Processes for Producing a Fermantation Product

Abstract

The present invention relates to processes for producing a fermentation product, such as ethanol, from milled starch-containing material comprising (a) saccharifying the milled starch-containing material with a glucoamylase having an amino acid sequence shown in SEQ ID NO: 2, or a glucoamylase being at least 70% identical thereto, at a temperature below the initial gelatinization temperature of said starch-containing material, (b) fermenting using a fermenting organism.

Description

REFERENCE TO A SEQUENCE LISTING

[0001] This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] 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 in the presence of glucoamylase.

[0004] 2. Description of Related Art

[0005] 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.

[0006] A conventional enzymatic liquefaction process may be carried out as a three-step hot slurry process. The slurry is heated to between 80-85.degree. C. and thermostable alpha-amylase added to initiate liquefaction. The slurry is then jet-cooked at a temperature between 105-125.degree. C. to complete gelatinization of the slurry, cooled to 60-95.degree. C. and, generally, additional alpha-amylase is added to finalize hydrolysis. The liquefaction process is generally carried out at pH between 5 and 6. Milled and liquefied whole grains are known as mash.

[0007] During saccharification, the dextrins from the liquefaction are further hydrolyzed to produce low molecular sugars DP.sub.1-3 that can be metabolized by a fermenting organism, such as yeast. The hydrolysis is typically accomplished using glucoamylase, alternatively or in addition to glucoamylases, alphaglucosidases and/or acid alpha-amylases can be used. A full saccharification step typically last up to 72 hours, however, it is common only to do a pre-saccharification of, e.g., 40-90 minutes at a temperature above 50.degree. C., followed by a complete saccharification during fermentation in a process known as simultaneous saccharification and fermentation (SSF).

[0008] 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.degree. 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.degree. C.

[0009] 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.

[0010] 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.

[0011] U.S. Pat. No. 4,316,956 provides a fermentation process for conversion of granular starch into ethanol.

[0012] European Patent No. 140410 provides an enzyme composition for starch hydrolysis.

[0013] WO 2004/081193 concerns a method of producing high levels of alcohol during fermentation of plant material. The method includes i) preparing the plant material for saccharification, ii) converting the prepared plant material to sugar without cooking, and iii) fermenting the sugars.

[0014] The object of the present invention is to provide improved processes for conversion of milled starch-containing material, such as granular starch, into a fermentation product, such as ethanol.

SUMMARY OF THE INVENTION

[0015] The present invention provides processes of producing a fermentation product from starch-containing material without gelatinization of said starch-containing material using glucoamylase.

[0016] In the first aspect, the invention provides a process for producing a fermentation product from milled starch-containing material comprising:

[0017] (a) saccharifying milled starch-containing material with a glucoamylase having an amino acid sequence shown in SEQ ID NO: 2, or a glucoamylase being at least 70% identical thereto, at a temperature below the initial gelatinization temperature of said starch-containing material,

[0018] (b) fermenting using a fermenting organism.

[0019] Steps (a) and (b) may be carried out sequentially or simultaneously.

[0020] Preferably, a slurry comprising water and milled starch-containing material is prepared before step (a). The dry solid content (DS) lies in the range from 20-55 wt.-%. 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. The process of the invention may be carried out for a period of 1 to 250 hours. The pH during saccharification and/or fermentation may be in the range from between 3 and 7. During fermentation the glucose concentration may be kept at a level of below about 3 wt.-%. In a preferred embodiment saccharification and fermentation is carried out simultaneously. According to a preferred embodiment the glucoamylase is derived from a strain of Athelia, preferably a strain of Athelia rolfsii. The glucoamylase is present in an amount of 0.001 to 10 AGU/g DS. In a preferred embodiment an acid alpha-amylase is present as well. The acid alpha-amylase may be a fungal or bacterial alpha-amylase, preferably a fungal alpha-amylase derived from a strain of Aspergillus, especially A. niger or A. oryzae. The acid alpha-amylase may be present in a concentration of 0.1 to 10 AFAU/g DS. The ratio between acid alpha-amylase and glucoamylase may be between 0.1 and 10 AGU/AFAU. Optionally the fermentation product, such as ethanol, is recovered after fermentation. Other ingredients and enzyme activities may also be present during the process of the invention. Examples of other enzyme activities are xylanase, cellulase, and phytase activity.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides processes for producing a fermentation product from starch-containing material without gelatinization of said starch-containing material. In one embodiment only a glucoamylase is needed during saccharification and fermentation. According to the invention the desired fermentation product, such as ethanol, can be produced without liquefying the aqueous slurry containing the starch-containing material. If the aqueous slurry containing starch-containing material is heated to above the gelatinization temperature liquefaction is necessary. In general a process of the invention includes saccharifying milled starch-containing material below the gelatinization temperature in the presence of a glucoamylase having the sequence shown in SEQ ID NO: 2, or homologues thereto, to produce sugars that can be fermented into the desired fermentation product by a suitable fermenting organism.

[0022] The inventors have found that when producing ethanol from uncooked milled corn using the glucoamylase derived from Athelia rolfsii shown in SEQ ID NO: 2 a significantly higher ethanol yield is obtained compared to a corresponding process using glucoamylase derived from Aspergillus niger or Talaromyces emersonii. When adding fungal acid alpha-amylase from Aspergillus niger (SEQ ID NO: 3) to the process the. performance is still significantly higher, i.e., compared to a corresponding process using the Aspergillus niger glucoamylase and fungal acid alpha-amylase derived from Aspergillus niger.

[0023] Accordingly, in the first aspect the invention relates to a process for producing a fermentation product from milled starch-containing material comprising:

[0024] (a) saccharifying milled starch-containing material with a glucoamylase having an amino acid sequence shown in SEQ ID NO: 2, or a glucoamylase being at least 70% identical thereto, at a temperature below the initial gelatinization temperature of said starch-containing material,

[0025] (b) fermenting using a fermenting organism.

[0026] Steps (a) and (b) of the process of the invention may be carried out sequentially or simultaneously.

[0027] Before step (a), a slurry of starch-containing material, such as granular starch, having 20-55 wt.-% dry solids, preferably 25-40 wt.-% dry solids, more preferably 30-35% 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 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.

[0028] The milled starch-containing material may be prepared by milling starch-containing material to a particle size of 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 of the starch-containing material is converted into a soluble starch hydrolysate.

[0029] The process of the invention is conducted at a temperature below the initial gelatinization temperature. Preferably the temperature at which step (a) is carried out is between 30-75.degree. C., preferably between 45-60.degree. C.

[0030] In a preferred embodiment, steps (a) and (b) are carried out as a simultaneous saccharification and fermentation process. In such preferred embodiment the process is typically carried at a temperature between 28.degree. C. and 36.degree. C., such as between 29.degree. C. and 35.degree. C., such as between 30.degree. C. and 34.degree. C., such as around 32.degree. C. According to the invention the temperature may be adjusted up or down during fermentation.

[0031] In an embodiment simultaneous saccharification and fermentation is carried out so that the sugar level, such as 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.-%.

[0032] 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.

Starch-Containing Materials

[0033] Any suitable starch-containing starting material, including granular starch, may be used according to the present invention. The starting material is generally selected based on the desired fermentation product. 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.

[0034] 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.degree. C. to 75.degree. 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 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 hydrolysate 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.

[0035] The starch-containing material is milled in order to expose more surface. In an embodiment the particle size 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, preferably 0.1-0.5 mm screen.

[0036] 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.degree. C. and 75.degree. 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. S. and Lii. C., Starch/Starke, Vol. 44 (12) pp. 461-466 (1992).

Fermentation Product

[0037] 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., H.sub.2 and CO.sub.2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B.sub.12, 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.

Fermenting Organism

[0038] "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 Star.TM./Lesaffre Ethanol Red (available from Red Star/Lesaffre, USA) FALI (available from Fleischmann's Yeast, a division of Bums Philp Food Inc., USA), SUPERSTART (available from Alltech), GERT STRAND (available from Gert Strand AB, Sweden) and FERMIOL (available from DSM Specialties).

Glucoamylase

[0039] The term "glucoamylase activity" means a glucan 1,4-alpha-glucosidase which hydrolyses 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.

[0040] The glucoamylase used in a process of the invention has the amino acid sequence shown in SEQ ID NO: 2 (amino acid residues 1 to 561), 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: 2 (amino acid residues 1 to 561). The glucoamylase derived from Athelia rolfsii, the amino acid sequence of which is available as SPTREMBL:Q12596, is almost identical to the one shown in SEQ ID NO: 2, except for one amino acid residue corresponding to the amino acid residue in position 97 of SEQ ID NO: 2, which in the database sequence is a serine, whereas in SEQ ID NO: 2 it is a proline. The annotation of the database sequence identifies amino acid residues 1-18 as a signal peptide, and residues 19-579 (i.e., 561 amino acid residues) as the mature glucoamylase enzyme, with residues 472-482 serving as a linker between the glucoamylase domain and the starch-binding domain comprised in residues 483-579.

