL database under the primary accession no. P56271 and described in more detail in WO 89/01969 {Example 3). The acid Aspergillus niger acid aipha-amyiase is also shown as SEQ ID NO: 1 in WO 2004/080923 (Novozymes) which is hereby incorporated by reference. ASso variants of said acid fungai amylase having at least 70% identity, such as at ieast 80% or even at ieast 90% identify, such as at ieast 95%, at ieast 96%, at least 97%, at least 98%. or at least 99% identity to SEQ SD NO; 1 in WO 2004/080923 are contemplated. A suitable commercialiy avaiiable acid fungai alpha- amyiase derived from Aspergillus niger is SP288 (available from Novozymes A/S, Denmark). In a preferred embodiment the aipha-amylase is derived from Aspergillus kawachii and disclosed by Kaneko et al. (J, Ferment. Bioeng, 81 :292-298 (1996) "Molecuiar-cioning and determination of the nucleotide-sequence of a gene encoding an acid-stabie alpha- amylase from Aspergillus kawachii") and further as EMBL;#AB008370.

The fungai acid aipha-amylase may also be a wiSd-fype enzyme comprising a carbohydrate-binding module (CBM) and an aipha-amylase catalytic domain (i.e., a none- hybrid), or a variant thereof. In an embodiment the wild-type acid alpha-amyiase is derived from a strain of Aspergillus kawachii

Fungal Hybrid Alpha-Amylases

In a preferred embodiment the fungai acid aipha-amylase is a hybrid aipha-amylase.

Preferred examples of fungai hybrid alpha-amyiases include the ones disciosed in WO 2005/003311 or U.S. Patent Publication no. 2005/0054071 (Novozymes) or U.S. appSication no. 60/638,614 (Novozymes) which is hereby incorporated by reference. A hybrid alpha- amylase may comprise an alpha-amylase catalytic domain (CD) and a carbohydrate-binding domain/module (CBM) and optional a Sinker.

Specific examples of contemplated hybrid alpha-amyiases include those disclosed in U.S. application no, 80/638,814 including Fungamyl variant with catalytic domain JA118 and Athelia rolfsii SBD (SEQ ID NO: 100 in U.S. appiication no, 60/638,614), RNzomucor pusillus alpha-amyiase with Athelia rσlfsii AMG Sinker and SBD (SEQ ID NO: 101 in U.S. application no. 60/638,614) and Mβήpilus giganteus aSpha-amylase with Aihβlia rolfsii glucoamylase Sinker and SBD (SEQ IO NO: 102 in U.S. application no. 60/638,614),

Other specific exampSβs of contemplated hybrid aSpha-amylases include those disclosed in U, S, Application Publication no. 2005/0054071 , including those disclosed in Table 3 on page 15, such as Aspergillus niger alpha-amylase with Aspergillus kawachil linker anύ starch binding domain.

Commercial Alpha-Amylasβ Products Commercia! compositions comprising alpha-amylase include MYCOLASE™ from

DSSVl (Gist Brocades), BAN™, TERSV1ASV1YL™ SC₁ FUNGAMYl™, LSQUOZYME™ X and SAN™ SUPER, SAN™ EXTRA L (Novozymes A/S) and CLARASE™ L-40,000, DEX-LQ™, SPEZYME™ FRED, SPEZYME™ AA, SPEZYME™ HPA and SPEZYME™ DELTA AA (Genencor Int.), and the acid fungal aSpha-amylase sold under the trade name SP288 {avaiiable from Novozymes A/S, Denmark).

An alpha-amylase may according to the invention be added in an amount of 0.1 to 10 AFAU/g DS, preferably 0,10 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS or or 0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS.

When measured in KNU units the aipha-amyiase activity is preferably present in an amount of 0.0005-5 KNU per g DS₁ preferably 0.001-1 KNU per g DS, such as around 0.050 KNU per g DS.

Puiiuianase

The puiiuianase may be any puiiuianase, preferably of bacteria! origin. Puilulanases (E.G. 3.2.1.41 , puilulan 6-glucano~hydro!ase), are de-branching enzymes characterized by their ability to hydroiyze the alpha-1 ,8-glycosιdic bonds in, for example, amyiopectin and puliuian.

