# Patent

## Title
Process for producing a starch hydrolyzate

## Abstract

The present invention relates to processes for producing a starch hydrolyzate and optionally a fermentation product, such as ethanol.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This applications claims priority or the benefit of U.S. provisional application No. 60/813,941 filed Jun. 15, 2006, the contents of which are fully incorporated herein by reference.


REFERENCE TO A SEQUENCE LISTING
This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to an improved process for production of a starch hydrolyzate, e.g., for use in a fermentation process.
BACKGROUND OF THE INVENTION
The present invention relates to processes for production of a fermentation product from milled starch-containing material, such as granular starch, at a temperature below the initial gelatinization temperature of the milled starch-containing material.
Grains, cereals or tubers of plants contain starch. The starch is in the form of microscopic granules, which are insoluble in water at room temperature. When an aqueous starch slurry is heated, the granules swell and eventually burst, dispersing the starch molecules into the solution. During this “gelatinization” process, there is a dramatic increase in viscosity. Because the solids level in a typical industrial process is around 30-40%, the starch has to be thinned or “liquefied” so that it can be handled. This reduction in viscosity is generally accomplished by enzymatic degradation in a process referred to as liquefaction. During liquefaction, the long-chained starch is degraded into smaller branched and linear chains of glucose units (dextrins) by an alpha-amylase.
A conventional enzymatic liquefaction process may be carried out as a three-step hot slurry process. The slurry is heated to between 80-85° C. and thermostable alpha-amylase added to initiate liquefaction. The slurry is then jet-cooked at a temperature between 105-125° C. to complete gelatinization of the slurry, cooled to 60-95° C. and, generally, additional alpha-amylase is added to finalize hydrolysis. The liquefaction process is generally carried out at pH between 5 and 6.
During saccharification, the dextrins from the liquefaction are further hydrolyzed to produce low molecular sugars DP1-3 that can be metabolized by a fermenting organism, such as yeast. The hydrolysis is typically accomplished using glucoamylase, alternatively or in addition to glucoamylases, alpha-glucosidases and/or acid alpha-amylases can be used. A full saccharification step typically lasts up to 72 hours, however, it is common to do a pre-saccharification of, e.g., 40-90 minutes at a temperature above 50° C., followed by a complete saccharification during fermentation in a process known as simultaneous saccharification and fermentation (SSF).
Fermentation is performed using a fermenting organism, such as yeast, which is added to the mash. Then the fermentation product is recovered. For ethanol, e.g., fuel, potable, or industrial ethanol, the fermentation is carried out, for typically 35-60 hours at a temperature of typically around 32° C. When the fermentation product is beer, the fermentation is carried out, for typically up to 8 days at a temperature of typically around 14° C.
Following fermentation, the mash may be used, e g., as a beer, or distilled to recover ethanol. The ethanol may be used as, e.g., fuel ethanol, drinking ethanol, and/or industrial ethanol.
It will be apparent from the above discussion that the starch hydrolysis in a conventional process is very energy consuming due to the different temperature requirements during the various steps. Several patent applications address the issue by providing processes for converting granular starch into ethanol without the energy consuming gelatinisation step.
U.S. Pat. No. 4,316,956 and WO 2004/113551 provide fermentation processes for conversion of granular starch into ethanol.
The applications WO 2005/003311 and PCT/US05/46725 provide fungal alpha-amylases useful for conversion of granular starch into fermentable sugars, e.g., for ethanol production.
The application PCT/DK2005/000819 provides bacterial alpha-amylases useful for conversion of granular starch into fermentable sugars, e.g., for ethanol production.
The object of the present invention is to provide improved processes for conversion of milled starch-containing material, such as granular starch.
SUMMARY OF THE INVENTION
The present invention provides processes for producing a starch hydrolyzate from starch-containing material without gelatinization of said starch-containing material. The starch hydrolyzate may be used, e.g., as a sweetener or in the production of a fermentation product, such as ethanol. Surprisingly the inventors have discovered that by combining the action of a bacterial alpha-amylase comprising a carbohydrate-binding module and the action of a fungal alpha-amylase comprising a carbohydrate-binding module an increase in yield is achieved compared to using an increased amount of either a bacterial alpha-amylase comprising a carbohydrate-binding module or an increased amount of a fungal alpha-amylase comprising a carbohydrate-binding module.
Accordingly in a first aspect, the invention provides a process comprising saccharification of a granular starch with: a) a glucoamylase, b) a bacterial alpha-amylase comprising a carbohydrate-binding module (CBM), and, c) a fungal alpha-amylase comprising a CBM, to produce a starch hydrolyzate.
In a preferred embodiment the starch hydrolyzate of the first aspect is further contacted with a fermenting organism to produce a fermentation product, preferably ethanol. Preferably saccharification and fermentation is carried out simultaneously.
In a second aspect, the invention provides compositions of a bacterial alpha-amylase comprising a carbohydrate-binding module and the action of a fungal alpha-amylase comprising a carbohydrate-binding module. In a preferred embodiment the composition furthermore comprises a glucoamylase. Preferably the ratio between fungal acid alpha-amylase activity (AFAU) per glucoamylase activity (AGU) (AFAU per AGU) is at least 0.1, in particular at least 0.16, such as in the range from 0.12 to 0.50 or even higher.