[0041] The glucoamylase may in an embodiment be added in an amount of 0.001 to 10 AGU/g DS, preferably from 0.01 to 5 AGU/g DS, such as around 0.1, 0.3, 0.5, 1 or 2 AGU/g DS, especially 0.1 to 0.5 AGU/g DS or 0.02-20 AGU/g DS, preferably 0.1-10 AGU/g DS.

Alpha-Amylase

[0042] In a preferred embodiment an alpha-amylase may be added to the process of the invention. The alpha-amylase may according to the invention be of any origin. Preferred are alpha-amylases of fungal or bacterial origin.

[0043] 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

[0044] According to the invention the bacterial alpha-amylase may be derived from the genus Bacillus.

[0045] In a preferred embodiment the Bacillus alpha-amylase is 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-amylases include the Bacillus lichenifornis alpha-amylase (BLA) shown in SEQ ID NO: 5, the Bacillus amyloliquefaciens alpha-amylase (BAN) shown in SEQ ID NO: 6, and the Bacillus stearothermophilus alpha-amylase (BSG) shown in SEQ ID NO: 7. In an embodiment of the invention the alpha-amylase is 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% identity to any of the sequences shown in SEQ ID NO: 5, 6, or 7 of the present application or SEQ ID NO: 1, 2 or 3 in WO 99/19467.

[0046] 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. Pat. Nos. 6,093,562, 6,187,576, and 6,297,038 (hereby incorporated by reference) and include Bacillus stearothermophilus alpha-amylase (BSG alpha-amylase) variants having a double deletion disclosed in WO 1996/023873--see e.g., page 20, lines 1-10 (hereby incorporated by reference), preferably corresponding to delta(181-182) compared to the wild-type BSG 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-amylases, especially Bacillus stearothermophilus 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: 7 of the present application and SEQ ID NO: 3 disclosed in WO 99/19467.

[0047] The alpha-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 alpha-amylase from Bacillus stearothermophilus strain NCIB 11837 is commercially available from Novozymes A/S, Denmark. The maltogenic alpha-amylase is described in U.S. Pat. Nos. 4,598,048, 4,604,355 and 6,162,628, which are hereby incorporated by reference.

Bacterial Hybrid Alpha-Amylases

[0048] A hybrid alpha-amylase specifically contemplated comprises 445 C-terminal amino acid residues of the Bacillus licheniformis alpha-amylase (shown in SEQ ID NO: 5) and the 37 N-terminal amino acid residues of the alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQ ID NO: 6), with one or more, especially all, of the following substitution:

G48A+T491+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S

(using the Bacillus licheniformis numbering). 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).

[0049] The bacterial alpha-amylase may be added in amounts as are well-known in the art. When measured in KNU units (described below in the Materials & Methods"-section) the alpha-amylase activity is preferably present in an amount of 0.5-5,000 NU/g of DS, in an amount of 1-500 NU/g of DS, or more preferably in an amount of 5-1,000 NU/g of DS, such as 10-100 NU/g DS.

Fungal Alpha-Amylases

[0050] Fungal acid alpha-amylases include acid alpha-amylases derived from a strain of the genus Aspergillus, such as, Aspergillus oryzae and Aspergillus niger alpha-amylases.

[0051] A preferred acid 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. more than 70%, more than 75%, more than 80%, more than 85% more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or even 100% identity to the mature part of the amino acid sequence shown in SEQ ID NO: 10 in WO 96/23874 or shown as SEQ ID NO: 4 of the present application.

[0052] Another preferred acid alpha-amylase 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: 3. Also variants of said acid fungal amylase having at least 70% identity, such as at least 80% or even at least 90% identity, such as at least 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NO: 3 are contemplated. A commercially available acid fungal alpha-amylase derived from Aspergillus niger is SP288 (available from Novozymes A/S, Denmark).

[0053] The fungal acid alpha-amylase may also be a wild-type enzyme comprising a carbohydrate-binding module (CBM) and an alpha-amylase catalytic domain (i.e., none-hybrid), or a variant thereof. In an embodiment the wild-type acid alpha-amylase is derived from a strain of Aspergillus kawachi, in particular the alpha-amylase shown in SEQ ID NO: 31. Also variants of said fungal acid amylase 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: 31 are contemplated.

Fungal Hybrid Alpha-Amylases

[0054] 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 PCT/US2004/020499 (Novozymes), which is hereby incorporated by reference. A hybrid alpha-amylase may comprise an alpha-amylase catalytic domain (CD) and a carbohydrate-binding module (CBM) and optional a linker.

[0055] Hybrid enzymes or a genetically modified wild-type enzymes, as referred to herein, include species comprising an amino acid sequence of an alpha-amylase enzyme (EC 3.2.1.1) linked (i.e., covalently bound) to an amino acid sequence comprising a carbohydrate-binding module (CBM).

[0056] CBM-containing hybrid enzymes, as well as detailed descriptions of the preparation and purification, thereof, are known in the art [see, e.g. WO 90100609, WO 94/24158 and WO 95/16782, as well as Greenwood et al. Biotechnology and Bioengineering 44 (1994) pp. 1295-1305]. They may, e.g., be prepared by transforming into a host cell a DNA construct comprising at least a fragment of DNA encoding the carbohydrate-binding module ligated, with or without a linker, to a DNA sequence encoding the enzyme of interest, and growing the transformed host cell to express the fused gene. The resulting recombinant product (hybrid enzyme)--often referred to in the art as a "fusion protein--may be described by the following general formula:

A-CBM-MR-X

[0057] In the latter formula, A-CBM is the N-terminal or the C-terminal region of an amino acid sequence comprising at least the carbohydrate-binding module (CBM) per se. MR is the middle region (the "linker"), and X is the sequence of amino acid residues of a polypeptide encoded by a DNA sequence encoding the enzyme (or other protein) to which the CBM is to be linked.

[0058] The moiety A may either be absent (such that A-CBM is a CBM per se, i.e. comprises no amino acid residues other than those constituting the CBM) or may be a sequence of one or more amino acid residues (functioning as a terminal extension of the CBM per se). The linker (MR) may be a bond, or a short linking group comprising from about 2 to about 100 carbon atoms, in particular of from 2 to 40 carbon atoms. However, MR is preferably a sequence of from about 2 to about 100 amino acid residues, more preferably of from 2 to 40 amino acid residues, such as from 2 to 15 amino acid residues.

[0059] The moiety X may constitute either the N-terminal or the C-terminal region of the overall hybrid enzyme.

[0060] It will thus be apparent from the above that the CBM in a hybrid enzyme of the type in question may be positioned C-terminally, N-terminally or internally in the hybrid enzyme.

Linker Sequence

[0061] The optional linker sequence may be any suitable linker sequence. In preferred embodiments the linker sequence is derived from the Athelia rolfsii glucoamylase, the A. niger glucoamylase or the A. kawachii alpha-amylase such as a linker sequence selected from the group consisting of A. niger glucoamylase linker:

TGGTTTTATPTGSGSVTSTSKTTATASKTSTSTSSTSA (SEQ ID NO: 8), A. kawachii alpha-amylase linker: TTTTTTAAATSTSKATTSSSSSSAAATTSSS (SEQ ID NO: 9), Athelia rolfsii glucoamylase linker: GATSPGGSSGS (SEQ ID NO: 10), and the PEPT linker: PEPTPEPT (SEQ ID NO: 11). In another preferred embodiment the hybrid enzymes has a linker sequence which differs from the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 in no more than 10 positions, no more than 9 positions, no more than 8 positions, no more than 7 positions, no more than 6 positions, no more than 5 positions, no more than 4 positions, no more than 3 positions, no more than 2 positions, or even no more than 1 position.

Carbohydrate-Binding Modules

[0062] A carbohydrate-binding module (CBM), or as often referred to, a carbohydrate-binding domain (CBD), is a polypeptide amino acid sequence which binds preferentially to a poly- or oligosaccharide (carbohydrate), frequently--but not necessarily exclusively--to a water-insoluble (including crystalline) form thereof.