Specifically contemplated puiluianases include pulluianases from the genus Bacillus, preferably Bacillus amyioderamificans disclosed in U.S. Patent No. 4,560,651 (hereby incorporated by reference), the puiiuianase disclosed as SEQ ID NO: 2 in WO 01/151620

(hereby incorporated by reference), the Bacillus deramifscans disclosed as SEQ ID NO: 4 in WO 01/151620 (hereby incorporated by reference), and the puiiuianase from Baaiius aάdopullutyiicus disclosed as SEQ ID NO: 6 in WO 01/151620 (hereby incorporated by reference) and also described in FEMS Mic. Let. (1994) 115, 97-106,

Suitable commercially available puliuianase products include PROMOZYIVSE D, PROMOZYME™ D2 (Novozymes A/S, Denmark), OPTiMAX L-30G (Genencor Int., USA), and AMANO 8 (Amanα, Japan).

The puliuSanase may according to the invention be added in an effective amount wiiich include the preferred range from between 1-100 micro g per g DS, especialiy from 10- 60 micro g per g DS, Puliuianase activity may be determined as NPUN. An Assay for determination of NPUN is described in the "Materials & Methods" -section beiow.

Compositions

In the ftnai aspect the invention relates to a composition comprising a combination of an aipha-amylase as described above and a carbohydrate-source generating enzyme. More specifically the invention relates to a composition comprising i) a carbohydrate-source generating enzyme; and ii) an alpha-amyiase seiected from the group consisting of. (v) the aipha-amylase shown in SEQ SD NO: 2, or i) an alieiSc variant thereof having alpha-amyiase activity, or ii) a fragment thereof having alpha-amyiase activity,

(x) the aipha-amylase having an amino acid sequence which has at ieast 60% identity with amino acids 1 to 435 of SEQ !D NO: 2;

(y) the alpha-amyiase which is encoded by a nucleotide sequence (i) which hybridizes under at ieast Sow stringency conditions with nucieotides 4 to 1308 of SEQ ID NO: 1 , or (ii) a complementary strand of (i); or

(z) a variant comprising a conservative substitution, deletion, and/or insertion of one or more amino acids in positions 1 to 435 of SEQ SD NO: 2, The alpha-amyiase may in a preferred embodiment have at ieast 65% identity with the mature part of amino acids 1-435 of SEQ SD NO: 2, preferably at ieast 70% identity, preferably at ieast 80% identity, at least 85% identity, at least 90% identity, at ieast 95%, preferably at least 96% , more preferably at ieast 97%, more preferably at least 98% identity, or more preferably at ieast 99% identity with the mature part of amino acids 1-435 of SEQ ID NO: 2.

The carbohydrate-source generating enzyme may be any carbohydrate-source generating enzymes, preferabiy the ones mentioned in the "Carbohydrate-Source generating enzyme" section above. Especially contemplated are gSucoamySases selected from the group consisting of glucoamylases derived from the genus Aspergillus, preferably a strain of Aspergillus niger, Aspergillus oryzaβ, Aspergillus awamorL or the genus Athelia, preferably a strain of Athelia rolfsii, the genus Talaromyces, preferably a strain the Talaromyces emersonii, or the genus RNzopus, such as a strain of Rhizopus nivius, or of the genus Hutnicola, preferably a strain of Humicola grisea var, thermoidea, or a strain of the genus Trametes, preferably a strain of Tramβtøs cingulata.

In a preferred embodiment the amount to glucoamyiase is adjusted so that the composition provides an amount during use of 0.001 to 10 AGU/g DS, preferably from 0,01 to 5 AGU/g DS_S especiaily 0,1 to 0,5 AGU/g DS,

In a preferred embodiment the amount of aipha-amylase is adjusted so that the composition provides an amount during use of 0.01 to 10 AFAU/g DS, preferably 0.1 to 5 AFAU/g DS, especiaiiy 0.3 to 2 AFAU/g DS or in an amount of 0,0005-5 KNU per g DS, preferably 0,001-1 KNU per g DS, such as around 0.050 KNU per g DS.. In a preferred embodiment the aipha-amylase and carbohydrate-source generating enzyme, preferably giucoamylase is present in the composition in a ratio of between 0.1 and 10 AGU/AFAU, preferably 0.30 and 5 AFAU/ AGU, especiaiSy between 0.5 and 3 AFAU/AGU.

