WO2011100161A1 - Addition of alpha - glucosidase and cobalt for producing fermentation products from starch - Google Patents

Addition of alpha - glucosidase and cobalt for producing fermentation products from starch Download PDF

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Publication number
WO2011100161A1
WO2011100161A1 PCT/US2011/023675 US2011023675W WO2011100161A1 WO 2011100161 A1 WO2011100161 A1 WO 2011100161A1 US 2011023675 W US2011023675 W US 2011023675W WO 2011100161 A1 WO2011100161 A1 WO 2011100161A1
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alpha
process
amylase
preferably
starch
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PCT/US2011/023675
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French (fr)
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Chee-Leong Soong
Jeremy Saunders
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Novozymes North America, Inc.
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Publication of WO2011100161A1 publication Critical patent/WO2011100161A1/en

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    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405Glucanases
    • C12N9/2408Glucanases acting on alpha -1,4-glucosidic bonds
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    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405Glucanases
    • C12N9/2408Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411Amylases
    • C12N9/2414Alpha-amylase (3.2.1.1.)
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    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405Glucanases
    • C12N9/2408Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411Amylases
    • C12N9/2425Beta-amylase (3.2.1.2)
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    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405Glucanases
    • C12N9/2408Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411Amylases
    • C12N9/2428Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/20Preparation of compounds containing saccharide radicals produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/02Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06Ethanol, i.e. non-beverage
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y302/00Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/0102Alpha-glucosidase (3.2.1.20)
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02EREDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00Technologies for the production of fuel of non-fossil origin
    • Y02E50/10Biofuels
    • Y02E50/17Grain bio-ethanol

Abstract

The present invention relates to a method of increasing fermentation product yield in processes for producing fermentation products from un-gelatinized starch-containing material, and compositions utilized in such processes.

Description

ADDITION OF ALPHA-GLUCOSIDASE AND COBALT FOR PRODUCING FERMENTATION PRODUCTS FROM STARCH

REFERENCE TO A SEQUENCE LISTING

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

FIELD OF THE INVENTION

The present invention relates to a method of increasing fermentation product yield in processes for producing fermentation products from un-gelatinized starch-containing material, and compositions utilized in such processes.

BACKGROUND OF THE INVENTION

Production of fermentation products, such as ethanol, from starch-containing material is well-known in the art. Generally two different kinds of processes are used. The most commonly used process, often referred to as a "conventional process", includes liquefying gelatinized starch at high temperature using typically a bacterial alpha-amylase, followed by simultaneous saccharification and fermentation carried out in the presence of a glucoamylase and a fermenting organism. Another well known process, often referred to as a "raw starch hydrolysis"-process (RSH process) includes simultaneously saccharifying and fermenting granular starch below the initial gelatinization temperature typically in the presence of an acid fungal alpha-amylase and a glucoamylase.

WO 2005/1 13785 and WO 2007/076388 disclose the use of an alpha-glucosidase during processes for producing fermentation products from starch-containing material.

WO 2006/005032 discloses isolated polypeptides with alpha-glucosidase activity and isolated polynucleotide sequences encoding the same.

Despite the growth of the ethanol production industry, the overall costs of such production are relatively high. Therefore, there is still a desire and need for providing improved processes for producing fermentation products, such as ethanol, from starch-containing material and a continued need to improve the associated process economics.

SUMMARY OF THE INVENTION

The present invention relates to methods of increasing fermentation product yield in processes for producing fermentation products from un-gelatinized starch-containing material.

In the first aspect the invention relates to processes for producing fermentation products from starch-containing material comprising simultaneously saccharifying and fermenting starch- containing material using an alpha-amylase, a carbohydrate-source generating enzyme and a fermenting organism at a temperature below the initial gelatinization temperature of said starch- containing material and in the presence of alpha-glucosidase activity and cobalt (Co2+) ions.

In a second aspect the invention relates to composition comprising alpha-glucosidase activity and Co2+.

In a third aspect the invention relates to uses of compositions comprising alpha- glucosidase activity and Co2+ in processes for producing fermentation products from starch- containing material comprising simultaneously saccharifying and fermenting starch-containing material using an alpha-amylase, a carbohydrate-source generating enzyme and a fermenting organism at a temperature below the initial gelatinization temperature of said starch-containing material.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that when adding Co2+ and alpha-glucosidase activity in a raw starch hydrolysis process (RSH process), the ethanol yield is higher than compared to adding alpha-glucosidase activity without Co2+ to the same process. The Co2+ may be added before, during, or after the alpha-glucosidase is added to the RSH process. Treating the composition comprising alpha-glucosidase activity with Co2+ before the treated composition is added to the RSH process is preferred.

Not being bound by any particular theory, it is believed that the Co2+ may have a positive effect on alpha-glucosidase activity and/or enzyme protein stability. By way of example, the inventors have shown that ethanol yield in a process for producing ethanol from un-gelatinized starch materials is higher when alpha-glucosidase activity and Co2+ are present, as compared to the yield from the same process wherein alpha-glucosidase activity is present but Co2+ is not present. The increased yield effect is shown, by way of example, when alpha-glucosidase activity and Co2+ are added directly to the fermentation in the RSH process, but a much higher concentration of Co2+ in the fermentation medium is required to achieve the same result that can be achieved when the Co2+ is used to treat a composition comprising alpha-glucosidase activity prior to adding the treated composition to the RSH process. Little to no effect is seen on ethanol yield when the Co2+ is added to the RSH process without the addition of alpha- glucosidase activity.

Alpha-Glucosidase

Alpha-glucosidases (EC 3.2.1.20) hydrolyze terminal, non-reducing alpha-1 ,4-linked glucose residues in various substrates, releasing glucose. They degrade disaccharides and oligosaccharides quickly while polysaccharides are attacked slowly if at all. Maltose, maltose derivatives, sucrose, aryl-alpha-glucosides, and alkyl-alpha-glucosides can act as substrates. The term "alpha-glucosidase activity" is defined herein as an alpha-D-glucoside glucohydrolase activity (E.C. 3.2.1.20) which catalyzes the exohydrolysis of terminal, non- reducing 1 ,4-linked alpha-D-glucose residues with the release of alpha-D-glucose. Natural substrates of the enzyme activity include, for example, maltose, maltotriose, maltotetraose, maltopentaose, starch (soluble), amylose, amylopectin, isomaltose, Kojibiose, sucrose, nigerose, turanose, melizitose, and glycogen. For purposes of the present invention, alpha- glucosidase activity is determined with maltose as substrate in 0.1 M sodium acetate buffer pH 4.3 at 25°C. One unit of alpha-glucosidase activity is defined as 1.0 micromole of glucose produced per minute at 25°C, pH 4.3 from maltose as substrate in sodium acetate buffer.