[0063] CBMs derived from starch degrading enzymes are often referred to as starch-binding modules or SBMs (CBMs which may occur in certain amylolytic enzymes, such as certain glucoamylases, or in enzymes such as cyclodextrin glucanotransferases, or in alpha-amylases). Likewise, other sub-classes of CBMs would embrace, e.g., cellulose-binding modules (CBMs from cellulolytic enzymes), chitin-binding modules (CBMs which typically occur in chitinases), xylan-binding modules (CBMs which typically occur in xylanases), mannan-binding modules (CBMs which typically occur in mannanases). SBMs are often referred to as SBDs (Starch Binding Domains).

[0064] CBMs are found as integral parts of large polypeptides or proteins consisting of two or more polypeptide amino acid sequence regions, especially in hydrolytic enzymes (hydrolases) which typically comprise a catalytic module containing the active site for substrate hydrolysis and a carbohydrate-binding module (CBM) for binding to the carbohydrate substrate in question. Such enzymes can comprise more than one catalytic module and one, two or three CBMs, and optionally further comprise one or more polypeptide amino acid sequence regions linking the CBM(s) with the catalytic module(s), a region of the latter type usually being denoted a "linker". Examples of hydrolytic enzymes comprising a CBM--some of which have already been mentioned above--are cellulases, xylanases, mannanases, arabinofuranosidases, acetylesterases and chitinases. CBMs have also been found in algae, e.g., in the red alga Porphyra purpurea in the form of a non-hydrolytic polysaccharide-binding protein.

[0065] In proteins/polypeptides in which CBMs occur (e.g., enzymes, typically hydrolytic enzymes), a CBM may be located at the N or C terminus or at an internal position.

[0066] That part of a polypeptide or protein (e.g., hydrolytic enzyme) which constitutes a CBM per se typically consists of more than about 30 and less than about 250 amino acid residues.

[0067] The "Carbohydrate-Binding Module of Family 20" or a CBM-20 module is in the context of this invention defined as a sequence of approximately 100 amino acids having at least 45% identity to the Carbohydrate-Binding Module (CBM) of the polypeptide disclosed in FIG. 1 by Joergensen et al (1997) in Biotechnol. Lett. 19:1027-1031. The CBM comprises the last 102 amino acids of the polypeptide, i.e., the subsequence from amino acid 582 to amino acid 683. The numbering of Glycoside Hydrolase Families applied in this disclosure follows the concept of Coutinho, P. M. & Henrissat, B. (1999) CAZy--Carbohydrate-Active Enzymes server at URL: afmb.cnrs-mrs.fr/.about.cazy/CAZY/index.html or alternatively Coutinho, P. M. & Henrissat, B. 1999; The modular structure of cellulase's and other carbohydrate-active enzymes: an integrated database approach. In "Genetics, Biochemistry and Ecology of Cellulose Degradation", K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita and T. Kimura eds., Uni Publishers Co., Tokyo, pp. 15-23, and Bourne, Y. & Henrissat, B. 2001; Glycoside hydrolases and glycosyltransferases: families and functional modules, Current Opinion in Structural Biology 11:593-600.

[0068] Examples of enzymes which comprise a CBM suitable for use in the context of the invention are alpha-amylases, maltogenic alpha-amylases, cellulases, xylanases, mannanases, arabinofuranosidases, acetylesterases and chitinases. Further CBMs of interest in relation to the present invention include CBMs deriving from glucoamylases (EC 3.2.1.3) or from CGTases (EC 2.4.1.19).

[0069] CBMs deriving from fungal, bacterial or plant sources will generally be suitable for use in the context of the invention. Preferred are CBMs of fungal origin, more preferably from Aspergillus sp., Bacillus sp., Klebsiella sp., or Rhizopus sp. In this connection, techniques suitable for isolating the relevant genes are well known in the art.

[0070] Preferred for the invention is CBMs of Carbohydrate-Binding Module Family 20. CBMs of Carbohydrate-Binding Module Family 20 suitable for the invention may be derived from glucoamylases of Aspergillus awamori (SWISSPROT Q12537), Aspergillus kawachii (SWISSPROT P23176), Aspergillus niger (SWISSPROT P04064), Aspergillus oryzae (SWISSPROT P36914), from alpha-amylases of Aspergillus kawachii (EMBL:#AB008370), Aspergillus nidulans (NCBI AAF17100.1), from beta-amylases of Bacillus cereus (SWISSPROT P36924), or from CGTases of Bacillus circulans (SWISSPROT P43379). Preferred is a CBM from the alpha-amylase of Aspergillus kawachii (EMBL:#AB008370) as well as CBMs having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98% or even at least 99% identity to the CBM of the alpha-amylase of Aspergillus kawachii (EMBL:#AB008370), i.e., a CBM having at least 50%, 60%, 70%, 80%, 95%, 96%, 97%, 98% or even at least 99% identity to the amino acid sequence of SEQ ID NO: 12. Also preferred for the invention are the CBMs of Carbohydrate-Binding Module Family 20 having the amino acid sequences shown in SEQ ID NO: 14 (Bacillus flavothermus CBM), SEQ ID NO: 15 (Bacillus sp. CBM), and SEQ ID NO: 16 (Alcaliphilic Bacillus CBM) and disclosed in International application PCT/DK2004/000456 as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively. Further preferred CBMs include the CBMs of the glucoamylase from Hormoconis sp. such as from Hormoconis resinae (Syn. Creosote fungus or Amorphotheca resinae) such as the CBM of SWISSPROT:Q03045 (SEQ ID NO: 17), from Lentinula sp. such as from Lentinula edodes (shiitake mushroom) such as the CBM of SPTREMBL:Q9P4C5 (SEQ ID NO: 18), from Neurospora sp. such as from Neurospora crassa such as the CBM of SWISSPROT:P14804 (SEQ ID NO: 19), from Talaromyces sp. such as from Talaromyces byssochlamydioides such as the CBM of NN005220 (SEQ ID NO: 20), from Geosmithia sp. such as from Geosmithia cylindrospora, such as the CBM of NN48286 (SEQ ID NO: 21), from Scorias sp. such as from Scorias spongiosa such as the CBM of NN007096 (SEQ ID NO: 22), from Eupenicillium sp. such as from Eupenicillium ludwigii such as the CBM of NN005968 (SEQ ID NO: 23), from Aspergillus sp. such as from Aspergillus japonicus such as the CBM of NN001136 (SEQ ID NO: 24), from Penicillium sp. such as from Penicillium cf. miczynskii such as the CBM of NN48691 (SEQ ID NO: 25), from Mz1 Penicillium sp. such as the CBM of NN48690 (SEQ ID NO: 26), from Thysanophora sp. such as the CBM of NN48711 (SEQ ID NO: 27), and from Humicola sp. such as from Humicola grisea var. thermoidea such as the CBM of SPTREMBL:Q12623 (SEQ ID NO: 28). Most preferred CBMs include the CBMs of the glucoamylase from Aspergillus sp. such as from Aspergillus niger , such as SEQ ID NO: 29, and Athelia sp. such as from Athelia rolfsii, such as SEQ ID NO: 30.

[0071] Preferably the hybrid enzyme comprises a CBM sequence having at least 50%, at least 60%, at least 70%, at least 80% at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or even at least 99% identity to any of the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 10, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30. In yet another preferred embodiment the CBM sequence has an amino acid sequence which differs from the amino acid sequence amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 14 10, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30 in no more than 10 amino acid positions, no more than 9 positions, no more than 8 positions, no more than 7 positions, no more than 6 positions, no more than 5 positions, no more than 4 positions, no more than 3 positions, no more than 2 positions, or even no more than 1 position. In a most preferred embodiment the hybrid enzyme comprises a CBM derived from a glucoamylase from Athelia rolfsii, such as the glucoamylase from A. rolfsii AHU 9627 described in U.S. Pat. No. 4,727,026.

[0072] Further suitable CBMs of Carbohydrate-Binding Module Family 20 may be found at URL: afmb.cnrs-mrs.fr/.about.cazy/CAZY/index.html).

[0073] Once a nucleotide sequence encoding the substrate-binding (carbohydrate-binding) region has been identified, either as cDNA or chromosomal DNA, it may then be manipulated in a variety of ways to fuse it to a DNA sequence encoding the enzyme of interest. The DNA fragment encoding the carbohydrate-binding amino acid sequence, and the DNA encoding the enzyme of interest are then ligated with or without a linker. The resulting ligated DNA may then be manipulated in a variety of ways to achieve expression.

Catalytic Domain in Hybrid

[0074] Alpha-amylases (in particular acid alpha-amylases) which are appropriate as the basis for CBM/amylase hybrids of the types employed in the context of the present invention include those of fungal origin. Preferred examples are the ones described above in the "fungal alpha-amylases"-section, which include acid alpha-amylases from Aspergillus niger shown as SEQ ID NO: 3 or Aspergillus oryzae shown in SEQ ID NO: 4.