The above composition is suitabie for use in a fermentation product producing process of the invention.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as iiiustrations of severa! aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and de-scribed herein wtil become apparent to those skilled in the art from the foregoing description. Such modifications are aiso intended to fall within the scope of the appended claims. In the case of conflict, the present disciosure inciuding definitions wiil control.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. The present invention is further described by the following exampSβs which should not be construed as limiting the scope of the invention.

Materials & Methods

Glucoamylases: Glucoamyiase AN; Giucoamyiase derived from Aspergillus niger disclosed in Boe! et ai. (EIvI BO J. 3(5): 1097-1102 (1984)) and availabie from Novozymes A/S, Denmark. Giucoamyiase TC: Giucoamyiase derived from Trametes cingulata disclosed in WO 2006/069289 and disclosed in SEQ ID NQ; 4 herein. The enzyme is aiso available from Novozymes /VS, Denmark on request.

Giucoa mγiase TjE ; Giucoamyiase derived from Talaromyc&s emersonii and disciosed as SEQ ID NO: 7 in WO 99/28448.

ΔJPM-_.Amyjase A; Hybrid aipha-amylase disclosed in SEQ !D NO; 2 and further disclosed in table 1 of Richardson et al, (The Journal of θioiogical Chemistry, Vo!. 277, No 29, pp. 26501-28507 (2002)) as BD5088.

Yeast: RED STAR™ available from Red Star/Lesaffre, USA

Mgd_.ia_.an_cj__reag_ents;

Chemicais used as buffers and substrates were commercSa! products of at ieast reagent grade.

PDA: 39 g/L Potato Dextrose Agar, 20 g/L agar, 50 ml/L glyceroi

Methods

Unless otherwise stated, DNA manipulations and transformations were performed using standard methods of moiecular biology as described in Sambrook et a!., 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.) "Moiecular Biological Methods for Bacillus".

John Wiiey and Sons, 1990.

Giucoamyiase activity

Giucoamyiase activity may be measured in AGI units or in Giucoamyiase Units (AGU).

GJ_.M_.coamyjase_.actjvjty_..(AG!)

Giucoamyiase (equivalent to amyloglυcosidase) converts starch into glucose. The amount of giucose is determined here by the glucose oxidase method for the activity determination. The method described in the section 76-11 Starch — Giucoamyiase Method with Subsequent Measurement of Glucose with Giucose Oxidase in "Approved methods of the American Association of Cereal Chemists", Voi,1~2 AACC, from American Association of Cereal Chemists, 2000; ISBN; 1-891 127-12-8.

One glucoamyiase unit (AGI) is the quantity of enzyme which will form 1 micro mole of glucose per minute under the standard conditions of the method.

Standard conditions/reaction conditions:

Substrate: Soluble starch, concentration approx. 16 g dry maiter/L

Buffer: Acetate, approx. 0.04 M, pH=4,3 pH; 4.3

Incubation temperature: 60^aC

Reaction time: 15 minutes

Termination of the reaction: NaOH to a concentration of approximately 0,2 g/L (pH~9)

Enzyme concentration: 0,15-0.55 AAUZmL

The starch should be Lintner starch, which is a thin-boiling starch used in the laboratory as colorimetric indicator. Lintner starch is obtained by dilute hydrochloric acid treatment of native starch so that it retains the ability to color blue with iodine.

GSucoamySase activity (AGU)

The Novo Giucoamyiase Unit (AGU) is defined as the amount of enzyme, which hydroiyzes 1 micromole maltose per minute under the standard conditions 37⁰C₁ pH 4.3, substrate: maltose 23,2 mM, buffer, acetate 0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. IVSutarotase is added to the glucose dehydrogenase reagent so that any alpha-D-glucose present is turned into beta-D-glucose.

Glucose dehydrogenase reacts specifically with beta-D-glucose in the reaction mentioned above, forming NADH which is determined using a photometer at 340 nm as a measure of the original giucose concentration.

A folder (EB-SM-PI.3.1 ,02/0.1) describing this analytical method in more detail is available on request from Novozymes A/S, Denmark, which folder is hereby included by reference.