According to the invention, the alpha-glucosidase activity may be any alpha- glucosidase, In a preferred embodiment the alpha-glucosidase is from an alpha-glucosidase derived from rice, corn, the genus Bacillus or the genus Aspergillus. In one embodiment, the alpha-glucosidase activity is from an alpha-glucosidase derived from Aspergillus fumigatus. In another embodiment, the alpha-glucosidase activity is from an alpha-glucosidase derived from Bacillus stearot ermop ilus. In a preferred embodiment, the alpha-glucosidase activity is from an alpha-glucosidase derived from Aspergillus fumigatus that is expressed in Trichoderma reesei, and has the amino acid sequence disclosed as amino acids 20-988 shown in SEQ ID NO: 1 , or an alpha-glucosidase being at least 85% identical thereto, or at least about 87%, or at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least 99% identical thereto.

Processes for producing fermentation products from un-gelatinized starch-containing material

In this aspect the invention relates to processes for producing fermentation products from starch-containing material without gelatinization (i.e., without cooking) of the starch- containing material. According to the invention the desired fermentation product, such as ethanol, can be produced without liquefying the aqueous slurry comprising the starch- containing material and water. In one embodiment a process of the invention includes saccharifying (e.g., milled) starch-containing material, e.g., granular starch, below the initial gelatinization temperature, preferably in the presence of alpha-amylase and/or carbohydrate- source generating enzyme(s) to produce sugars that can be fermented into the desired fermentation product by a suitable fermenting organism.

In this embodiment the desired fermentation product, preferably ethanol, is produced from un-gelatinized (i.e., uncooked), preferably dry milled cereal grains, such as corn.

Accordingly, in the first aspect the invention relates to processes for producing fermentation products from starch-containing material comprising simultaneously saccharifying and fermenting starch-containing material using an alpha-amylase, a carbohydrate-source generating enzyme and a fermenting organism at a temperature below the initial gelatinization temperature of said starch-containing material and in the presence of alpha-glucosidase activity and cobalt (Co2+) ions.

The fermentation product, such as especially ethanol, may optionally be recovered after fermentation, e.g., by distillation. Suitable starch-containing starting materials are listed in the "Starch-Containing Materials" section below. Typically alpha-amylase(s) and/or carbohydrate- source generating enzymes such as glucoamylase(s), is(are) present during fermentation.

Examples of carbohydrate-source generating enzymes such as glucoamylases can be found below and include raw starch hydrolyzing glucoamylases.

Examples of alpha-amylase(s) include acid alpha-amylases, preferably acid fungal alpha-amylases.

Examples of fermenting organisms include yeast, preferably a strain of Sacc aromyces cerevisiae. Other suitable fermenting organisms are listed in the "Fermenting Organisms" section.

The term "initial gelatinization temperature" means the lowest temperature at which starch gelatinization commences. In general, starch heated in water begins to gelatinize between about 50°C and 75°C; the exact temperature of gelatinization depends on the specific starch and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In context of this invention the initial gelatinization temperature of a given starch-containing material may be determined as the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein et al., 1992, Starch/Starke 44(12): 461-466.

In one embodiment, a slurry of starch-containing material, such as granular starch, preferably granular corn starch, having 10-55 w/w-% dry solids (DS), preferably 25-45 w/w-% dry solids, more preferably 30-40 w/w-% dry solids of starch-containing material may be prepared. The slurry may include water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants. Because the process of the invention is carried out below the initial gelatinization temperature, and thus no significant viscosity increase takes place, high levels of stillage may be used if desired. In an embodiment the aqueous slurry contains from about 1 to about 70 vol.-%, preferably 15-60% vol.-%, especially from about 30 to 50 vol.-% water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants, or combinations thereof, or the like.

The starch-containing material may be prepared by reducing the particle size, e.g., by milling, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably 0.1-0.5 mm. After being subjected to a process of the invention at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 preferably at least 99% of the dry solids in the starch-containing material are converted into a soluble starch hydrolysate.

A process in this aspect of the invention is conducted at a temperature below the initial gelatinization temperature, which means that the temperature typically lies in the range between 30-75°C, preferably between 45-60°C.

In a preferred embodiment the process is carried at a temperature from 25°C to 40°C, such as from 28°C to 35°C, such as from 30°C to 34°C, preferably around 32°C.

A skilled person in the art can easily determine which doses/quantities of enzyme and fermenting organism to use.

The amount of alpha-glucosidase used according to the invention is an effective amount. Accordingly, in one embodiment the amount of alpha-glucosidase for use in a process of the invention is between 0.01 to 10 AGU/g dry solids. In another embodiment, the amount of alpha-glucosidase for use in a process of the invention is between 0.05 to 5.00 AGU/g dry solids.

The amount of cobalt to be used according to the invention is an effective amount. Accordingly, in one embodiment, the amount of cobalt used in the process of the invention is between 0.5 - 200 nmol/g mash.

In an embodiment the process is carried out so that the sugar level, such as glucose level, is kept at a low level, such as below 6 w/w-%, such as below about 3 w/w-%, such as below about 2 w/w-%, such as below about 1 w/w-%., such as below about 0.5 w/w-%, or below 0.25 w/w-%, such as below about 0.1 w/w-%. Such low levels of sugar may be accomplished by employing adjusted quantities of enzyme and fermenting organism.

The employed quantities of enzyme and fermenting organism may also be selected to maintain low concentrations of maltose in the fermentation broth. For instance, the maltose level may be kept below about 0.5 w/w-%, such as below about 0.2 w/w-%.

The process of the invention may be carried out at a pH from about 3 and 7, preferably from pH 3.5 to 6, or more preferably from pH 4 to 5. In an embodiment fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.