[0075] Even more preferred is an embodiment wherein the hybrid enzyme comprises an alpha-amylase sequence derived from the A. oryzae acid alpha-amylase (Fungamyl.TM., SEQ ID NO: 4), and/or a linker sequence derived from the A. kawachii alpha-amylase (SEQ ID NO: 9 or the A. rolfsii glucoamylase (SEQ ID NO: 10), and/or a CBM derived from the A. kawachii alpha-amylase (SEQ ID NO: 13) or the A. rolfsii glucoamylase (SEQ ID NO: 30).

[0076] Also preferred is an embodiment wherein the hybrid enzyme comprises an alpha-amylase sequence derived from the A. niger acid alpha-amylase (SP288) catalytic module having the sequence shown in SEQ ID NO: 3, and/or a linker sequence derived from the A. kawachii alpha-amylase (SEQ ID NO: 9) or the A. rolfsii glucoamylase (SEQ ID NO: 10), and/or the CBM derived from the A. kawachii alpha-amylase (SEQ ID NO: 12), the A. rolfsii glucoamylase (SEQ ID NO: 30) or the A. niger glucoamylase (SEQ ID NO: 29). In a particularly preferred embodiment the hybrid enzyme comprises the A. niger acid alpha-amylase (SP288) catalytic module having the sequence shown in SEQ ID NO: 3 and the A. kawachii alpha-amylase linker (SEQ ID NO: 9) and CBM (SEQ ID NO: 12).

[0077] In a specific embodiment the hybrid enzyme is the mature part of the amino acid sequence shown in SEQ ID NO: 33 (A. niger acid alpha-amylase catalytic domain-A. kawachii alpha-amylase linker-A. niger glucoamylase CBM), SEQ ID NO: 35 (A. niger acid alpha-amylase catalytic domain-A. kawachii alpha-amylase linker-A. rolfsii glucoamylase CBM), or SEQ ID NO: 37 (A. oryzae acid alpha-amylase catalytic domain-A. kawachii alpha-amylase linker-A. kawachii alpha-amylase CBM), or SEQ ID NO: 39 (A. niger acid alpha-amylase catalytic domain-A. rolfsii glucoamylase linker-A. rolfsii glucoamylase CBM), or SEQ ID NO: 41 (A. oryzae acid alpha-amylase catalytic domain-A. rolfsii glucoamylase linker-A. rolfsii glucoamylase CBM) or the hybrid consisting of A. niger acid alpha-amylase catalytic domain (SEQ ID NO: 3)-A. kawachii alpha-amylase linker (SEQ ID NO: 9)-A. kawachii alpha-amylase CBM (SEQ ID NO: 13) or a hybrid enzyme that has an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or even at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of the afore mentioned amino acid sequences.

[0078] In another preferred embodiment the hybrid enzyme has an amino acid sequence which differs from the amino acid sequence amino acid sequence shown in SEQ ID NO: 33 (A. niger acid alpha-amylase catalytic domain-A. kawachii alpha-amylase linker-A. niger glucoamylase CBM), SEQ ID NO: 35 (A. niger acid alpha-amylase catalytic domain-A. kawachii alpha-amylase linker-A. rolfsii glucoamylase CBM), SEQ ID NO: 37 (A. oryzae acid alpha-amylase catalytic domain-A. kawachii alpha-amylase linker-A. kawachii alpha-amylase CBM), SEQ ID NO: 39 (A. niger acid alpha-amylase catalytic domain-A. rolfsii glucoamylase linker-A. rolfsii glucoamylase CBM) or SEQ ID NO: 41 (A. oryzae acid alpha-amylase catalytic domain-A. rolfsii glucoamylase linker-A. rolfsii glucoamylase CBM) or the hybrid consisting of A. niger acid alpha-amylase catalytic domain (SEQ ID NO: 3)-A. kawachii alpha-amylase linker (SEQ ID NO: 9)-A. kawachii alpha-amylase CBM (SEQ ID NO: 13) in no more than 10 positions, no more than 9 positions, no more than 8 positions, no more than 7 positions, no more than 6 positions, no more than 5 positions, no more than 4 positions, no more than 3 positions, no more than 2 positions, or even no more than 1 position.

Commercial Alpha-Amylase Products

[0079] Preferred commercial compositions comprising alpha-amylase include MYCOLASE from DSM (Gist Brocades), BAN.TM., TERMAMYL.TM. SC, FUNGAMYL.TM., LIQUOZYME.TM. X and SAN.TM. SUPER, SAN.TM. EXTRA L (Novozymes A/S) and CLARASE.TM. L-40,000, DEX-LO.TM., SPEYME FRED, SPEZYME.TM. AA, and SPEZYME.TM. DELTA AA (Genencor Int.), and the acid fungal alpha-amylase sold under the trade name SP288 (available from Novozymes A/S, Denmark).

[0080] The acid alpha-amylases may according to the invention be added in an amount of 0.1 to 10 AFAU/g DS, preferably 0.10 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS.

Combination of Glucoamylase and Acid Alpha-Amylase

[0081] Even though the presence of acid alpha-amylase is not mandatory according to the invention the activities of acid alpha-amylase and glucoamylase may be present in a ratio of between 0.3 and 5.0 AFAU/AGU. More preferably the ratio between acid alpha-amylase activity and glucoamylase activity is at least 0.35,. at least 0.40, at least 0.50, at least 0.60, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.85, or even at least 1.9 AFAU/AGU. However, the ratio between acid alpha-amylase activity and glucoamylase activity should preferably be less than 4.5, less than 4.0, less than 3.5, less than 3.0, less than 2.5, or even less than 2.25 AFAU/AGU. In AUU/AGI the activities of acid alpha-amylase and glucoamylase are preferably present in a ratio of between 0.4 and 6.5 AUU/AGI. More preferably the ratio between acid alpha-amylase activity and glucoamylase activity is at least 0.45, at least 0.50, at least 0.60, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, or even at least 2.5 AUU/AGI. However, the ratio between acid alpha-amylase activity and glucoamylase activity is preferably less than 6.0, less than 5.5, less than 4.5, less than 4.0, less than 3.5, or even less than 3.0 AUU/AGI.

Protease

[0082] According to the process of the invention a protease may be present during saccharification and/or fermentation as well.

[0083] In a preferred embodiment the protease is an acid protease of microbial origin, preferably of fungal or bacterial origin.

[0084] 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.

[0085] Contemplated acid fungal proteases include fungal proteases derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra, Irpex, Penicillium, Scierotiumand 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.

[0086] 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.

[0087] 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 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% identity.

[0088] 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).

[0089] 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

[0090] 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

[0091] The fermentation product, such as ethanol, may optionally be recovered after fermentation. The recovery may be performed by any conventional manner such as, e.g., distillation.

[0092] 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.

[0093] Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

MATERIALS AND METHODS

Glucoamylases:

[0094] Glucoamylase derived from Athelia rolfsii disclosed in SEQ ID NO: 2 and available from Novozymes A/S. [0095] Glucoamylase derived from Aspergillus niger disclosed in Boel et al. (1984), EMBO J. 3 (5) p. 1097-1102 and available from Novozymes A/S. [0096] Glucoamylase derived from Talaromyces emersonii disclosed in WO 99/28448 and available from Novozymes A/S. [0097] Acid fungal alpha-amylase is derived from Aspergillus niger consisting of the Aspergillus niger acid alpha-amylase catalytic domain (SEQ ID NO: 3), Aspergillus kawachii alpha-amylase linker (SEQ ID NO: 9)-Aspergillus kawachii alpha-amylase CBM (SEQ ID NO: 13).

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

Homology/Identity

[0098] In context of the present invention "homology" means the degree of identity between two amino acid sequences. The homology 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, Wisconsin, 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.

Alpha-Amylase Activity (KNU)

[0099] 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.

[0100] One Kilo Novo alpha amylase Unit (KNU) is defined as the amount of enzyme which, under standard conditions (i.e. at 37.degree. C.+/-0.05; 0.0003 M Ca.sup.2+; and pH 5.6) dextrinizes 5260 mg starch dry substance Merck Amylum solubile.

[0101] 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

[0102] 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 MU (Acid Alpha-amylase Units).

Acid Alpha-Amylase Units (MU)

[0103] The acid alpha-amylase activity can be measured in MU (Acid Alpha-amylase Units), which is an absolute method. One Acid Amylase Unit (MU) 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.

TABLE-US-00001 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.degree.-20.degree.dH (German degree hardness) pH: 4.2 Incubation temperature: 30.degree. 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

[0104] The starch should be Lintner starch, which is a thin-boiling starch used in the laboratory as calorimetric 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)

[0105] 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.