Alpha-amyiase activity (KNU)

The alpha-amyiase activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. initially, a blackish-blue color is formed, but during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which is compared to a colored glass standard.

One Kilo Novo aipha amyiase Unit (KNU) is defined as the amount of enzyme which, under standard conditions (i.e., at 37⁰C +/- 0.05; 0,0003 M Ca²'; and pH 5.6) dexfrinizes 5260 mg starch dry substance Merck Amylurπ solubile,

A folder EB-SM-0009.02/01 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.

Acid aJpha-amytase activity

When used according to the present invention the activity of any acid alpha-amyiase may be measured in AFAU (Acid Fungal Alpha-amyiase Units), Alternatively activity of acid alpha-amyiase may be measured in AAU (Acid Aipha-amylase Units).

Acid Alpha-amyiase Units fAAU)

The acid alpha-amyiase activity can be measured in AAU (Acid Alpha-amyiase Units), which is an absolute method. One Acid Amyiase Unit (AAU) is the quantity of enzyme converting 1 g of starch (100% of dry matter) per hour under standardized conditions into a product having a transmission at 620 nm after reaction with an iodine solution of known strength equal to the one of a color reference.

Standard conditions/reaction conditions;

Substrate: SoSuble starch. Concentration approx, 20 g DS/L

Buffer: Citrate, approx. 0.13 M₁ pH=4.2 iodine solution: 40.176 g potassium iodide + 0.088 g iodine/L

City water 15°-20^vdH (German degree hardness) pH: 4.2 incubation temperature: 3CPC

Reaction time: 11 minutes

Wavelength: 620 nm

Enzyme concentration: 0.13-0.19 AAU/rnL

Enzyme working range' 0.13-0.19 AAU/mL

The starch should be Lintner starch, which is a thin-boiling starch used in the laboratory as colorimetric indicator. Lintner starch is obtained by dilute hydrochloric acid treatment of native starch so that it retains the ability to color bSuβ with iodine. Further details can be found in EP 0140410 B2, which disclosure is hereby included by reference.

Determination of FAU-F

FAU(F) Fungal ASpha- Amylase .Units (Fungamyl) is measured relative to an enzyme standard of a declared strength.

A foider (EB-SM-0216 02) describing this standard method in more detai! is available on request from Novozymes A/S, Denmark, which folder is hereby included by reference. Acid alpha-amySase activity (AFAU)

Acici alpha-amylase activity may be measured in AFAU (Acid Fungal Alpha-amySase Units), whicn are determined relative to an enzyme standard, 1 AFAU is defined as the amount of enzyme which degrades 5,260 mg starch dry matter per hour under the below mentioned standard conditions.

Acid aipha-amylase, an endo-aipha-amylase (1 ,4-aipha-D-giucan-glucanohydroiase, E.C. 3.2,1.1} hydrolyzes alpha- 1 ,4-glucosidic bonds in the inner regions of the starch molecυiθ to form dβxtrins and oligosaccharides with different chain iengths. The intensity of color formed with iodine is directly proportional to the concentration of starch, Amyiase activity is determined using reverse colorimetry as a reduction in the concentration of starch under the specified anaSytieai conditions,

ALPHA - AMYLASE

STARCH + IODINE xr-^τ→ DEXTRJNS + OLl GOS ACCHARf DES

Λ =590nni blue/violet t - 23 sec. decoloration

Standard conditions/reaction conditions:

Substrate: Soluble starch, approx. 0.17 g/L

Buffer: Citrate, approx. 0.03 M

Iodine (I2): 0.03 g/L

CaC!2: 1.85 mM pH: 2.50 ± 0.05

Incubation temperature: 40⁰C

Reaction time: 23 seconds

Waveiength: 590nm

Enzyme concentration: 0,025 AFAU/mL

Enzyme working range: 0.01 -0,04 AFAU/mL

A foicfer E8-SM-0259.02/01 describing this anaiyticai method in more detail is avaiiable upon request to Novozyrnβs A/S, Denmark, which folder is hereby included by reference.

Determination of [^allogenic Amylase activity (MANU)

One MANU (Myogenic Amylase Novo Unit) may be defined as the amount of enzyme required to release one micro mole of maltose per minute at a concentration of 10

2K mg of maStotriose (Sigma M 8378} substrate per mi of 0,1 M citrate buffer, pH 5.0 at 37⁰G for 30 minutes.