DETAILED DESCRIPTION OF THE INVENTION
Before saccharification a slurry of starch-containing material, such as granular starch, having 20-55 weight % dry solids, preferably 25-40 weight % dry solids, more preferably 30-35% dry solids of starch-containing material is 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 other fermentation product plant process water. Because the process of the invention is carried out below the 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. % stillage, preferably 15-60% vol. % stillage, especially from about 30 to 50 vol. % stillage.
In order to expose more surface of the starch-containing material it is milled. In an embodiment the particle size is between 0.05-3.0 mm, or at least 30% of the milled starch-containing material fit through a sieve with a 0.05 to 3.0 mm screen. 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 of the starch-containing material or of the starch of the starch-containing material is converted into a soluble starch hydrolyzate.
The process of the invention is conducted at a temperature below the initial gelatinization temperature. In a preferred embodiment, the process is carried out as a simultaneous saccharification and fermentation process. In such preferred embodiment the process is typically carried out at a temperature between 28° C. and 36° C., such as between 29° C. and 35° C., such as between 30° C. 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 simultaneous saccharification and fermentation is carried out so that the sugar level, such as the glucose level, is kept at a low level such as below about 3 wt. %, preferably below about 2 wt. %, more preferred below about 1 wt. %., even more preferred below about 0.5%, or even more preferred below about 0.1 wt. %. Such low levels of sugar can be accomplished by simply employing adjusted quantities of enzyme and fermenting organism. A skilled person in the art can easily determine which quantities of enzyme and fermenting organism to use. 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 wt. % or below about 0.2 wt. %.
The process of the invention may be 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.
Any suitable starch-containing starting material comprising granular starch may be used according to the present invention. Examples of starch-containing starting materials, suitable for use in the processes of present invention, include tubers, roots, stems, whole grains, corns, cobs, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice peas, beans, or cereals, sugar-containing raw materials, such as molasses, fruit materials, sugar, cane or sugar beet, potatoes, and cellulose-containing materials, such as wood or plant residues. 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 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 material comprising milled whole grain including non-starch fractions such as germ residues and fibers.
The starch-containing raw material, such as whole grain, is milled in order to open up the structure and allowing for further processing. Two milling 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 hydrolyzate is used in production of syrups. Both dry and wet milling is well known in the art of starch processing and is equally contemplated for the process of the invention. In an embodiment the particle size after milling is between 0.05 to 3.0 mm, or so that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the milled starch-containing material fit through a sieve with a 0.05 to 3.0 mm screen, and preferably with a 0.1 ̃0.5 mm screen.
The term “initial gelatinization temperature” means the lowest temperature at which gelatinization of the starch commences. Starch heated in water begins to gelatinize between 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 the context of this invention the initial gelatinization temperature of a given starch-containing material is the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein and Lii, 1992, Starch/Stärke, 44(12): 461-466.
The term “starch hydrolyzate” is understood as the soluble degradation products of the hydrolysis processes of the invention. The starch hydrolysis may comprise mono-, di-, and oligosaccharides, such as glucose, maltose, maltodextrins, cyclodextrins and any mixture of these. Preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% or at least 98% of the dry solids of the granular starch is converted into a soluble starch hydrolyzate.
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, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); 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, as are well known in the art. Preferred fermentation processes are anaerobic fermentation processes, as are well known in the art.
The term “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 the Saccharomyces spp., and in particular, Saccharomyces cerevisiae. Commercially available yeast include, e.g., Red StarTM/Lesaffre Ethanol Red (available from Red Star/Lesaffre, USA) FALI (available from Fleischmann's Yeast, a division of Burns Philp Food Inc., USA), SUPERSTART (available from Alltech), GERT STRAND (available from Gert Strand AB, Sweden) and FERMIOL (available from DSM Specialties).
Fungal and Bacterial Alpha-Amylase Comprising a CBM