[0106] 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.

##STR00001##

TABLE-US-00002 Standard conditions/reaction conditions: Substrate: Soluble starch, approx. 0.17 g/L Buffer: Citrate, approx. 0.03 M Iodine (I2): 0.03 g/L CaCl.sub.2: 1.85 mM pH: 2.50 .+-. 0.05 Incubation temperature: 40.degree. C. Reaction time: 23 seconds Wavelength: 590 nm Enzyme concentration: 0.025 AFAU/mL Enzyme working range: 0.01-0.04 AFAU/mL

[0107] 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

[0108] Glucoamylase activity may be measured in AGI units or in AmyloGlucosidase Units (AGU).

Glucoamylase Activity (AGI)

[0109] 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.

[0110] 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.

TABLE-US-00003 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.degree. 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.

[0111] 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)

[0112] The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute under the standard conditions 37.degree. C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes.

[0113] 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.

TABLE-US-00004 AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1 M pH: 4.30 .+-. 0.05 Incubation 37.degree. C. .+-. 1 temperature: Reaction time: 5 minutes Enzyme working range: 0.5-4.0 AGU/mL

TABLE-US-00005 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 37.degree. C. .+-. 1 temperature: Reaction time: 5 minutes Wavelength: 340 nm

[0114] 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)

[0115] 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.

[0116] One Anson Unit (AU) is defined as the amount of enzyme which under standard conditions (i.e. 25.degree. 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.

[0117] 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

Evaluation of Athelia Rolfsii Glucoamylase in `One-Step` Fuel Ethanol Fermentations

[0118] The relative performance of Athelia rolfsii glucoamylase to Aspergillus niger glucoamylase and Talaromyces emersonii glucoamylase was evaluated via mini-scale fermentations. About 380 g of milled corn (ground in a pilot scale hammer mill through a 1.65 mm screen) was added to about 620 g tap water. This mixture was supplemented with 3 mL 1 g/L penicillin. The pH of this slurry was adjusted to 5.0 with 40% H.sub.2SO.sub.4. The dry solid (DS) level was determined in triplicate to be about 32%. Approximately 5 g of this slurry was added to 15 mL tubes.

[0119] A two dose dose-response was conducted with each enzyme. Dosages used were 0.3 and 0.6 nmol/ g DS. Six replicates of each treatment were run.

[0120] After dosing the tubes were inoculated with 0.04 mL/g mash of yeast propagate (Red Star.TM. yeast) that had been grown for 22.5 hours on corn mash. Tubes were capped with a screw on top which had been punctured with a small needle to allow gas release and vortexed briefly before weighing and incubation at 32.degree. C. 70 hours fermentations were carried out and ethanol yields were determined by weighing the tubes. Tubes were vortexed briefly before weighing. The result of the experiment is shown in Table 1.

[0121] It can be seen from Table 1 the ethanol yield per gram DS is significantly higher when using the Athelia rolfsii glucoamylase compared to yields for the wild-type Aspergillus niger and Talaromyces emersonii glucoamylases.

TABLE-US-00006 TABLE 1 Glucoamylase nmol/g DS Ethanol yields Athelia rolfsii 0.3 96.4 Aspergillus niger 47.2 Talaromyces emersonii 30.5 Athelia rolfsii 0.6 121.9 Aspergillus niger 87.2 Talaromyces emersonii 43.4

EXAMPLE 2

Evaluation of Athelia Rolfsii Glucoamylase in Combination with Acid Fungal Alpha-Amylase in `One-Step` Saccharification

[0122] The glucose concentration after one step saccharification with Athelia rolfsii glucoamylase alone and in combination with a fungal acid alpha-amylase activity (Aspergillus niger acid alpha-amylase hybrid with starch-binding domain from Aspergillus kawachii alpha-amylase), respectively, was compared with Aspergillus niger glucoamylase alone and in combination the same fungal acid alpha-amylase under the same conditions and at the same dose levels.

[0123] The evaluation was made by mini-scale saccharification very similar to the mini-scale fermentation used in Example 1, except for the fact that no yeast was added and a buffer was used to hold the pH at 4.5.

[0124] Briefly, 194 g of milled corn was mixed with 306 g of 37 mM NaOAc, 0.025% sodium azide, 20 mM CaCl.sub.2, pH 4.5 to yield a slurry of approximately 35% DS. The pH of this slurry was adjusted to 4.5 with 40% H.sub.2SO4 (initial pH, before adjustment, was around 4.9). The slurry was allowed to hydrate while stirring at room temperature for one hour. Approximately 5 g of this slurry was added to a 20 mL vial for each reaction. The vials containing corn slurry were then pre-incubated at 32.degree. C. for one hour prior to dosing. Each vial was dosed with the appropriate amount of enzyme, capped and vortexed immediately. Actual dosages were based on the exact weight of corn slurry in each vial. Three replicates were run for each reaction. Vials were incubated at 32.degree. C. Each vial was vortexed after 4 hours and the reactions were stopped by addition of 50 microL of 40% H.sub.2SO4 and prepped for HPLC analysis. The HPLC preparation consisted of centrifuging, and filtering through a 0.45 micro m filter. Samples awaiting HPLC analysis were stored at 4.degree. C.

[0125] Table 2 shows the glucose concentration after 4 hours of saccharification.

TABLE-US-00007 4 hour Glucose Treatment (g/L) 0.263 mg/g DS Aspergillus niger glucoamylase 33.2 0.263 mg/g DS Aspergillus niger glucoamylase + 0.034 mg/g 40.4 DS Aspergillus niger acid alpha- amylase with Aspergillus kawachii alpha-amylase linker and CBM 0.263 mg/g DS Athelia rolfsii glucoamylase 45.6 0.263 mg/g DS Athelia rolfsii glucoamylase + 0.034 mg/g 63.2 DS Aspergillus niger acid alpha- amylase with Aspergillus kawachii alpha-amylase linker and CBM

[0126] The obtained glucose level after saccharification correlates with the ethanol fermentation yield that would be obtained if fermented by Saccharomyces yeast. Consequently, the above results show that Athelia rolfsii glucoamylase alone and in combination with a fungal acid alpha-amylase performs better than Aspergillus niger glucoamylase alone and in combination with a fungal acid alpha-amylase under the same conditions.