Determination of pulluianase activity (NPUN) Endø-puϋuianase activity in NPUN is measured relative to a Novozyrnes puliuianase standard. One puliuianase unit (NPUN) is defined as the amount of enzyme that reieases 1 micro moi gSucose per minute under the standard conditions {0.7% red puliuSan (Megazyme), pH 5, 40^*C, 20 minutes), The activity is measured in NPUN/ml using red puilulan.

1 mi diluted sampie or standard is incubated at 40^aC for 2 minutes, 0.5 mi 2% red puliuian, 0,5 U KCi₁ 50 mU citric acid, pH 5 are added and mixed. The tubes are incubated at 40"C for 20 minutes and stopped by adding 2.5 mi 80% ethanoi. The tubes are left standing at room temperature for 10-80 minutes foiiowed by centrifugation 10 minutes at 4000 rpm. OD of the supematants is then measured at 510 nm and the activity caicuiated using a standard curve.

EXAMPLES Example 1 Yeast Propagation

Yeast is propagated prior to fermentation. Corn is ground to pass through #45 mesh screen. 200 mi tap water and 1 g urea are mixed with 300 g corn mash. PenSciliin is added to 3 mg/iiter. in 50 g of the mash slurry, 6.4 microL Giucoamyiase AN and 0.024 g dry yeast (RED STAR™) are added and the pH is adjusted to around 5.0. The yeast slurry is incubated at 32°C with constant stirring at 300 rpm for 7 hours in a partiaiiy open flask.

One-Step.... ferfneπtatjon yslπ.fl....Aip.ha-Amy|ase....A..J.lscjgsM....in SEQ [D NO: 2....and

Giucoamyiase TC

Ai! one step ground com to ethanoi treatments are evaluated via mini-scale fermentations. Briefly, 410 g of ground corn (with particie size around 0.5 mm) is added to 590 g tap water. This mixture is supplemented with 3.0 mi 1 g/L penicillin and 1 g of urea. The pH of this sSurry is adjusted to 4.5 with 5 N NaOH or diluted H₂SO..!. DS ievel is adjusted to around 35 wt-%. Approximately 5 g of this slurry is added to 20 mi vials. Each vial is dosed with the appropriate amount of enzyme as set out in the table beiow followed by addition of 200 microL yeast propagate per 5 g siurry. Actual enzyme dosages are based on the exact weight of corn siurry in each vial. Viais are closed and incubated at 32"C immediately. 9 repSicate fermentations of each treatment are run. Three repiicates are seiected for 24 hours, 48 hours and 70 hours time point anaiysis. Vials are vortexed at 24, 48 and 70 hours and analyzed by HPLC. The HPLC preparation consists of stopping the reaction by addition of 50 microL of 40% HjSO₄, centrifuging, and filtering through a 0.45 micrometer filter. Samptes are stored at 4°C prior to analysis,

Agilent™ 1100 HPLC system coupled with R! detector is used to determine ethanol and sugars. The HPLC system consists of a degasser, quat-pump, cooied autosampler and heated column compartment. The separation column may be aminex HPX-87H ion exciusion column (300 mm x 7.8 mm) from BioRad™, which Sinks to 30 mm x 4.8 mm micro- guard cation-H cartridge guard column. A 10 microL sample is injected at the flow rate of 0.6 ml/min. The mobile phase is 5 mM H₂SO^. The column is kept at 65"C and R! detector at 5O⁰C. The total run time is 25 minutes per sampSe,

The ethanoi yields after 24, 48 and 70 hours are determined.

Example 2 Liquefaction and SSF using Alpha- Amyiase A disclosed in SEQ ID NO: 2

Ground corn is used to make a 30 wt-% slurry with tap water. The pH is adjusted to approximateiy 5.0 using NaOH or diiuted H₂SO^. 50 NU/ g DS Alpha-Amylase A is added and kept at 85°C for 1.5 hours,

SSF is done as mini-scaie fermentations, if needed, the pH is adjusted to 5.0, e.g. _r with diiuted H^SO*. Mash is adjusted to a 0.5 g/L concentration Urea and 3 mg/L Peniciln,