Fermentation Medium

"Fermentation media" or "fermentation medium" refers to the environment in which fermentation is carried out and which includes the fermentation substrate, that is, the carbohydrate source that is metabolized by the fermenting organism.

The fermentation medium may comprise nutrients and growth stimulator(s) for the fermenting organism(s). Nutrient and growth stimulators are widely used in the art of fermentation and include nitrogen sources, such as ammonia; urea, vitamins and minerals, or combinations thereof. The fermentation medium may also include enzymes such as amylases and/or other carbohydrate source generating enzymes, especially wherein the process of the invention is carried out as a simultaneous saccharification and fermentation process or a RSH process.

Fermenting Organisms

The term "fermenting organism" refers to any organism, including bacterial and fungal organisms, suitable for use in a fermentation process and capable of producing the desired fermentation product, such as ethanol. 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 Sacc aromyces spp., in particular, Sacc aromyces cerevisiae.

In one embodiment the fermenting organism is added to the fermentation medium so that the viable fermenting organism, such as yeast, count per ml_ of fermentation medium is in the range from 105 to 1012, preferably from 107 to 1010, especially about 5x107.

Commercially available yeast includes, e.g., RED STAR™ and ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast (available from Ethanol Technology, Wl, 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).

Starch-Containing Materials

Any suitable starch-containing material may be used according to the present invention.

The starting material is generally selected based on the desired fermentation product. Examples of starch-containing materials, suitable for use in a process of the invention, 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. Contemplated are also waxy and non-waxy types of corn and barley. In a preferred embodiment the starch-containing material is corn.

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 50°C to 75°C the swelling may be reversible. However, with higher temperatures an irreversible swelling called "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 least 99.5% pure or it may be a more crude starch-containing materials comprising (e.g., milled) whole grains including non-starch fractions such as germ residues and fibers. The raw material, such as whole grains, may be reduced in particle size, e.g. , by milling, in order to open up the structure and allowing for further processing. Two processes are preferred according to the invention: wet and dry milling. In dry milling 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, e.g. , syrups. Both dry and wet milling is well known in the art of starch processing and is equally contemplated for a process of the invention. In an embodiment the particle size is reduced to between 0.05 to 3.0 mm, preferably 0.1 -0.5 mm, or so that at least 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. In a preferred embodiment the granular starch is granular corn starch. 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., 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., H2 and C02); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B12, beta-carotene); and hormones. In a preferred embodiment the fermentation product is ethanol, e.g. , fuel ethanol; drinking 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 comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer. Preferred fermentation processes used include alcohol fermentation processes. In a preferred embodiment the fermentation product is ethanol. The fermentation product, such as ethanol, obtained according to the invention, may preferably be used as fuel. However, in the case of ethanol it may also be used as potable ethanol.

Recovery

Subsequent to fermentation the fermentation product may be separated from the fermentation medium. The slurry may be distilled to extract the desired fermentation product or the desired fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques. Alternatively the fermentation product may be recovered by stripping. Methods for recovery are well known in the art. ENZYMES

Even if not specifically mentioned in context of a process of the invention, it is to be understood that enzyme(s) is(are) used in an effective amount.

Alpha-Amylase

According to the invention any alpha-amylase may be used, such as of fungal, bacterial or plant origin. In a preferred embodiment the alpha-amylase is an acid alpha-amylase, e.g., acid fungal alpha-amylase or acid bacterial alpha-amylase. The term "acid alpha-amylase" means an alpha-amylase (E.C. 3.2.1.1 ) which added in an effective amount has activity optimum at a pH in the range of 3 to 7, preferably from 3.5 to 6, or more preferably from 4-5.

Bacterial Alpha-Amylase

According to the invention a bacterial alpha-amylase is preferably derived from the genus Bacillus.

In a preferred embodiment the Bacillus alpha-amylase is derived from a strain of Bacillus lic eniformis, Bacillus amyloliquefaciens, Bacillus subtilis or Bacillus stearothermophilus, but may also be derived from other Bacillus sp. Specific examples of contemplated alpha-amylases include the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4 in WO 99/19467, the Bacillus amyloliquefaciens alpha-amylase SEQ ID NO: 5 in WO 99/19467 and the Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 in WO 99/19467 (all sequences hereby incorporated by reference). In an embodiment the alpha- amylase may be an enzyme having a degree of identity of at least 60%, preferably at least 70%, more preferred at least 80%, even more preferred at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to any of the sequences shown in SEQ ID NOS: 1 , 2 or 3, respectively, in WO 99/19467.

The Bacillus alpha-amylase may also be a variant and/or hybrid, especially 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 alpha-amylase variants are disclosed in U.S. Patent No. 6,093,562, 6,187,576, or 6,297,038 (which are hereby incorporated by reference) and include Bacillus stearothermophilus alpha- amylase (BSG alpha-amylase) variants having a deletion of one or two amino acid in positions R179 to G182, preferably a double deletion disclosed in WO 96/23873 - see, e.g., page 20, lines 1-10 (hereby incorporated by reference), preferably corresponding to delta(181-182) compared to the wild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467 or deletion of amino acids R179 and G180 using SEQ ID NO:3 in WO 99/19467 for numbering (which reference is hereby incorporated by reference). Even more preferred are Bacillus alpha-amylases, especially Bacillus stearot ermop ilus 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- amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467.

Bacterial Hybrid Alpha-Amylase

A hybrid alpha-amylase specifically contemplated comprises 445 C-terminal amino acid residues of the Bacillus licheniformis alpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues of the alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467), with one or more, especially all, of the following substitutions:

G48A+T49I+G107A+H156Y+A181T+N190F+I201 F+A209V+Q264S (using the 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- amylase backbones): H154Y, A181T, N190F, A209V and Q264S and/or deletion of two residues between positions 176 and 179, preferably deletion of E178 and G179 (using the SEQ ID NO: 5 numbering of WO 99/19467).

In an embodiment the bacterial alpha-amylase is dosed 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.

Fungal Alpha-Amylase

Fungal alpha-amylases include alpha-amylases derived from a strain of the genus Aspergillus, such as, Aspergillus oryzae, Aspergillus niger and Aspergillis kawachii alpha- amylases.

A preferred acidic fungal alpha-amylase is a Fungamyl-like alpha-amylase which is derived from a strain of Aspergillus oryzae. According to the present invention, the term "Fungamyl-like alpha-amylase" indicates an alpha-amylase which exhibits a high identity, 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 mature part of the amino acid sequence shown in SEQ ID NO: 10 in WO 96/23874.