Sequence CWU 1

1

4112427DNAAthelia rolfsiisig_peptide(1)..(54)CDS(1)..(208)mat_peptide(55)..(2427)Intron(209- )..(283)CDS(284)..(354)Intron(355)..(410)misc_feature(367)..(367)n is a, c, g or t. 1atg ttt cgt tca ctc ctg gcc ttg gct gcg tgt gca gtc gcc tct gta 48Met Phe Arg Ser Leu Leu Ala Leu Ala Ala Cys Ala Val Ala Ser Val -15 -10 -5tct gca cag tct gcg tct gcg aca gca tat ctt acc aag gaa tct gca 96Ser Ala Gln Ser Ala Ser Ala Thr Ala Tyr Leu Thr Lys Glu Ser Ala -1 1 5 10gtt gcc aag aat ggc gta ctt tgc aac att ggt agc cag gga tgc atg 144Val Ala Lys Asn Gly Val Leu Cys Asn Ile Gly Ser Gln Gly Cys Met15 20 25 30tct gag ggt gcc tat agc ggt att gtg atc gca tct ccc tct aaa act 192Ser Glu Gly Ala Tyr Ser Gly Ile Val Ile Ala Ser Pro Ser Lys Thr 35 40 45agc cct gac tat ctc t gtgagtatta tttgtaaagt agcctcactg atagtacatt 248Ser Pro Asp Tyr Leu 50ttctgagttc tgttacaacc ctggtattat aatag at acc tgg act cgc gac 300 Tyr Thr Trp Thr Arg Asp 55tcg tcg ctc gtc ttc aag atg tta att gac caa tac aca aat ggc ctg 348Ser Ser Leu Val Phe Lys Met Leu Ile Asp Gln Tyr Thr Asn Gly Leu 60 65 70gat acg gtatgtggca tcngcgttcc ggctcgcctc aaagatgnaa aattgatgtt 404Asp Thr 75tcttag aca ctg cgc act ctc att gac gag ttt gtc tct gcg gaa gcc 452 Thr Leu Arg Thr Leu Ile Asp Glu Phe Val Ser Ala Glu Ala 80 85acc att caa caa acc agt aac cca tct ggt acc gtc tct acc ggt ggt 500Thr Ile Gln Gln Thr Ser Asn Pro Ser Gly Thr Val Ser Thr Gly Gly90 95 100 105ctc ggc gaa ccc aaa ttc aat atc gac gag acg gca ttt acg ggc gca 548Leu Gly Glu Pro Lys Phe Asn Ile Asp Glu Thr Ala Phe Thr Gly Ala 110 115 120tgg ggt cgt gtaagctacc aatacacaat caaaatcgac catctgtatt 597Trp Gly Argtactatctat aatttctag ccc caa cgt gat ggt ccc gcc ctc cgt gca acc 649 Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr 125 130 135gca atc atg acc tat gcg acg tat ctg tac aac aat ggc aac act tcc 697Ala Ile Met Thr Tyr Ala Thr Tyr Leu Tyr Asn Asn Gly Asn Thr Ser 140 145 150tac gtg acc aac acc ctt tgg cct atc atc aag ctc gac ctt gac tat 745Tyr Val Thr Asn Thr Leu Trp Pro Ile Ile Lys Leu Asp Leu Asp Tyr 155 160 165gtc aac tcg gac tgg aac cag acc a gtaagcgaat ttctaggggg 790Val Asn Ser Asp Trp Asn Gln Thr 170 175acttatctaa aacagcatat tcaaccagta aatag cg ttt gac ctc tgg gaa 842 Thr Phe Asp Leu Trp Glu 180gaa gtt gac tcg tct tct ttc ttt acg act gcc gtt cag cac cgt gct 890Glu Val Asp Ser Ser Ser Phe Phe Thr Thr Ala Val Gln His Arg Ala 185 190 195ctt gtt cag ggc gca gcc ttt gct acc ctc atc ggc caa act tcg tct 938Leu Val Gln Gly Ala Ala Phe Ala Thr Leu Ile Gly Gln Thr Ser Ser 200 205 210gct tcg act tac tcc gcc acg gcc cct agc att ctc tgc ttc ttg cag 986Ala Ser Thr Tyr Ser Ala Thr Ala Pro Ser Ile Leu Cys Phe Leu Gln 215 220 225gtgagataaa aatctttcta tgtaattggt ttttcccctc aaattgaaat tgacatattt 1046gcgatccaat ag tct tac tgg aac acc aac gga tac tgg acg gcc aac act 1097 Ser Tyr Trp Asn Thr Asn Gly Tyr Trp Thr Ala Asn Thr 230 235 240ggt ggc gga cgt tcc ggc aag gac gcc aac acc ata ctc gct tct atc 1145Gly Gly Gly Arg Ser Gly Lys Asp Ala Asn Thr Ile Leu Ala Ser Ile 245 250 255cac aca ttt gac gcc agc gcc ggc tgc tct gct gcc acg tct caa cca 1193His Thr Phe Asp Ala Ser Ala Gly Cys Ser Ala Ala Thr Ser Gln Pro 260 265 270tgc tct gac gta gca ttg gcc aac ctg aag gta tac gtt gac tct ttc 1241Cys Ser Asp Val Ala Leu Ala Asn Leu Lys Val Tyr Val Asp Ser Phe275 280 285 290cgt agt att tat acg atc aac agc ggt att tcc tct acc tcg ggt gtt 1289Arg Ser Ile Tyr Thr Ile Asn Ser Gly Ile Ser Ser Thr Ser Gly Val 295 300 305gct act ggt cgc tac ccc gaa gat tcg tat tac aat ggc aac 1331Ala Thr Gly Arg Tyr Pro Glu Asp Ser Tyr Tyr Asn Gly Asn 310 315 320gtacgtattt atctaatttt tccaagacag tcaaagttta tgttcatctg ccccctttta 1391cctgtacatt caaaatag ccc tgg tac ctc tgc aca ctc gcc gtc gcc gag 1442 Pro Trp Tyr Leu Cys Thr Leu Ala Val Ala Glu 325 330cag ctc tat gat gct ctc atc gta tgg aag gct gcc ggg gag ctc aac 1490Gln Leu Tyr Asp Ala Leu Ile Val Trp Lys Ala Ala Gly Glu Leu Asn 335 340 345gtc acc tcc gtc tcg ctc gcg ttc ttc cag caa ttc gac tcg agc atc 1538Val Thr Ser Val Ser Leu Ala Phe Phe Gln Gln Phe Asp Ser Ser Ile 350 355 360acc gcc ggc act tac gcc tcc tcg tcg agc gta tac act tcg ctc atc 1586Thr Ala Gly Thr Tyr Ala Ser Ser Ser Ser Val Tyr Thr Ser Leu Ile 365 370 375tct gac atc cag gcg ttc gca gac gag ttt gtt gac att gtt gcc aag 1634Ser Asp Ile Gln Ala Phe Ala Asp Glu Phe Val Asp Ile Val Ala Lys380 385 390 395tac acg cct tcg tct ggc ttc ttg tct gag cag tat gat aag tcc acg 1682Tyr Thr Pro Ser Ser Gly Phe Leu Ser Glu Gln Tyr Asp Lys Ser Thr 400 405 410ggt gct cag gat tcg gct gct aac ttg act t gtaagtcatc tatttgttca 1733Gly Ala Gln Asp Ser Ala Ala Asn Leu Thr 415 420ttctattcct tttcaaaaaa aaaagtgatg ctaatgattt ttggcggaaa ccag gg 1789 Trptcc tat gct gct gct atc acc gct tac caa gcc cgc aat ggc ttc aca 1837Ser Tyr Ala Ala Ala Ile Thr Ala Tyr Gln Ala Arg Asn Gly Phe Thr 425 430 435ggt gct tcg tgg ggt gct aag gga gtt tct acc tcc tgc tcg act ggt 1885Gly Ala Ser Trp Gly Ala Lys Gly Val Ser Thr Ser Cys Ser Thr Gly 440 445 450gct aca agc ccg ggt ggc tcc tcg ggt agt gtc gag gtc act ttc gac 1933Ala Thr Ser Pro Gly Gly Ser Ser Gly Ser Val Glu Val Thr Phe Asp455 460 465 470gtt tac gct acc aca gta tat ggc c gtaagcactt gactagcttc 1978Val Tyr Ala Thr Thr Val Tyr Gly 475aaaccatact tcatcatgct gataaacaaa aaaatgaaac ag ag aac atc tat 2031 Gln Asn Ile Tyr 480atc acc ggt gat gtg agt gag ctc ggc aac tgg aca ccc gcc aat ggt 2079Ile Thr Gly Asp Val Ser Glu Leu Gly Asn Trp Thr Pro Ala Asn Gly 485 490 495gtt gca ctc tct tct gct aac tac ccc acc tgg agt g gtaagttgac 2126Val Ala Leu Ser Ser Ala Asn Tyr Pro Thr Trp Ser 500 505 510ccttaccagt atcttgacag acattgatat tgacttccgc aatacag cc acg atc 2181 Ala Thr Ilegct ctc ccc gct gac acg aca atc cag tac aag tat gtc aac att gac 2229Ala Leu Pro Ala Asp Thr Thr Ile Gln Tyr Lys Tyr Val Asn Ile Asp 515 520 525ggc agc acc gtc atc tgg gag gat gct atc agc aat cgc gag atc acg 2277Gly Ser Thr Val Ile Trp Glu Asp Ala Ile Ser Asn Arg Glu Ile Thr530 535 540 545acg ccc gcc agc ggc aca tac acc gaa aaa gac act tgg gat gaa tct 2325Thr Pro Ala Ser Gly Thr Tyr Thr Glu Lys Asp Thr Trp Asp Glu Ser 550 555 560taaactgctg aacttgaacg gcttgcaaaa gcgaatggtg tagaaaataa acgaagattt 2385tgattgcttt gttttgtttc tcttcctatc ttgtttctct ag 24272579PRTAthelia rolfsii 2Met Phe Arg Ser Leu Leu Ala