3«) Approximately 4 grams of mash is added to 18 m! polystyrene tubes (Falcon 352025), Tubes are then dosed with the appropriate amount of Giucoamyiase TE {0,3 AGU/g DS). After dosing the tubes with enzyme, they are inoculated with 0.04 rnl/g mash of yeast propagate {RED STAR™) that is grown for 21 hours on com mash. Vials are capped with a screw on Hd which is punctured with a needle to allow gas release and vortexed briefly before weighing and incubation at 32°C. Fermentation progress is followed by weighing the tubes over time. Tubes are vortexed briefly before each weighing. Weight loss values are converted to ethanol yield {g ethanol/g DS) by the following formula:

er 70 hours of fermentation, replicates from fermentation are sacrificed for HPLC analysis for residual sugar and glycerol concentrations. The reactions are stopped by adding 30 MicroL 40% H₂SO₄ to each. The tubes are centήfuged at 3000 rpm for 15 minutes to clear the supernatant, and then 1 ml of cleared supernatant is passed through a 0.45 micron syringe filter and placed in HPLC viais. The samples are analyzed by using an Agilent HPLC System using analytical BiO-RAD Aminex HPX-87H column and a BIO-RAD Cation H reftil guard column. HPLC run conditions are; 0. QQSIVi H-SO₄ mobile phase, flow rate of 0.6 ml/mSn, column temperature at 85⁰C, Ri detector (Refractive Index) at 5O'^;C, injection volume of 10 ml. and a 25 min run time.

Claims

Claims

1. A process for producing a fermentation product from starch-containing material comprising the steps of:

5 (a) liquefying starch-containing material with an alpha-amyiase;

(fa) saccharifying the liquefied materia! using a carbohydrate- source generating enzyme;

(C) fermenting using a fermenting organism. wherein the aipha-amylase used in the liquefaction step (a) is selected from the group I O consisting of;

(v) the aipha-amylase shown in SEQ SD NO: 2, or i) an alieiic variant thereof having alpha-amyiase activity, or ii) a fragment thereof having aipha-amylase activity, (x) an alpha-arnySase having an amino acid sequence which has at ieast 15 60% identity with amino acids 1 to 435 of SEQ SD NO: 2;

(y) an aipha-amylase which is encoded by a nucieotide sequence (i) which hybridizes under at ieast Sow stringency conditions with nucieotides 4 to 1308 of SEQ ID NO: 1 , or (ii) a complementary strand of (i); or

(z) a variant comprising a conservative substitution, deletion, and/or 0 insertion of one or more amino acids in positions 1 to 435 of SEQ SD NO: 2.

2. The process of ciaim 1 , wherein one or more carbohydrases selected from the group of aipha-amylase, puiiulanase, beta-amyiase, or a combination thereof, is introduced during step (a). 5

3. The process of claim 1 or 2, wherein the aipha-amylase comprises an amino acid sequence which has at least 65% identity with amino acids 1-435 of SEQ SD NO: 2« preferably at ieast 70% identity, preferably at least 80% identity, at least 85% identity, at least 90% identity, at least 95%, preferabiy at least 96%, more preferably at least 97%, more 0 preferably at ieast 98% identity, or more preferably at least 99% identity with amino acids 1- 435 of SEQ SD NO: 2.

5. The process of any of cSaims 1-3, wherein the alpha-amyiase is derived from a microorganism of the order Thermococcales,

6. The process of any of claims 1-5. wherein the carbohydrate-source generating enzyme is a giucoamylase, beta-amyiase or maltogenic amylase, or a mixture thereof.

7. The process of any of claims 1 -6, wherein the fermentation product is an alcohol, preferably ethanoi, especialiy fuel ethanoi, potable ethanoi and/or industrial ethanoi

8. The process of any of claims 1-7, wherein the step (b) and (c) are carried out sequentially or simultaneously (i.e., SSF process).

9. A process for producing a fermentation product from starch-containing material comprising:

(a) saccharifying starch-containing material with an aipha-amySase at a temperature below the initia! geiatiπization temperature of said starch-containing material,

(b) fermenting using a fermenting organism, wherein the alpha-amyiase used in liquefaction saccharification step (a) or simultaneous saccharification anύ fermentation in combined step (a) and (b) is selected from the group consisting of.