Another preferred acid alpha-amylase is derived from a strain of Aspergillus niger. In a preferred embodiment the acid fungal alpha-amylase is the one from Aspergillus niger disclosed as "AMYA_ASPNG" in the Swiss-prot TeEMBL database under the primary accession no. P56271 and described in WO 89/01969 (Example 3 - incorporated by reference). A commercially available acid fungal alpha-amylase derived from Aspergillus niger is SP288 (available from Novozymes A/S, Denmark). Other contemplated wild-type alpha-amylases include those derived from a strain of the genera Rhizomucor and Meripilus, preferably a strain of Rhizomucor pusillus (WO 2004/055178 incorporated by reference) or Meripilus giganteus.

In a preferred embodiment the alpha-amylase is derived from Aspergillus kawachii and disclosed by Kaneko et al., 1996, J. Ferment. Bioeng. 81 : 292-298 "Molecular-cloning and determination of the nucleotide-sequence of a gene encoding an acid-stable alpha-amylase from Aspergillus kawachiP' and further as EMBL: #AB008370.

The fungal alpha-amylase may also be a wild-type enzyme comprising a starch-binding domain (SBD) and an alpha-amylase catalytic domain (i.e., non-hybrid), or a variant thereof. In an embodiment the wild-type alpha-amylase is derived from a strain of Aspergillus kawachii.

Fungal Hybrid Alpha-Amylase

In a preferred embodiment the fungal acid alpha-amylase is a hybrid alpha-amylase. Preferred examples of fungal hybrid alpha-amylases include the ones disclosed in WO 2005/00331 1 or U.S. Application Publication no. 2005/0054071 (Novozymes) or WO 2006/069290 (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), such as a starch binding domain, and optional a linker.

Specific examples of contemplated hybrid alpha-amylases include those disclosed in Table 1 to 5 of the examples in WO 2006/069290, including Fungamyl variant with catalytic domain JA118 and Athelia rolfsii SBD (SEQ ID NO: 100 in WO 2006/069290), Rhizomucor pusillus alpha-amylase with Athelia rolfsii AMG linker and SBD (SEQ ID NO: 101 in WO 2006/069290), Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD (which is disclosed in Table 5 as a combination of amino acid sequences SEQ ID NO:20, SEQ ID NO:72 and SEQ ID NO:96 in US publication no. US 2006/0148054) or as V039 in Table 5 in WO 2006/069290, and Meripilus giganteus alpha-amylase with Athelia rolfsii glucoamylase linker and SBD (SEQ ID NO: 102 in WO 2006/069290). Other specifically contemplated hybrid alpha-amylases are any of the ones listed in Tables 3, 4, 5, and 6 in Example 4 in US publication no. US Application Publication no. 2006/0148054 and WO 2006/069290 (hereby incorporated by reference).

Other specific examples of contemplated hybrid alpha-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 kawachii linker and starch binding domain.

Contemplated are also alpha-amylases which exhibit a high identity to any of above mention alpha-amylases, 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 mature enzyme sequences.

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

Commercial Alpha-Amylase Products

Preferred commercial compositions comprising alpha-amylase include MYCOLASE™ from DSM, BAN™, TERMAMYL™ SC, FUNGAMYL™, LIQUOZYME™ X, LIQUOZYME™ SC and SAN™ SUPER, SAN™ EXTRA L (Novozymes A/S) and CLARASE™ L-40,000, DEX- LO™, SPEZYME™ FRED, SPEZYME™ AA, and SPEZYME™ DELTA AA, SPEZYME™ RSL (Genencor Int.), FUELZYME™-LF (Verenium Inc), and the acid fungal alpha-amylase sold under the trade name SP288 (available from Novozymes A/S, Denmark) and STARGEN™ 001 (Genencor Int).

Carbohydrate-Source Generating Enzyme

The term "carbohydrate-source generating enzyme" includes glucoamylase (being a glucose generator), beta-amylase and maltogenic amylase (being maltose generators) and also pullulanases. 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 mixture of carbohydrate-source generating enzymes may be used. A preferred carbohydrate-source generating enzyme is glucoamylase.

In another embodiment of the invention, at least one alpha-amylase and one glucoamylase are used. In a preferred embodiment, the alpha-amylase is a fungal alpha- amylase.

The ratio between glucoamylase activity (AGU) and fungal alpha-amylase activity (FAU- F) (i.e., AGU per FAU-F) may in a preferred embodiment of the invention be between 0.1 and 100 AGU/FAU-F, in particular between 2 and 50 AGU/FAU-F, such as in the range from 10-40 AGU/FAU-F, especially when doing one-step fermentation (raw starch hydrolysis - RSH), i.e., when saccharification and fermentation are carried out simultaneously without a liquefaction step. Glucoamylase

A glucoamylase used according to the invention may be derived from any suitable source, e.g., derived from a microorganism or a plant. Preferred glucoamylases are of fungal or bacterial origin, selected from the group consisting of Aspergillus glucoamylases, in particular Aspergillus niger G1 or G2 glucoamylase (Boel et al., 1984, EMBO J. 3(5): 1097-1 102), or variants thereof, such as those disclosed in WO 92/00381 , WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A. 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 ); disulphide bonds, A246C (Fierobe et al., 1996, Biochemistry 35: 8698-8704; and introduction of Pro residues in position A435 and S436 (Li et al., 1997, Protein Eng. 10: 1 199- 1204.

Other glucoamylases include Athelia rolfsii (previously denoted Corticium rolfsii) glucoamylase (see U.S. 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 glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (U.S. Patent No. RE 32,153), Talaromyces duponti, Talaromyces thermophilus (U.S. Patent No. 4,587,215).

Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO 86/01831 ) and Trametes cingulata, Pachykytospora papyracea; and Leucopaxillus giganteus all disclosed in WO 2006/069289; or Peniophora rufomarginata disclosed in WO 2007/124285; or a mixture thereof. Also hybrid glucoamylase are contemplated according to the invention. Examples of hybrid glucoamylases are 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).

Contemplated are also glucoamylases which exhibit a high identity to any of above mention 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 mature enzymes sequences mentioned above.