Leu Ala Ala Cys Ala Val Ala Ser Val -15 -10 -5Ser Ala Gln Ser Ala Ser Ala Thr Ala Tyr Leu Thr Lys Glu Ser Ala -1 1 5 10Val Ala Lys Asn Gly Val Leu Cys Asn Ile Gly Ser Gln Gly Cys Met15 20 25 30Ser Glu Gly Ala Tyr Ser Gly Ile Val Ile Ala Ser Pro Ser Lys Thr 35 40 45Ser Pro Asp Tyr Leu Tyr Thr Trp Thr Arg Asp Ser Ser Leu Val Phe 50 55 60Lys Met Leu Ile Asp Gln Tyr Thr Asn Gly Leu Asp Thr Thr Leu Arg 65 70 75Thr Leu Ile Asp Glu Phe Val Ser Ala Glu Ala Thr Ile Gln Gln Thr 80 85 90Ser Asn Pro Ser Gly Thr Val Ser Thr Gly Gly Leu Gly Glu Pro Lys95 100 105 110Phe Asn Ile Asp Glu Thr Ala Phe Thr Gly Ala Trp Gly Arg Pro Gln 115 120 125Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Ile Met Thr Tyr Ala Thr 130 135 140Tyr Leu Tyr Asn Asn Gly Asn Thr Ser Tyr Val Thr Asn Thr Leu Trp 145 150 155Pro Ile Ile Lys Leu Asp Leu Asp Tyr Val Asn Ser Asp Trp Asn Gln 160 165 170Thr Thr Phe Asp Leu Trp Glu Glu Val Asp Ser Ser Ser Phe Phe Thr175 180 185 190Thr Ala Val Gln His Arg Ala Leu Val Gln Gly Ala Ala Phe Ala Thr 195 200 205Leu Ile Gly Gln Thr Ser Ser Ala Ser Thr Tyr Ser Ala Thr Ala Pro 210 215 220Ser Ile Leu Cys Phe Leu Gln Ser Tyr Trp Asn Thr Asn Gly Tyr Trp 225 230 235Thr Ala Asn Thr Gly Gly Gly Arg Ser Gly Lys Asp Ala Asn Thr Ile 240 245 250Leu Ala Ser Ile His Thr Phe Asp Ala Ser Ala Gly Cys Ser Ala Ala255 260 265 270Thr Ser Gln Pro Cys Ser Asp Val Ala Leu Ala Asn Leu Lys Val Tyr 275 280 285Val Asp Ser Phe Arg Ser Ile Tyr Thr Ile Asn Ser Gly Ile Ser Ser 290 295 300Thr Ser Gly Val Ala Thr Gly Arg Tyr Pro Glu Asp Ser Tyr Tyr Asn 305 310 315Gly Asn Pro Trp Tyr Leu Cys Thr Leu Ala Val Ala Glu Gln Leu Tyr 320 325 330Asp Ala Leu Ile Val Trp Lys Ala Ala Gly Glu Leu Asn Val Thr Ser335 340 345 350Val Ser Leu Ala Phe Phe Gln Gln Phe Asp Ser Ser Ile Thr Ala Gly 355 360 365Thr Tyr Ala Ser Ser Ser Ser Val Tyr Thr Ser Leu Ile Ser Asp Ile 370 375 380Gln Ala Phe Ala Asp Glu Phe Val Asp Ile Val Ala Lys Tyr Thr Pro 385 390 395Ser Ser Gly Phe Leu Ser Glu Gln Tyr Asp Lys Ser Thr Gly Ala Gln 400 405 410Asp Ser Ala Ala Asn Leu Thr Trp Ser Tyr Ala Ala Ala Ile Thr Ala415 420 425 430Tyr Gln Ala Arg Asn Gly Phe Thr Gly Ala Ser Trp Gly Ala Lys Gly 435 440 445Val Ser Thr Ser Cys Ser Thr Gly Ala Thr Ser Pro Gly Gly Ser Ser 450 455 460Gly Ser Val Glu Val Thr Phe Asp Val Tyr Ala Thr Thr Val Tyr Gly 465 470 475Gln Asn Ile Tyr Ile Thr Gly Asp Val Ser Glu Leu Gly Asn Trp Thr 480 485 490Pro Ala Asn Gly Val Ala Leu Ser Ser Ala Asn Tyr Pro Thr Trp Ser495 500 505 510Ala Thr Ile Ala Leu Pro Ala Asp Thr Thr Ile Gln Tyr Lys Tyr Val 515 520 525Asn Ile Asp Gly Ser Thr Val Ile Trp Glu Asp Ala Ile Ser Asn Arg 530 535 540Glu Ile Thr Thr Pro Ala Ser Gly Thr Tyr Thr Glu Lys Asp Thr Trp 545 550 555Asp Glu Ser 5603484PRTAspergillus nigermat_peptide(1)..(484) 3Leu Ser Ala Ala Ser Trp Arg Thr Gln Ser Ile Tyr Phe Leu Leu Thr1 5 10 15Asp Arg Phe Gly Arg Thr Asp Asn Ser Thr Thr Ala Thr Cys Asn Thr 20 25 30Gly Asn Glu Ile Tyr Cys Gly Gly Ser Trp Gln Gly Ile Ile Asp His 35 40 45Leu Asp Tyr Ile Glu Gly Met Gly Phe Thr Ala Ile Trp Ile Ser Pro 50 55 60Ile Thr Glu Gln Leu Pro Gln Asp Thr Ala Asp Gly Glu Ala Tyr His65 70 75 80Gly Tyr Trp Gln Gln Lys Ile Tyr Asp Val Asn Ser Asn Phe Gly Thr 85 90 95Ala Asp Asn Leu Lys Ser Leu Ser Asp Ala Leu His Ala Arg Gly Met 100 105 110Tyr Leu Met Val Asp Val Val Pro Asp His Met Gly Tyr Ala Gly Asn 115 120 125Gly Asn Asp Val Asp Tyr Ser Val Phe Asp Pro Phe Asp Ser Ser Ser 130 135 140Tyr Phe His Pro Tyr Cys Leu Ile Thr Asp Trp Asp Asn Leu Thr Met145 150 155 160Val Glu Asp Cys Trp Glu Gly Asp Thr Ile Val Ser Leu Pro Asp Leu 165 170 175Asp Thr Thr Glu Thr Ala Val Arg Thr Ile Trp Tyr Asp Trp Val Ala 180 185 190Asp Leu Val Ser Asn Tyr Ser Val Asp Gly Leu Arg Ile Asp Ser Val 195 200 205Leu Glu Val Gln Pro Asp Phe Phe Pro Gly Tyr Asn Lys Ala Ser Gly 210 215 220Val Tyr Cys Val Gly Glu Ile Asp Asn Gly Asn Pro Ala Ser Asp Cys225 230 235 240Pro Tyr Gln Lys Val Leu Asp Gly Val Leu Asn Tyr Pro Ile Tyr Trp 245 250 255Gln Leu Leu Tyr Ala Phe Glu Ser Ser Ser Gly Ser Ile Ser Asn Leu 260 265 270Tyr Asn Met Ile Lys Ser Val Ala Ser Asp Cys Ser Asp Pro Thr Leu 275 280 285Leu Gly Asn Phe Ile Glu Asn His Asp Asn Pro Arg Phe Ala Lys Tyr 290 295 300Thr Ser Asp Tyr Ser Gln Ala Lys Asn Val Leu Ser Tyr Ile Phe Leu305 310 315 320Ser Asp Gly Ile Pro Ile Val Tyr Ala Gly Glu Glu Gln His Tyr Ala 325 330 335Gly Gly Lys Val Pro Tyr Asn Arg Glu Ala Thr Trp Leu Ser Gly Tyr 340 345 350Asp Thr Ser Ala Glu Leu Tyr Thr Trp Ile Ala Thr Thr Asn Ala Ile 355 360 365Arg Lys Leu Ala Ile Ala Ala Asp Ser Ala Tyr Ile Thr Tyr Ala Asn 370 375 380Asp Ala Phe Tyr Thr Asp Ser Asn Thr Ile Ala Met Ala Lys Gly Thr385 390 395 400Ser Gly Ser Gln Val Ile Thr Val Leu Ser Asn Lys Gly Ser Ser Gly 405 410 415Ser Ser Tyr Thr Leu Thr Leu Ser Gly Ser Gly Tyr Thr Ser Gly Thr 420 425 430Lys Leu Ile Glu Ala Tyr Thr Cys Thr Ser Val Thr Val Asp Ser Ser 435 440 445Gly Asp Ile Pro Val Pro Met Ala Ser Gly Leu Pro Arg Val Leu Leu 450 455 460Pro Ala Ser Val Val Asp Ser Ser Ser Leu Cys Gly Gly Ser Gly Arg465 470 475 480Leu Tyr Val Glu4498PRTAspergillus oryzaeSIGNAL(1)..(19)mat_peptide(20)..(498) 4Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala Ala -15 -10 -5Pro Ala Leu Ala Ala Thr Pro Ala Asp Trp Arg Ser Gln Ser Ile Tyr -1 1 5 10Phe Leu Leu Thr Asp Arg Phe Ala Arg Thr Asp Gly Ser Thr Thr Ala 15 20 25Thr Cys Asn Thr Ala Asp Gln Lys Tyr Cys Gly Gly Thr Trp Gln Gly30 35 40 45Ile Ile Asp Lys Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile 50 55 60Trp Ile Thr Pro Val Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly 65 70 75Asp Ala Tyr His Gly Tyr Trp Gln Gln Asp Ile Tyr Ser Leu Asn Glu 80 85 90Asn Tyr Gly Thr Ala Asp Asp Leu Lys Ala Leu Ser Ser Ala Leu His 95 100 105Glu Arg Gly Met Tyr Leu Met Val Asp Val Val Ala Asn His Met Gly110 115 120 125Tyr Asp Gly Ala Gly Ser Ser Val Asp Tyr Ser Val Phe Lys Pro Phe 130 135 140Ser Ser Gln Asp Tyr Phe His Pro Phe Cys Phe Ile Gln Asn Tyr Glu 145 150 155Asp Gln Thr Gln Val Glu Asp Cys Trp Leu Gly Asp Asn Thr Val Ser 160 165 170Leu Pro Asp Leu Asp Thr Thr Lys Asp Val Val Lys Asn Glu Trp Tyr 175 180 185Asp Trp Val Gly Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly Leu Arg190 195 200 205Ile Asp Thr Val Lys His Val Gln Lys Asp Phe Trp Pro Gly Tyr Asn 210 215 220Lys Ala Ala Gly