(v) the aipha-amylase shown in SEQ ID NO: 2, or i) an allelic variant thereof having alpha-amyiase activity, or ii) a fragment thereof having alpha-amylase activity,

(x) an alpha-amylase having an amino acid sequence which has at least 60% identity with amino acids 1 to 435 of SEQ iD NO: 2;

(y) an aipha-amylase which is encoded by a nucieotide sequence (i) which hybridizes under at least low stringency conditions with nucleotides 4 to 1308 of SEQ ID NO: 1 , or (ii) a complementary strand of (i); or

(z) a variant comprising a conservative substitution, deletion, and/or insertion of one or more amino acids in positions 1 to 435 of SEQ !D NO: 2.

10. A process of claim 9, further wherein an acid alpha-amyiasβ, such as an acid fungal alpha-amylase, or a piant aipha-amylase, is introduced during fermentation or simultaneous saccharification anά fermentation.

11. The process of claim 9 or 10, wherein the alpha-amylase comprises an amino acid sequence which has at least 65% identity with amino acids 1-435 of SEQ SD NO; 2, preferably at least 70% identity, preferably at least 80% identity, at least 85% identity, at least 90% identity, at least 95%, preferably at least 96%, more preferably at least 97%, more preferably at least 98% identity, or more preferably at ieast 99% identity with the amino acids 1-435 of SEQ ID NO: 2,

12. The process of any of claims 9-11 , wherein the aipha-amylase is derived from a microorganism of the order Thermococcalβs.

13, The process of any of claims 9-12, wherein the sacchahfication and fermentation is carried out sequentially or simultaneously (i.e., one-step fermentation),

14. The process of any of claims 9-13, wherein the temperature during saccharification in step (a) is in the range from 30"C to 75'⁵C, preferabiy between 45 and 6O⁰C.

15. The process of any of claims 9-14, wherein the temperature during simultaneous saccharification and fermentation, or fermentation in step (b) is between 28¹⁵C and 36"C, such as between 29'⁵C and 3S⁵C, such as between 30"C and 34"C, such as around 32"C,

16. The process of any of claims 9-15, wherein a carbohydrate-source generating enzyme is present during saccharification in step (a) or simultaneous saccharification and fermentation in combined steps (a) and (b).

17. The process of any of claims 9-16, wherein the starch-containing material is uncooked granuiar starch.

18. The process of any of claims 9-17. wherein the fermentation product is an alcohol, preferably ethanol, especiaily fuel ethanol, potabie ethanol and/or industriai ethanol.

19. A composition comprising i) a carbohydrate-source generating enzyme; &nά ii) an alpba-amylase selected from the group consisting of: (V) the aSpha-arnylase shown in SEQ ID NO: 2, or i) an alSβiϊε variant thereof having alpha-amyiase activity, or ii) a fragment thereof having alpha-amylase activity, (x) an alpha-amylase having an amino acid sequence which has at ieast 60% identity with amino acids 1 to 435 of SEQ ID NO: 2; (y) an alpha-amylase which is encoded by a nucleotide sequence (i) which hybridizes under at least Sow stringency conditions with nucleotides 4 to 1308 of SEQ ID NO: 1 , or (ii) a complementary strand of (i); or

(z) a variant comprising a conservative substitution, deletion, and/or insertion of one or more amino acids in positions 1 to 435 of SEQ SD NO: 2.

20, The composition of claim 19, wherein the alpha-amylase comprises an amino acid sequence which has at least 65% identity with amino acids 1-435 of SEQ SD NO: 2« preferably at least 70% identity, preferably at least 80% identity, at least 85% identity, at least 90% identity, at least 95%, preferably at least 96%, more preferably at least 97%, more preferably at least 98% identity, or more preferably at least 99% identity with amino acids 1- 435 of SEQ ID NO: 2.

21 The composition of claim 19 or 20, wherein the alpha-amylase is derived from a microorganism of the order Thermococcales.

22. The composition of any of claims 19-21 , further wherein the composition comprises a second aSpha-amyjase and/or a puϋulanase.

23. The composition of any of claims 19-22, wherein the carbohydrate-source generating enzyme is a giucoamylase, beta-amyiase or maltogenic amylase, or a mixture thereof.

24. Use of a composition of any of claims 19-23 in a process of claims 1-18.