In a preferred embodiment the glucoamylase is the Trametes cingulata glucoamylase disclosed in WO 2006/069289 (available from Novozymes).

Commercially available compositions comprising glucoamylase include AMG 200L;

AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME EXCEL and AMG™ E (from Novozymes A/S); OPTIDEX™ 300, GC480, GC417 (from Genencor Int.); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™, GC019, and G990 ZR (from Genencor Int.).

Glucoamylases may in an embodiment be added in an amount of 0.0001-20 AGU/g DS, preferably 0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2 AGU/g DS.

Beta-amylase

A beta-amylase (E.C 3.2.1.2) is the name traditionally given to exo-acting maltogenic amylases, which catalyze the hydrolysis of 1 ,4-alpha-glucosidic linkages in amylose, 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 released has the beta anomeric configuration, hence the name beta-amylase.

Beta-amylases have been isolated from various plants and microorganisms (Fogarty et al., 1979, Progress in Industrial Microbiology 15: 1 12-115). These beta-amylases are characterized by having optimum temperatures in the range from 40°C to 65°C and optimum pH in the range from 4.5 to 7. A commercially available beta-amylase from barley is NOVOZYM™ WBA from Novozymes A S, Denmark and SPEZYME™ BBA 1500 from Genencor Int., USA.

Maltogenic Amylase

The amylase may also be a maltogenic alpha-amylase. A "maltogenic alpha-amylase" (glucan 1 ,4-alpha-maltohydrolase, E.C. 3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose in the alpha-configuration. A maltogenic amylase from Bacillus stearot ermop ilus strain NCIB 1 1837 is commercially available from Novozymes A/S. Maltogenic alpha-amylases are described in U.S. Patent Nos. 4,598,048, 4,604,355 and 6, 162,628, which are hereby incorporated by reference.

The maltogenic 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.

Pullulanase

Pullulanases (E.C. 3.2.1.41 , pullulan 6-glucano-hydrolase), are debranching enzymes characterized by their ability to hydrolyze the alpha-1 ,6-glycosidic bonds in, for example, amylopectin and pullulan.

Specifically contemplated pullulanases according to the present invention include the pullulanases from Bacillus amyloderamificans disclosed in U.S. Patent No. 4,560,651 (hereby incorporated by reference), the pullulanase disclosed as SEQ ID NO: 2 in WO 01/151620 (hereby incorporated by reference), the Bacillus deramificans disclosed as SEQ ID NO: 4 in WO 01/151620 (hereby incorporated by reference), and the pullulanase from Bacillus acidopullulyticus disclosed as SEQ ID NO: 6 in WO 01/151620 (hereby incorporated by reference) and also described in FEMS Mic. Let. (1994) 1 15, 97-106.

Additional pullulanases contemplated according to the present invention included the pullulanases from Pyrococcus woesei, specifically from Pyrococcus woesei DSM No. 3773 disclosed in WO 92/02614, and the mature protein sequence disclosed as SEQ ID No: 6 herein.

The pullulanase may according to the invention be added in an effective amount which include the preferred amount of about 0.0001-10 mg enzyme protein per gram DS, preferably 0.0001-0.10 mg enzyme protein per gram DS, more preferably 0.0001-0.010 mg enzyme protein per gram DS. Pullulanase activity may be determined as NPUN. An Assay for determination of NPUN is described in the "Materials & Methods"-section below.

Suitable commercially available pullulanase products include PROMOZYME D,

PROMOZYME™ D2 (Novozymes A/S, Denmark), OPTIMAX L-300 (Genencor Int., USA), and AMANO 8 (Amano, Japan).

Composition comprising Alpha-Glucosidase Activity and Co2+

According to this aspect, the invention relates to compositions comprising alpha- glucosidase activity and Co2+. In one embodiment, the ratio of Co2+ to alpha-glucosidase activity in the composition is between 0.10 nmol/AGU - 15000 nmol/AGU. In another embodiment, the ratio of Co2+ to alpha-glucosidase activity is between 0.5 nmol/AGU - 500.0 nmol/AGU. In another embodiment, the ratio of Co2+ to alpha-glucosidase activity is between 1.0 nmol/AGU - 10.0 nmol/AGU.

The source of Co2+ can be any suitable source. In one embodiment, the Co2+ is selected from cobalt carbonate, cobalt chloride, cobalt citrate, cobalt naphthenate, cobalt nitrate, cobalt succinate, and cobalt sulfate.

The source of alpha-glucosidase activity can be from any suitable source, including the ones listed in the "Alpha-Glucosidase" section above. In a preferred embodiment, the alpha- glucosidase activity is from an alpha-glucosidase derived from the strain Aspergillus fumigatus and has the amino acid sequence disclosed as amino acids 20-988 in SEQ ID NO: 1 , or an alpha-glucosidase being at least 85% identical thereto, or at least about 87%, or at least about 90%, or at least about 93%, or at least about 95%, or at least about 97% thereto.

The above composition of the invention is suitable for use in a process for producing fermentation products, such as ethanol, according to the invention. In one embodiment, the composition is used in a process for producing fermentation products in addition to a carbohydrate-source generating enzyme and an alpha-amylase, preferably a glucoamylase and an acid alpha-amylase, respectively.

The carbohydrate-source generating enzyme may be any carbohydrate-source generating enzyme, including the ones listed in the "Carbohydrate-Source Generating Enzymes" section above. In a preferred embodiment the carbohydrate-source generating enzyme is a glucoamylase selected from the group derived from a strain of Aspergillus, preferably Aspergillus niger or Aspergillus awamori, a strain of Talaromyces, especially Talaromyces emersonii; or a strain of At elia, especially At elia rolfsii; a strain of Trametes, preferably Trametes cingulata; a strain of the genus Pachykytospora, preferably a strain of Pachykytospora papyracea; or a strain of the genus Leucopaxillus, preferably Leucopaxillus giganteus; or a strain of the genus Peniophora, preferably a strain of the species Peniophora rufomarginata; or a mixture thereof.