According to the invention a fungal alpha-amylase comprising a CBM and a bacterial alpha-amylase comprising a CBM are applied in the process of the invention. In a preferred embodiment the alpha-amylase is an acid alpha-amylase, e.g., fungal acid alpha-amylase or bacterial acid 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-Amylases Comprising a CBM
By the term a “bacterial alpha-amylase comprising a CBM” is understood an enzyme comprising a catalytic domain and a CBM, said catalytic domain and said CBM both derived from a bacterial source. The bacterial alpha-amylase comprising a CBM may be a wild-type bacterial enzyme, a variant of such a wild-type bacterial enzyme, or a hybrid enzyme comprising a bacterial alpha-amylase catalytic domain and a bacterial CBM. In a preferred embodiment of the invention the bacterial alpha-amylase catalytic domain and/or bacterial CBM is derived from the genus Bacillus or the genus Anoxybacillus. 
Preferred for the invention is any bacterial alpha-amylase comprising a CBM, both hybrids and wild-types, wherein the CBM has the sequence shown as amino acids 521 ̃619 in SEQ ID NO: 1 herein or the CBM has a sequence homologous to said sequence.
Also preferred for the invention is any bacterial alpha-amylase comprising a CBM, both hybrids and wild-types, Wherein the catalytic domain has the sequence shown as amino acids 32-520 in SEQ ID NO: 1 herein or the catalytic domain has a sequence homologous to said sequence.
In another preferred embodiment the bacterial alpha-amylase comprising a CBM comprises a catalytic domain derived from a strain of B. licheniformis, B. amyloliquefaciens, B. subtilis or B. stearothermophilus, but may also be derived from other Bacillus sp. Specific examples of contemplated alpha-amylase catalytic domains include a amylase catalytic domain having at least 60%, at least 70%, at least 80% or even at least 90% identity to the amylase from Bacillus licheniformis (BLA) shown in SEQ ID NO: 35, the B. licheniformis variant LE429 shown in SEQ ID NO: 41, the amylase from B. stearothermophilus (BSG) shown in SEQ ID NO: 36, the amylase from B. amyloliquefaciens (BAN) shown in SEQ ID NO: 37, the amylase from B. halodurance SP722 shown in SEQ ID NO: 38, the amylase SP690 shown in SEQ ID NO: 39, the amylase AA560 shown in SEQ ID NO: 40 in patent application PCT/DK2005/000819 (hereby incorporated by reference). The catalytic domain may also be derived from an amylase from Pseudomonas saccharophilia, such as from the amylase disclosed as SEQ ID NO: 1 in WO 04/111217 (hereby incorporated by reference).
In an embodiment of the invention the bacterial alpha-amylase comprising a CBM is an enzyme comprising a catalytic domain having a degree of identity of at least 70%, preferably at least 80%, more preferred at least 85%, even more preferred at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to any of the sequences shown in SEQ ID NO: 1, 2 or 3 in WO 99/19467 (hereby incorporated by reference). The Bacillus alpha-amylase catalytic domain may also be a variant and/or hybrid sequence, 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 of which are hereby incorporated by reference). Specifically contemplated alpha-amylase variants are disclosed in U.S. Pat. Nos. 6,093,562, 6,187,576, and 6,297,038 (all of which are hereby incorporated by reference) and include Bacillus stearothermophilus alpha-amylase (BSG alpha-amylase) variants having 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: 7 disclosed in WO 99/19467 (hereby incorporated by reference). Even more preferred are Bacillus alpha-amylase catalytic domains, especially Bacillus stearothermophilus alpha-amylase, which have a double deletion corresponding to delta(181-182) and further comprise a N193F substitution (also denoted |181*+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.
In a particularly preferred embodiment the bacterial alpha-amylase comprising a CBM is a wild-type bacterial alpha-amylase derived from a strain of Anoxybacillus contaminans. Preferably the bacterial alpha-amylase comprising a CBM has the sequence shown in SEQ ID NO: 1 herein. Also preferred are polypeptides having at least 70% identity, such as at least 80% or even at least 90% identity, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1 herein. In another embodiment the bacterial alpha-amylase comprising a CBM is one of the hybrid enzymes disclosed in PCT/DK2005/000819 as SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 (hereby incorporated by reference) or an amino acid sequence homologous to any of these sequences.
The bacterial alpha-amylase may be used in an amount of 0.001 to 1.0 mg/g DS, preferably in an amount of 0.01 to 0.5 mg/g DS, more preferably in an amount of 0.02 to 0.2 mg/g DS. Measured in AFAU the bacterial alpha-amylase may be used in an amount of 0.01-10 AFAU/g of DS, in an amount of 0.05-2.5 AFAU/g of DS, or more preferably in an amount of 0.1-1 AFAU/g of DS, such as approximately 0.5 AFAU/g DS.
Measured in KNU the bacterial alpha-amylase may be used in an amount of 0.001-10 KNU/g of DS, in an amount of 0.005-2 KNU/g of DS, or more preferably in an amount of 0.01-0.2 KNU/g of DS, such as approximately 0.035 KNU/g DS.