The alpha-amylase may be any alpha-amylase, including the ones mentioned in the "Alpha-Amylases" section above. In a preferred embodiment the alpha-amylase is an acid alpha-amylase, especially an acid fungal alpha-amylase. In a preferred embodiment the alpha- amylase is selected from the group of fungal alpha-amylases. In a preferred embodiment the alpha-amylase is derived from the genus Aspergillus, especially a strain of A. niger, A. oryzae, A. awamori, or A. kawachii, or of the genus Rhizomucor, preferably a strain of Rhizomucor pusillus, or the genus Meripilus, preferably a strain of Meripilus giganteus, or the genus Bacillus, preferably a strain of Bacillus stearothermophilus.

The ratio between glucoamylase activity (AGU) and acid fungal alpha-amylase activity (FAU-F) (i.e., AGU per FAU-F) may in a preferred embodiment of the invention be between 0.1 and 100 AGU/FAU-F, in particular between 2 and 50 AGU/FAU-F, such as in the range from 10-40 AGU/FAU-F glucoamylase and acid alpha-amylase is in the range between 0.3 and 5.0 AFAU/AGU.

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 illustrations of several 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 will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

The present invention is described in further detail in the following examples which are offered to illustrate the present invention, but not in any way intended to limit the scope of the invention as claimed. All references cited herein are specifically incorporated by reference for that which is described therein. Materials & Methods

Materials:

Glucoamylase A (AMG A): Glucoamylase derived from Trametes cingulata disclosed in SEQ ID NO: 2 in WO 2006/069289 and available from Novozymes A/S.

Alpha-Amylase A (AAA): Hybrid alpha-amylase consisting of R izomucor pusillus alpha- amylase with Aspergillus niger glucoamylase linker and SBD disclosed as V039 in Table 5 in WO 2006/069290 (Novozymes A/S)and available from Novozymes A/S.

Alpha-Glucosidase (AG): Alpha-glucosidase derived from Aspergillus fumigatus disclosed in SEQ ID NO: 6 in WO 2006/005032 (Novozymes, Inc.) and as SEQ ID NO: 1 herein, expressed in Tric oderma reesei RutC30 (Montenecourt and Eveleigh, 1979, Adv. C em. Ser. 181 : 289- 301 ).

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

Cobalt: Cobalt chloride hexahydrate is available from Sigma-Aldrich (St. Louis, MO). Methods

Identity

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".

For purposes of the present invention the degree of identity between two amino acid sequences, as well as the degree of identity between two nucleotide sequences, may be determined by the program "align" which is a Needleman-Wunsch alignment (i.e., a global alignment). The program is used for alignment of polypeptide, as well as nucleotide sequences. The default scoring matrix BLOSUM50 is used for polypeptide alignments, and the default identity matrix is used for nucleotide alignments. The penalty for the first residue of a gap is -12 for polypeptides and -16 for nucleotides. The penalties for further residues of a gap are -2 for polypeptides, and -4 for nucleotides.

"Align" is part of the FASTA package version v20u6 (see Pearson et al., 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 alignments use the Smith-Waterman algorithm with no limitation on gap size (see "Smith-Waterman algorithm", Smith et al., 1981 , J. Mol. Biol. 147:195-197).

Glucoamylase activity/Alpha-glucosidase activity (AGU)

Glucoamylase and alpha-glucosidase activity may be measured in Glucoamylase Units (AGU). The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute under the standard conditions 37°C, pH 4.3, substrate: maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase 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 measured using a photometer at 340 nm as a measure of the original glucose concentration.

Figure imgf000018_0001

Figure imgf000018_0002

Alpha-amylase activity (KNU)

The alpha-amylase 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 alpha-amylase Unit (KNU) is defined as the amount of enzyme which, under standard conditions (i.e., at 37°C +/- 0.05; 0.0003 M Ca2+; and pH 5.6) dextrinizes 5260 mg starch dry substance Merck Amylum solubile. Acid alpha-amylase activity (AFAU)

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

Acid Alpha-amylase Units (AAU)

The acid alpha-amylase activity can be measured in AAU (Acid Alpha-amylase Units), which is an absolute method. One Acid Amylase 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.

Figure imgf000019_0001

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. Further details can be found in EP 0140,410 B2, which disclosure is hereby incorporated by reference.

Determination of FAU-F

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

Temperature 37°C

pH 7.15

Wavelength 405 nm

Reaction time 5 min

Measuring time 2 min

Acid alpha-amylase activity (AFAU)

Acid alpha-amylase activity may be measured in AFAU (Acid Fungal Alpha-amylase Units), which 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 alpha-amylase, an endo-alpha-amylase (1 ,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1 ) hydrolyzes alpha-1 ,4-glucosidic bonds in the inner regions of the starch molecule to form dextrins and oligosaccharides with different chain lengths. The intensity of color formed with iodine is directly proportional to the concentration of starch. Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under the specified analytical conditions.

ALPHA - AMYLASE

STARCH + IODINE 40° , pH 2,5 DEXTRINS + OLIGOSACCHARIDES

2 = 590 nm

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 (l2): 0.03 g/L

CaCI2: 1.85 mM

pH: 2.50 ± 0.05

Incubation temperature: 40°C

Reaction time: 23 seconds

Wavelength: 590nm

Enzyme concentration: 0.025 AFAU/mL

Enzyme working range: 0.01-0.04 AFAU/mL EXAMPLES

Example 1

Effect of cobalt metal ion (Co2+) on alpha-amylase (AA 1) and glucoamylase (AMG A) combination in a one-step simultaneous saccharification and fermentation (SSF) process in the presence of alpha-glucosidase (AG)

Preparation of Alpha-Glucosidase: Alpha-glucosidase derived from Aspergillus fumigatus disclosed in SEQ ID NO: 6 in WO 2006/005032 (Novozymes, Inc.) and SEQ ID NO: 1 herein was expressed in Tric oderma reesei (RutC30). Shake flask broth can be purified using hydrophobic interaction chromatography. The broth is applied to a phenyl-sepharose 20 ml column at 0.1 M sodium acetate + 1.7 M ammonium sulfate solution. The elution consists of a linear gradient of 1.7 M to 0 M ammonium sulfate in 0.1 M sodium acetate (pH 4.4) over 15 column volumes. The fraction that corresponds to a major peak is collected and desalted using ultrafiltration (10 kDa membrane). A second purification step can be accomplished using a SP- Sepharose column (30 x 2.5 cm). The desalted fraction can be loaded on the column in 0.05 M sodium acetate, pH 4.4 and eluted from the resin using a linear gradient of 0-1 M NaCI in 0.05 M sodium acetate, pH 4.0 over 9 column volumes. The fraction with a major peak is collected. Alpha-glucosidase activity is determined as described above.