Fungal Alpha-Amylases Comprising a CBM
By the term a “fungal alpha-amylase comprising a CBM” is understood an enzyme comprising a catalytic domain and a CBM, said catalytic domain and said CBM both derived from a fungal source. The fungal alpha-amylase comprising a CBM may be a wild-type fungal enzyme, a variant of such a wild-type fungal enzyme, or a hybrid enzyme comprising a fungal alpha-amylase catalytic domain and a fungal CBM.
In an embodiment the wild-type acid alpha-amylase is derived from a strain of Aspergillus kawachi, in particular the polypeptide shown in SEQ ID NO: 41 in WO 2005/003311 or homologous sequences, e.g., variants of said polypeptide comprising one or more of the substitutions G33A, I36K, S74A, D75Y, E77D, P120A, I153D, D154N, W155Y, D156E, N157D, L158Q, Q162E, E166L, T169N, I170T, E199K, E199L, D232L, N233D, N235D, L238Y, D239T, W256Y, Q257P, E331Q, S336A, D339K, D339N, V340D, and Y342A. The most preferred variants comprise one or more of the following substitutions; S74A, E166L, E199L, D339K, and D156E. Yet more preferred is the variant having the multiple substitutions S74A/E166L/E199L.
In a preferred embodiment the fungal alpha-amylase comprising a CBM is a hybrid alpha-amylase. Fungal hybrid enzymes, as referred to herein, include species comprising an amino acid sequence of an alpha-amylase enzyme (EC 3.2.1.1) of fungal origin linked (i.e., covalently bound) to an amino acid sequence comprising a carbohydrate-binding module (CBM), preferably of fungal origin.
Fungal alpha-amylases catalytic domains suitable for use in a hybrid enzyme for use in the process of the invention include acid alpha-amylases derived from a strain of the genus Aspergillus, such as, Aspergillus oryzae and Aspergillus niger alpha-amylases. A preferred fungal alpha-amylase is a Fungamyl-like alpha-amylase which is preferably derived from a strain of Aspergillus oryzae. In the present disclosure, 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. A preferred hybrid enzyme comprises a Fungamyl-like alpha-amylase catalytic domain and a CBM derived from the A. rolfsii glucoamylase.
Another preferred acid alpha-amylase catalytic domain suitable for use in a hybrid enzyme for use in the process of the invention is derived from a strain Aspergillus niger. In a preferred embodiment the acid fungal alpha-amylase is the one from A. niger disclosed 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 alpha-amylase is also shown as SEQ ID NO: 8 in WO 2005/003311. Preferred acid alpha-amylase amylases catalytic domains also comprise variants of said acid fungal amylase having at least 70% identity, such as at least 80% or at least 90% identity, such as at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% identity to SEQ ID NO: 8 in WO 2005/003311. Preferably the fungal alpha-amylase comprising a COM is the variant JA01 disclosed in WO 2005/003311.
Another preferred acid alpha-amylase amylases catalytic domains suitable for use in a hybrid enzyme for use in the process of the invention is any alpha-amylase catalytic domain disclosed in patent application PCT/US05/46725 (hereby incorporated by reference), and preferably the catalytic domain is derived from any species disclosed therein, and in particular selected from the group consisting of Thermomyces lanuginosus; in particular a polypeptide having the amino acids 1-441 in SEQ ID NO: 14, Malbranchea sp.; in particular a polypeptide having the amino acids 1-471 in SEQ ID NO: 18, Rhizomucor pusillus; in particular a polypeptide having the amino acids 1-450 in SEQ ID NO: 20, Dichotomocladium hesseltinei; in particular a polypeptide having the amino acids 1-445 in SEQ ID NO: 22, Stereum sp.; in particular a polypeptide having the amino acids 1-498 in SEQ ID NO: 26, Trametes sp.; in particular a polypeptide having the amino acids 18-513 in SEQ ID NO: 28, Coriolus consors, in particular a polypeptide having the amino acids 1-507 in SEQ ID NO: 30, Dinemasporium sp.; in particular a polypeptide having the amino acids 1-481 in SEQ ID NO: 32, Cryptosporiopsis sp.; in particular a polypeptide having the amino acids 1-495 in SEQ ID NO: 34, Diplodia sp., in particular a polypeptide having the amino acids 1-477 in SEQ ID NO: 38, Gliocladium sp.; in particular a polypeptide having the amino acids 1-449 in SEQ ID NO: 42, Nectria sp.; in particular a polypeptide having the amino acids 1-442 in SEQ ID NO: 115, Fusarium sp.; in particular a polypeptide having the amino acids 1-480 in SEQ ID NO: 4, a polypeptide having the amino acids 1-478 in SEQ ID NO: 6, or a polypeptide having the amino acids 1-441 in SEQ ID NO: 117, Thermoascus auranticus; in particular a polypeptide having the amino acids 1-477 in SEQ ID NO: 125, Thamindium elegans; particular a polypeptide having the amino acids 1-446 in SEQ ID NO: 131, Absidia cristata; in particular a polypeptide having the amino acids 41-481 in SEQ ID NO: 157, Acremonium sp,; in particular a polypeptide having the amino acids 22-626 in SEQ ID NO: 159, Coniochaeta sp.; in particular a polypeptide having the amino acids 24-630 in SEQ ID NO: 161, Meńpilus giganteus, in particular a polypeptide having the amino acids 27-602 in SEQ ID NO: 163, Penicillium sp.; in particular a polypeptide having the amino acids 21-643 in SEQ ID NO: 165, Streptomyces limosus; in particular a polypeptide having the amino acids 29-566 in SEQ ID NO: 167, Subulispora procurvata; in particular a polypeptide having the amino acids 22-613 in SEQ ID NO: 169, Syncephalastrum racemosum; in particular a polypeptide having the amino acids 21-463 in SEQ ID NO: 171, Trametes currugata; in particular a polypeptide having the amino acids 21-587 in SEQ ID NO: 173, Trichophaea saccata; in particular a polypeptide having the amino acids 30-773 in SEQ ID NO: 175, Valsańa rubricosa, in particular a polypeptide having the amino acids 22-586 in SEQ ID NO: 177 and Valsaria spartii; in particular a polypeptide having the amino acids 20-582 in SEQ ID NO: 179 in PCT/US05/46725 (hereby incorporated by reference). Also preferred are alpha-amylase amylases catalytic domains having a sequence homologous to the aforementioned polypeptides.
Most preferably a hybrid comprises a CBM disclosed in patent application PCT/US05/46725 (hereby incorporated by reference), preferably the CBM is from a glucoamylase selected from the group consisting of the Pachykytospora papayracea (SEQ ID NO: 76), Trametes cingulata (SEQ ID NO: 78), Leucopaxillus gigantus (SEQ ID NO: 80), Athelia rolfsii (SEQ ID NO: 92), Aspergillus kawachii (SEQ ID NO: 94), Aspergillus niger (SEQ ID NO: 96) or from a alpha-amylase selected from the group consisting of Trichopheraea saccata (SEQ ID NO: 52), Subulispora provurvata (SEQ ID NO: 82), Valsaria rubricosa (SEQ ID NO: 84), Acremonium sp. (SEQ ID NO: 86), Meripilus giganteus (SEQ ID NO: 88), Bacillus flavothermus (Anoxybacillus contaminans) (SEQ ID NO: 90), Coniochaeta sp. (SEQ ID NO: 98), Coniochaeta sp. (SEQ ID NO: 137), Trametes corrugate (SEQ ID NO: 139), Valsario spartii (SEQ ID NO: 141) and Penicillium sp. (SEQ ID NO: 143) in PCT/US05/46725 (hereby incorporated by reference). Also preferred are sequences homologous to any of the aforementioned sequences.
Also preferred for the invention are any of the polypeptides V001, V002, V003, V004, V005, V006, V007, V008, V009, V010, V011, V012, V013, V014, V015, V016, V017, V018, V019, V021, V022, V023, V024, V025, V026, V027, V028, V029, V030, V031, V032, V033, V034, V035, V036, V037, V038, V039, V040, V041, V042, V043, V047, V048, V049, V050, V051, V052, V054, V055, V057, V059, V060, V061, V063, V064, V065, V066, V067, V068 and V069 disclosed in patent application PCT/US05/46725 and hereby incorporated by reference. Also preferred are sequences homologous to any of the aforementioned sequences.
More preferred are a fungal alpha-amylase comprising a CBM wherein the fungal alpha-amylase catalytic domain is an amino acid sequence having at least 70% homology to amino acids 13 to 450 of SEQ ID NO: 4, preferably derived from Rhizomucor pusillus and/or the fungal CBM is an amino acid sequence having at least 70% homology to amino acids 488 to 595 in SEQ ID NO: 2, preferably a sequence derived from a strain of Aspergillus niger, preferably a sequence derived from Aspergillus niger glucoamylase.
Most preferably the fungal alpha-amylase comprising a CBM is the hybrid V039 disclosed in PCT/US05/46725 comprising the CD from Rhizomucor pusillus alpha-amylase and the CBM and linker from Aspergillus niger glucoamylase and having the sequence shown as amino acids 13-595 in SEQ ID NO: 2 herein.
The fungal alpha-amylase may be used in amounts of 0.001 to 1.0 mg/g DS, preferably in an amount of 0.01 to 0.5 mg/g DS, more preferably in an amount of 0.02 to 0.2 mg/g DS. Measured in AFAU the fungal alpha-amylase may be used in an amount of 0.01-10 AFAU/g of DS, in an amount of 0.05-2.5 AFAU/g of DS, or more preferably in an amount of 0.1-1 AFAU/g of DS, such as approximately 0.5 AFAU/g DS.
Glucoamylase
The term “glucoamylase activity” means a glucan 1,4-alpha-glucosidase which hydrolyzes the terminal 1,4-linked alpha-D-glucose residues successively from non-reducing ends of the chains with release of beta-D-glucose belonging to the Enzyme Class EC 3.2.1.3.
The glucoamylase used in a process of the invention may have the amino acid sequence disclosed in SEQ ID NO: 2 in PCT/US05/46724, and shown herein as SEQ ID NO: 3 or an amino acid sequence that is at least 70%, preferably at least 75%, or at least 80%, or at least 85%, or 90%, or at least 95%, at least 96%, at least 97%, at least 98% or even at least 99% identical to SEQ ID NO: 3. The glucoamylase preferably is derived from Trametes cingulata. 
Alternatively the glucoamylase used in a process of the invention may have the amino acid sequence shown PCT/US05/01147 as amino acid residues 1 to 561 in SEQ ID NO: 2, or an amino acid sequence that is at least 70%, preferably at least 75%, or at least 80%, or at least 85%, or 90%, or at least 95%, at least 96%, at least 97%, at least 98% or even at least 99% identical to said SEQ ID NO: 2 (amino acid residues 1 to 561). The glucoamylase preferably is derived from Athelia rolfsii. 
Also preferred are the glucoamylase derived from Talaromyces emersonii disclosed in WO 99/28448 and the glucoamylase derived from Aspergillus niger disclosed in Boel et al. (1984), EMBO J. 3(5): 1097-1102. Aspergillus niger glucoamylase is available from Novozymes A/S as Sprizyme PlusTM and Sprizyme FuelTM.