Ethanol yields: All treatments were evaluated via mini-scale fermentations. 410 g of ground yellow dent corn (with an average particle size around 0.5 mm) was added to 590 g tap water. The mixture was supplemented with 3.0 ml 1 g/L penicillin and 1 g of urea. The pH of the slurry was adjusted to 4.5 with 40% H2S04. Dry solid (DS) level was determined to be 35 wt. %. Approximately 5 g of this slurry was added to 15 ml vials. Each vial was dosed with the appropriate amount of enzymes and/or Co2+ dosage as shown in Table 1 below followed by addition of 200 microliters yeast propagate/5 g slurry. Actual enzyme and Co2+ dosages were based on the exact weight of corn slurry in each vial. For samples l-lll and VI-VII, Co2+ and AG were added directly to the fermentation vial. For samples IV and V, the AG samples were treated in 0.10 mM and 0.25 mM Co2+ at 4°C for 1 hr and then the AG/Co2+ solution was added to the fermentation vials, resulting in final concentrations of Co2+ in the fermentation vials as 0.000613 micromol/g mash and 0.00153 micromol/g mash, respectively.

Nine replicate fermentations of each treatment group were prepared. Vials were incubated at 32°C for the time periods indicated. Samples from each treatment group were analyzed at 24 hours (1 vial), 48 hours (2 vials), 70 hours (3 vials) and 95 hours (3 vials). Vials were vortexed at 24, 48, 70 and 95 hours and analyzed by HPLC. The HPLC preparation consisted of stopping the reaction by addition of 50 microliters of 40% H2S04, centrifuging, and filtering through a 0.45 micrometer filter. Samples were stored at 4°C until analysis. Agilent™ 1100 HPLC system coupled with Rl detector was used to determine ethanol and oligosaccharides concentration. The separation column was aminex HPX-87H ion exclusion column (300 mm x 7.8 mm) from BioRad™. The results are summarized in Table 2.

Table 1

Figure imgf000022_0001

Table 2

Figure imgf000022_0002

Ethanol yield (g/L) with time under different treatment conditions. The present invention is further described in the following numbered paragraphs:

[1] A process for producing a fermentation product from starch-containing material comprising simultaneously saccharifying and fermenting starch-containing material using an alpha-amylase, a carbohydrate-source generating enzyme and a fermenting organism at a temperature below the initial gelatinization temperature of said starch-containing material and in the presence of alpha-glucosidase activity and Co2+.

[2] The process of paragraph 1 , wherein the alpha-glucosidase activity is from an alpha- glucosidase derived from rice, corn, the genus Bacillus or the genus Aspergillus. [3] The process of paragraph 2, wherein the alpha-glucosidase is derived from Aspergillus fumigatus and has the amino acid sequence disclosed as amino acids 20-988 in SEQ ID NO: 1 , or an alpha-glucosidase being at least 85% identical thereto, or at least about 87%, or at least about 90%, or at least about 93%, or at least about 95%, or at least about 97% or at least 99% identity thereto.

[4] The process of any of paragraphs 1-3, wherein the starch-containing material is granular starch. [5] The process of any of paragraphs 1-4, wherein the starch-containing material is derived from whole grain.

[6] The process of any of paragraphs 1-5, wherein the starch-containing material is derived from corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice or potatoes.

[7] The process of any of paragraphs 1-6, wherein fermentation is carried out at a pH in the range between 3 and 7, preferably from 3.5 to 6, or more preferably from 4 to 5.

[8] The process of any of paragraphs 1-7, wherein the process is carried out for between 1 to 96 hours, preferably is from 6 to 72 hours.

[9] The process of any of paragraphs 1-8, wherein the dry solid content of the starch- containing material is in the range from 20-55 w/w-%, preferably 25-40 w/w-%, more preferably 30-35 w/w-%.

[10] The process of any of paragraphs 1-9, wherein the sugar concentration is kept at a level below about 6 w/w-% during simultaneous saccharification and fermentation, preferably below about 3 w/w-%. [1 1] The process of any of paragraphs 1-10, wherein the starch-containing material is prepared by reducing the particle size of starch-containing material to a particle size of 0.1-0.5 mm.

[12] The process of paragraph 10, wherein the reduction of particle size of the starch- containing material is done by milling, preferably dry milling. [13] The process of any of paragraphs 1-12, wherein the temperature during simultaneous saccharification and fermentation is between 25°C and 40°C, such as between 28°C and 35°C, such as between 30°C and 34°C, such as around 32°C. [14] The process of any of paragraphs 1-13, wherein the alpha-amylase is an acid alpha- amylase, preferably an acid fungal alpha-amylase.

[15] The process of paragraph 14, wherein the alpha-amylase is a fungal alpha-amylase, preferably derived from the genus Aspergillus, especially a strain of A. niger, A. oryzae, A. awamori, or A. kawachii, or of the genus Rhizomucor, preferably a strain the Rhizomucor pusillus, or the genus Meripilus, preferably a strain of Meripilus giganteus.

[16] The process of paragraph 14 or 15, wherein the alpha-amylase is present in an amount of 0.001 to 10 AFAU/g DS, preferably 0.01 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS or 0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS.

[17] The process of any of paragraphs 1-16, wherein the carbohydrate-source generating enzyme is selected from the group consisting of glucoamylase, maltogenic amylase, and beta- amylase.

[18] The process of any of paragraphs 1-17, wherein the carbohydrase-source generating enzyme is glucoamylase and is present in an amount of 0.001 to 10 AGU/g DS, preferably from 0.01 to 5 AGU/g DS, especially 0.1 to 0.5 AGU/g DS. [19] The process of any of paragraphs 14-18, wherein the alpha-amylase and glucoamylase is added in a ratio of between 0.1 and 100 AGU/FAU-F, preferably 2 and 50 AGU/FAU-F, especially between 10 and 40 AGU/FAU-F.