The glucoamylase may be used in an amount of 0.01 to 2.0 mg/g DS, preferably in an amount of 0.05 to 1.0 mg/g DS, more preferably in an amount 0.1 to 0.5 mg/g DS. Measured in AGU the glucoamylase may be used in an amount of 0.01-10 AGU/g of DS, in an amount of 0.05-2.5 AGU/g of DS, or more preferably in an amount of 0.1-1 AGU/g of DS, such as approximately 0.5 AGU/g DS.
Protease
According to the process of the invention a protease may be present during saccharification and/or fermentation.
In a preferred embodiment the protease is an acid protease of microbial origin, preferably of fungal or bacterial origin.
Suitable proteases include microbial proteases, such as fungal and bacterial proteases. Preferred proteases are acidic proteases, i.e., proteases characterized by the ability to hydrolyze proteins under acidic conditions below pH 7, preferably from 3.5 to 6, or more preferably from 4 to 5.
Contemplated acid fungal proteases include fungal proteases derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra, Irpex, Penicillium, Sclerotium and Torulopsis. Especially contemplated are proteases derived from Aspergillus niger (see, e.g., Koaze et al., 1964, Agr. Biol. Chem. Japan, 28: 216), Aspergillus saitoi (see. e.g., Yoshida, 1954, J. Agr. Chem. Soc. Japan, 28: 66), Aspergillus awamori (Hayashida et al., 1977, Agric. Biol. Chem., 42(5): 927-933, Aspergillus aculeatus (WO 95/02044), or Aspergillus oryzae, such as the pepA protease; and acidic proteases from Mucor pusillus or Mucor miehei. 
Contemplated are also neutral or alkaline proteases, such as a protease derived from a strain of Bacillus. A particular protease contemplated for the invention is derived from Bacillus amyloliquefaciens and has the sequence obtainable at Swissprot as Accession No. P06832. Also contemplated are the proteases having at least 90% identity to amino acid sequence obtainable at Swissprot as Accession No. P06832 such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% identity.
Further contemplated are the proteases having at least 90% identity to amino acid sequence disclosed as SEQ. ID. NO: 1 in the WO 2003/048353 such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% identity.
Also contemplated are papain-like proteases such as proteases within E.C. 3.4.22.* (cysteine protease), such as EC 3.4.22.2 (papain), EC 3.4.22.6 (chymopapain), EC 3.4.22.7 (asclepain), EC 3.4.22.14 (actinidain), EC 3.4.22.15 (cathepsin L), EC 3.4.22.25 (glycyl endopeptidase) and EC 3.4.22.30 (caricain).
Proteases may be added in the amounts of 0.1-1000 AU/kg dm, preferably 1-100 AU/kg DS and most preferably 5-25 AU/kg DS.
Additional Ingredients
Additional ingredients may be present during saccharification and/or fermentation to increase the effectiveness of the process of the invention. For instance, nutrients (e.g., fermentation organism micronutrients), antibiotics, salts (e.g., zinc or magnesium salts), other enzymes such as phytase, cellulase, hemicellulase, exo and endoglucanase, and xylanases.
Recovery of Fermentation Product
The fermentation product, such as ethanol, may optionally be recovered after fermentation. The recovery may be performed by any conventional manner such as, erg., distillation.
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 described 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.
Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.
Materials and Methods
Glucoamylases:
Acid bacterial alpha-amylase (ABAA) derived from Anoxybacillus contaminans and having the sequence shown in SEQ ID NO: 1.
Acid fungal alpha-amylase (AFAA) comprising the Rhizomucor pusillus alpha-amylase catalytic domain and the Aspergillus niger glucoamylase linker and CBM and having the sequence shown in SEQ ID NO: 2.
Glucoamylase derived from Aspergillus niger disclosed in Boel et al., 1984, EMBO J., 3(5): 1097-1102 and available from Novozymes A/S.
Glucoamylase derived from Trametes cingulata and having the sequence shown in SEQ ID NO: 3.
Yeast: Red StarTM available from Red Star/Lesaffre, USA
Homology/Identity
In context of the present invention polypeptide “identity” means the degree of identity between two amino acid sequences. The identity may suitably be determined by computer programs known in the art, such as, GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., 1970, Journal of Molecular Biology, 48: 443-453. The following settings for polypeptide sequence comparison are used: GAP creation penalty of 3.0 and GAP extension penalty of 0.1. The term “homologous sequence” is used to characterize a sequence having an amino acid sequence that is at least 70%, preferably at least 75%, or at least 80%, or at least 85%, or 90%, or at least 95%, at least 96%, at least 97%, at least 98% or even at least 99% identical to a known sequence. The relevant part of the amino acid sequence for the homology determination is the mature polypeptide, i.e., without the signal peptide.
Alpha-Amylase Activity (KNU)
The amylolytic 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.
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 incorporated by reference.
Acid Alpha-Amylase Activity
When used according to the present invention the activity of any 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.