[20] The process of paragraph 18, wherein the glucoamylase is derived from a strain of Aspergillus, preferably Aspergillus niger or Aspergillus awamori, a strain of Talaromyces, especially Talaromyces emersonii; or a strain of Athelia, especially Athelia rolfsii; a strain of Trametes, preferably Trametes cingulata; a strain of the genus Pachykytospora, preferably a strain of Pachykytospora papyracea; or a strain of the genus Leucopaxillus, preferably Leucopaxillus giganteus; or a strain of the genus Peniophora, preferably a strain of the species Peniophora rufomarginata; or a mixture thereof. [21] The process of any of paragraphs 1-20, wherein the fermentation product is recovered after fermentation.

[22] The process of any of paragraphs 1-21 , wherein the fermentation product is an alcohol, preferably ethanol, especially fuel ethanol, potable ethanol or industrial ethanol.

[23] The process of any of paragraphs 1-22, wherein the fermenting organism is a yeast, preferably a strain of Sacc aromyces, especially a strain of Sacc aromyces cerevisae. [24] The process of any of paragraphs 1-23, wherein alpha-glucosidase activity and Co2+ are added to the process.

[25] The process of any of paragraphs 1-24, wherein the alpha-glucosidase activity and the Co2+ are combined in a composition before said composition comprising alpha-glucosidase activity and Co2+ is added to the process.

[26] A composition comprising alpha-glucosidase activity and Co2+.

[27] The composition of paragraph 26, wherein the alpha-glucosidase is derived from Aspergillus fumigatus and has the amino acid sequence disclosed as amino acids 20-988 in SEQ ID NO: 1 , or an alpha-glucosidase being at least 85% identical thereto, or at least about 87%, or at least about 90%, or at least about 93%, or at least about 95%, or at least about 97% thereto. [28] Use of a composition of paragraph 26 or 27 in a process for producing fermentation products comprising simultaneously saccharifying and fermenting starch-containing material using an alpha-amylase, a carbohydrate-source generating enzyme and a fermenting organism at a temperature below the initial gelatinization temperature of said starch-containing material.

Claims

What is claimed: 1. A process for producing a fermentation product from starch-containing material comprising simultaneously saccharifying and fermenting starch-containing material using an alpha-amylase, a carbohydrate-source generating enzyme and a fermenting organism at a temperature below the initial gelatinization temperature of said starch-containing material and in the presence of alpha-glucosidase activity and Co2+.
2. The process of claim 1 , wherein the alpha-glucosidase activity is from an alpha- glucosidase derived from rice, corn, the genus Bacillus or the genus Aspergillus.
3. The process of claim 2, wherein the alpha-glucosidase is derived from Aspergillus fumigatus and has the amino acid sequence disclosed as amino acids 20-988 in SEQ ID NO: 1 , or an alpha-glucosidase being at least 85% identical thereto, or at least about 87%, or at least about 90%, or at least about 93%, or at least about 95%, or at least about 97% or at least 99% identity thereto.
4. The process of any of claims 1-3, wherein fermentation is carried out at a pH in the range between 3 and 7, preferably from 3.5 to 6, or more preferably from 4 to 5.
5. The process of any of claims 1-4, wherein the process is carried out for between 1 to 96 hours, preferably is from 6 to 72 hours.
6. The process of any of claims 1-5, wherein the dry solid content of the starch-containing material is in the range from 20-55 w/w-%, preferably 25-40 w/w-%, more preferably 30-35 w/w- %.
7. The process of any of claims 1-6, wherein the alpha-amylase is an acid alpha-amylase, preferably an acid fungal alpha-amylase.
8. The process of claim 7, wherein the alpha-amylase is a fungal alpha-amylase, preferably derived from the genus Aspergillus, especially a strain of A. niger, A. oryzae, A. awamori, or A. kawachii, or of the genus Rhizomucor, preferably a strain the Rhizomucor pusillus, or the genus Meripilus, preferably a strain of Meripilus giganteus.
9. The process of any of claims 1-8, wherein the carbohydrate-source generating enzyme is selected from the group consisting of glucoamylase, maltogenic amylase, and beta-amylase.
10. The process of claim 9, wherein the glucoamylase is derived from a strain of Aspergillus, preferably Aspergillus niger or Aspergillus awamori, a strain of Talaromyces, especially Talaromyces emersonii; or a strain of Athelia, especially Athelia rolfsii; a strain of Trametes, preferably Trametes cingulata; a strain of the genus Pachykytospora, preferably a strain of Pachykytospora papyracea; or a strain of the genus Leucopaxillus, preferably Leucopaxillus giganteus; or a strain of the genus Peniophora, preferably a strain of the species Peniophora rufomarginata; or a mixture thereof.
11. The process of any of claims 1-10, wherein the fermentation product is an alcohol, preferably ethanol, especially fuel ethanol, potable ethanol or industrial ethanol.
12. The process of any of claims 1-11 , wherein alpha-glucosidase activity and Co2+ are added to the process.
13. The process of any of claims 1-12, wherein the alpha-glucosidase activity and the Co2+ are combined in a composition before said composition comprising alpha-glucosidase activity and Co2+ is added to the process.
14. A composition comprising alpha-glucosidase activity and Co2+.
15. The composition of claim 14, wherein the alpha-glucosidase is derived from Aspergillus fumigatus and has the amino acid sequence disclosed as amino acids 20-988 in SEQ ID NO: 1 , or an alpha-glucosidase being at least 85% identical thereto, or at least about 87%, or at least about 90%, or at least about 93%, or at least about 95%, or at least about 97% thereto.
16. Use of a composition as claimed in claim 14 or 15 in a process for producing fermentation products comprising simultaneously saccharifying and fermenting starch- containing material using an alpha-amylase, a carbohydrate-source generating enzyme and a fermenting organism at a temperature below the initial gelatinization temperature of said starch- containing material.
PCT/US2011/023675 2010-02-09 2011-02-04 Addition of alpha - glucosidase and cobalt for producing fermentation products from starch WO2011100161A1 (en)

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US9617527B2 (en) 2010-04-14 2017-04-11 Novozymes A/S Polypeptides having glucoamylase activity and polynucleotides encoding same
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CN103987850A (en) * 2011-10-11 2014-08-13 诺维信北美公司 Processes for producing fermentation products
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CN105874076A (en) * 2013-10-24 2016-08-17 丹尼斯科美国公司 Enhanced fermentation process using a transglycosidase
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