Standard conditions/reaction conditions:
















Substrate:
Soluble starch. Concentration approx.



20 g DS/L.


Buffer:
Citrate, approx. 0.13 M, pH = 4.2


Iodine solution:
40.176 g potassium iodide + 0.088 g iodine/L


City water:
15°-20°dH (German degree hardness)


pH:
4.2


Incubation temperature:
30° C.


Reaction time:
11 minutes


Wavelength:
620 nm


Enzyme concentration:
0.13-0.19 AAU/mL


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 blue with iodine. Further details can be found in European Patent No. 140410, which disclosure is hereby incorporated by reference.
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 FAU 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.














Standard conditions/reaction conditions:


















Substrate:
Soluble starch, approx. 0.17 g/L



Buffer:
Citrate, approx. 0.03 M



Iodine (I2):
0.03 g/L



CaCl2:
1.85 mM



pH:
2.50 ± 0.05



Incubation temperature:
40° C.



Reaction time:
23 seconds



Wavelength:
590 nm



Enzyme concentration:
0.025 AFAU/mL



Enzyme working range:
0.01-0.04 AFAU/mL









A folder EB-SM-0259.02/01 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby incorporated by reference.
Glucoamylase Activity
Glucoamylase activity may be measured in AGI units or in AmyloGlucosidase Units (AGU).
Glucoamylase-Activity (AGI)
Glucoamylase (equivalent to amyloglucosidase) converts starch into glucose. The amount of glucose is determined here by the glucose oxidase method for the activity determination. The method described in the section 76-11 Starch-Glucoamylase Method with Subsequent Measurement of Glucose with Glucose Oxidase in “Approved methods of the American Association of Cereal Chemists”. Vol. 1-2 AACC, from American Association of Cereal Chemists, 2000; ISBN: 1-891127-12-8.
One glucoamylase unit (AGI) is the quantity of enzyme which will form 1 micromol of glucose per minute under the standard conditions of the method.










Standard conditions/reaction conditions:
















Substrate:
Soluble starch, concentration approx. 16 g



dry matter/L.


Buffer:
Acetate, approx. 0.04 M, pH = 4.3


pH:
4.3


Incubation temperature:
60° C.


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 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 blue with iodine.
Glucoamylase Activity (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 determined using a photometer at 340 nm as a measure of the original glucose concentration.












AMG incubation:










Substrate:
maltose 23.2 mM



Buffer:
acetate 0.1 M



pH:
4.30 ± 0.05



Incubation temperature:
37° C. ± 1



Reaction time:
5 minutes



Enzyme working range:
0.5-4.0 AGU/mL







Color reaction:










GlucDH:
430 U/L



Mutarotase:
9 U/L



NAD:
0.21 mM



Buffer:
phosphate 0.12 M; 0.15 M NaCl



pH:
7.60 ± 0.05



Incubation temperature:
37° C. ± 1



Reaction time:
5 minutes



Wavelength:
340 nm









A folder (EB-SM-0131.02/01) describing this analytical method in more detail is available on request from Novozymes A/S, Denmark, which folder is hereby incorporated by reference.
Proteolytic Activity (AU)
The proteolytic activity may be determined with denatured hemoglobin as substrate. In the Anson-Hemoglobin method for the determination of proteolytic activity denatured hemoglobin is digested, and the undigested hemoglobin is precipitated with trichloroacetic acid (TCA). The amount of TCA soluble product is determined with phenol reagent, which gives a blue color with tyrosine and tryptophan.
One Anson Unit (AU) is defined as the amount of enzyme which under standard conditions (i.e., 25° C., pH 7.5 and 10 min. reaction time) digests hemoglobin at an initial rate such that there is liberated per minute an amount of TCA soluble product which gives the same color with phenol reagent as one milliequivalent of tyrosine.
A folder AF 4/5 describing the analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby incorporated by reference.
EXAMPLE 1 
A 35% DS slurry was prepared from 194.44 g ground corn (90% less than 0.5 mm), 305.56 g of 37 mM NaOAc, 0.025% sodium azide, 20 mM CaCl2, pH 4.5. The pH was adjusted to 4.5 with 5N NaOH and the slurry was incubated with stirring at room temperature for one hour. For each reaction 5 g slurry was added to a 20 ml vial,—and the vials were incubated at 32° C. for one hour prior to enzyme dosing. Each vial was dosed with the appropriate amount of enzyme as shown in Table 1, capped and vortexed immediately, Vials were incubated at 32° C. and vortexed at 0.5, 1, 2, 3, and 4 hours. The reactions were stopped at 4 hours by addition of 50 micrL of 40% H2SO4. Three replicates were run for each reaction. Sample preparation consisted of centrifuging and filtering the supernatant through a 0.45 micrometer filter. Samples awaiting HPLC analysis were stored at 4° C. The HPLC results are shown in Table 1 below.







TABLE 1







Sugar concentrations in hydrolyzate after incubation with


combinations of various glucoamylases (GA), bacterial


alpha-amylase (BAA) and fungal alpha-amylase (FAA).








Enzyme Dose
Sugar Concentration


(mg enzyme protein/gDS)
(g/l)














GA


Glu-




Glucoamylase
dose
BAA
FAA
cose
maltose
Maltotriose





A. niger GA
0.150
0.0036
—
33.4
0.87
0.42


A. niger GA
0.150
0.0073
—
35.2
0.91
0.46


A. niger GA
0.150
0.0219
—
38.4
0.99
0.57


A. niger GA
0.150
0.0657
—
42.6
1.12
0.79


T. cingulata GA
0.100
0.0036
—
30.4
1.02
0.44


T. cingulata GA
0.100
0.0073
—
32.4
1.18
0.50


T. cingulata GA
0.100
0.0219
—
39.7
2.33
0.84


—
—
0.0036
0.033
25.0
10.2
3.24


—
—
0.0073
0.033
25.5
10.8
3.57


—
—
0.0219
0.033
26.2
12.2
4.17


—
—
0.0657
0.033
26.5
13.8
5.07


A. niger GA
0.100
0.0036
0.033
44.2
1.91
0.63


A. niger GA
0.100
0.0073
0.033
45.1
2.05
0.69


A. niger GA
0.100
0.0219
0.033
47.6
2.24
0.84









EXAMPLE 2 
Ground corn, 410 g of (90% less than 0.5 mm) was mixed 590 g tap water, 3.0 mL 1 g/L penicillin and 1 g of urea. The pH was adjusted to 4.5 with 5 N NaOH. DS level was determined to be 35%. 5 g of this slurry was added to a 20 ml vials for each reaction. Each vial was dosed with the appropriate amount of enzyme according to tables 2 or 3, followed by addition of 200 microL yeast propagate/5 g fermentation before incubation at 32° C. 9 replicate fermentations of each treatment were run. Three replicates were selected for 24 hour, 48 hour and 70 hour time point analysis. Vials were vortexed at 24, 48 and 70 hours. The time point analysis consisted of weighing the vials and prepping the sample for HPLC. The HPLC preparation consisted of stopping the reaction by addition of 50 microL of 40% H2SO4, centrifuging, and filtering through a 0.45 um filter. Samples awaiting HPLC analysis were stored at 4° C. The HPLC results are shown in table 2.







TABLE 2







Ethanol yields after incubation with yeast and combinations


of T. cingulata glucoamylase (GA) bacterial alpha-amylase (BAA)


and fungal alpha-amylase (FAA).










Enzyme Dose
Ethanol Yield



(mg enzyme/g DS)
(% w/v)














T. cingulata GA
BAA
FAA
24 hr
48 hr
70 hr


















—
0.000
0.033
6.82
10.67
13.16



—
0.050
0.033
7.18
11.55
13.30



0.060
0.000
0.033
9.13
14.16
15.18



0.060
0.050
0.033
9.23
14.31
16.06



0.060
0.000
—
4.45
6.63
8.54



0.060
0.050
—
5.21
7.51
9.43



0.027
—
0.015
10.87
14.26
15.32



0.054
—
0.030
9.39
13.38
15.77



0.162
—
0.090
13.05
15.19
15.58

## Claims

1. A process for saccharifying a granular starch, comprising treating the granular starch with:
a) a glucoamylase,
b) a bacterial alpha-amylase comprising a carbohydrate-binding module, and
c) a fungal alpha-amylase comprising a carbohydrate-binding module, to produce a starch hydrolyzate.

2. The process of claim 1, further comprising contacting the starch hydrolyzate with a fermenting organism to produce a fermentation product.

3. The process of claim 2, wherein the saccharification and fermentation are carried out simultaneously.

4. The process of claim 2, further comprising recovering the fermentation product after fermentation.

5. The process of claim 4, wherein the fermentation product is an alcohol.

6. The process of claim 1, wherein the fungal alpha-amylase comprising a carbohydrate-binding module and/or the bacterial alpha-amylase comprising a carbohydrate-binding module is an acid fungal alpha-amylase and/or an acid bacterial alpha-amylase.

7. The process of claim 1, wherein the carbohydrate-binding module of the fungal alpha-amylase has an amino acid sequence of amino acids 488 to 595 in SEQ ID NO: 2.

8. The process of claim 1, wherein the fungal alpha-amylase comprising a carbohydrate-binding module has an amino acid sequence of amino acids 13 to 595 in SEQ ID NO: 2.

9. The process of claim 1, wherein the bacterial alpha-amylase comprises a catalytic domain which has an amino acid sequence having at least 90% homology to amino acids 32 to 520 in SEQ ID NO: 1.

10. The process of claim 1, wherein the bacterial alpha-amylase comprises a catalytic domain which has an amino acid sequence having at least 95% homology to amino acids 32 to 520 in SEQ ID NO: 1.

11. The process of claim 1, wherein the carbohydrate-binding module of the bacterial alpha-amylase has an amino acid sequence of amino acids 521 to 619 in SEQ ID NO: 1.

12. The process of claim 1, wherein the glucoamylase is an Aspergillus niger, Athelia rolfsii, or Trametes cingulata glucoamylase.

13. The process of claim 1, wherein the process is performed in the presence of a protease or a phytase.