Val Tyr Cys Ile Gly Glu Val Leu Asp Gly Asp Pro 225 230 235Ala Tyr Thr Cys Pro Tyr Gln Asn Val Met Asp Gly Val Leu Asn Tyr 240 245 250Pro Ile Tyr Tyr Pro Leu Leu Asn Ala Phe Lys Ser Thr Ser Gly Ser 255 260 265Met Asp Asp Leu Tyr Asn Met Ile Asn Thr Val Lys Ser Asp Cys Pro270 275 280 285Asp Ser Thr Leu Leu Gly Thr Phe Val Glu Asn His Asp Asn Pro Arg 290 295 300Phe Ala Ser Tyr Thr Asn Asp Ile Ala Leu Ala Lys Asn Val Ala Ala 305 310 315Phe Ile Ile Leu Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly Gln Glu 320 325 330Gln His Tyr Ala Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala Thr Trp 335 340 345Leu Ser Gly Tyr Pro Thr Asp Ser Glu Leu Tyr Lys Leu Ile Ala Ser350 355 360 365Ala Asn Ala Ile Arg Asn Tyr Ala Ile Ser Lys Asp Thr Gly Phe Val 370 375 380Thr Tyr Lys Asn Trp Pro Ile Tyr Lys Asp Asp Thr Thr Ile Ala Met 385 390 395Arg Lys Gly Thr Asp Gly Ser Gln Ile Val Thr Ile Leu Ser Asn Lys 400 405 410Gly Ala Ser Gly Asp Ser Tyr Thr Leu Ser Leu Ser Gly Ala Gly Tyr 415 420 425Thr Ala Gly Gln Gln Leu Thr Glu Val Ile Gly Cys Thr Thr Val Thr430 435 440 445Val Gly Ser Asp Gly Asn Val Pro Val Pro Met Ala Gly Gly Leu Pro 450 455 460Arg Val Leu Tyr Pro Thr Glu Lys Leu Ala Gly Ser Lys Ile Cys Ser 465 470 475Ser Ser5483PRTBacillus licheniformismat_peptide(1)..(483) 5Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro1 5 10 15Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75 80Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn 85 90 95Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro 130 135 140Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe145 150 155 160Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195 200 205Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215 220Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe225 230 235 240Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met 245 250 255Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn 260 265 270Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu 275 280 285His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met 290 295 300Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser305 310 315 320Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His385 390 395 400Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser 450 455 460Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr465 470 475 480Val Gln Arg6480PRTBacillus amyloliquefaciensmat_peptide(1)..(480) 6Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp1 5 10 15Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20 25 30Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser 35 40 45Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu 50 55 60Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu65 70 75 80Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr 85 90 95Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp 100 105 110Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser 115 120 125Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg 130 135 140Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly145 150 155 160Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg 165 170 175Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val 195 200 205Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser 210 215 220Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe225 230 235 240Leu Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met 245 250 255Phe Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn 260 265 270Tyr Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu 275 280 285His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met 290 295 300Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala305 310 315 320Val Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile 370 375 380Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His385 390 395 400Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr 435 440 445Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser 450 455 460Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr465 470 475 4807514PRTBacillus stearothermophilusmat_peptide(1)..(514) 7Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu1 5 10 15Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55 60Leu Gly Glu Phe Asn Gln Lys Gly Ala Val Arg Thr Lys Tyr Gly Thr65 70 75 80Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met 85 90 95Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly 100 105 110Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln 115 120 125Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe 130 135 140Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His145 150 155 160Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170 175Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185 190Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His 195 200 205Pro Glu Val Val Thr Glu Leu Lys Ser Trp Gly Lys Trp Tyr Val Asn 210 215 220Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys225 230 235 240Phe Ser Phe Phe Pro Asp Trp Leu Ser Asp Val Arg Ser Gln Thr Gly 245 250 255Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys 260 265 270Leu His Asn Tyr Ile Met Lys Thr Asn Gly Thr Met Ser Leu Phe Asp 275 280 285Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Thr 290 295 300Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro305 310 315 320Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln 325 330 335Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp 355 360 365Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile 370 375 380Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His385 390 395 400Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Val 405 410 415Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val 435 440 445Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser 450 455 460Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp465 470 475 480Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Trp Ser Ile Thr Thr 485 490 495Arg Pro Trp Thr Asp Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val 500 505 510Ala Trp838PRTAspergillus nigerMISC_FEATURE(1)..(38)linker sequence 8Thr Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val1 5 10 15Thr Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Ser 20 25 30Thr Ser Ser Thr Ser Ala 35931PRTAspergillus kawachiMISC_FEATURE(1)..(31)linker sequence 9Thr Thr Thr Thr Thr Thr Ala Ala Ala Thr Ser Thr Ser Lys Ala Thr1 5 10 15Thr Ser Ser Ser Ser Ser Ser Ala Ala Ala Thr Thr Ser Ser Ser 20 25 301011PRTAthelia rolfsiiMISC_FEATURE(1)..(11)linker sequence 10Gly Ala Thr Ser Pro Gly Gly Ser Ser Gly Ser1 5 10118PRTArtificialPEPT linker 11Pro Glu Pro Thr Pro Glu Pro Thr1 512396DNAAspergillus kawachiCDS(1)..(396)CBM 12act agt aca tcc aaa gcc acc acc tcc tct tct tct tct tct gct gct 48Thr Ser Thr Ser Lys Ala Thr Thr Ser Ser Ser Ser Ser Ser Ala Ala1 5 10 15gct act act tct tca tca tgc acc gca aca agc acc acc ctc ccc atc 96Ala Thr Thr Ser Ser Ser Cys Thr Ala Thr Ser Thr Thr Leu Pro Ile 20 25 30acc ttc gaa gaa ctc gtc acc act acc tac ggg gaa gaa gtc tac ctc 144Thr Phe Glu Glu Leu Val Thr Thr Thr Tyr Gly Glu Glu Val Tyr Leu 35 40 45agc gga tct atc tcc cag ctc gga gag tgg gat acg agt gac gcg gtg 192Ser Gly Ser Ile Ser Gln Leu Gly Glu Trp Asp Thr Ser Asp Ala Val 50 55 60aag ttg tcc gcg gat gat tat acc tcg agt aac ccc gag tgg tct gtt 240Lys Leu Ser Ala Asp Asp Tyr Thr Ser Ser Asn Pro Glu Trp Ser Val65 70 75 80act gtg tcg ttg ccg gtg ggg acg acc ttc gag tat aag ttt att aag 288Thr Val Ser Leu Pro Val Gly Thr Thr Phe Glu Tyr Lys Phe Ile Lys 85 90 95gtc gat gag ggt gga agt gtg act tgg gaa agt gat ccg aat agg gag 336Val Asp Glu Gly Gly Ser Val Thr Trp Glu Ser Asp Pro Asn Arg Glu 100 105 110tat act gtg cct gaa tgt ggg aat ggg agt ggg gag acg gtg gtt gat 384Tyr Thr Val Pro Glu Cys Gly Asn Gly Ser Gly Glu Thr Val Val Asp 115 120 125acg tgg agg tag 396Thr Trp Arg 13013131PRTAspergillus kawachi 13Thr Ser Thr Ser Lys Ala Thr Thr Ser Ser Ser Ser Ser Ser Ala Ala1 5 10 15Ala Thr Thr Ser Ser Ser Cys Thr Ala Thr Ser Thr Thr Leu Pro Ile 20 25 30Thr Phe Glu Glu Leu Val Thr Thr Thr Tyr Gly Glu Glu Val Tyr Leu 35 40 45Ser Gly Ser Ile Ser Gln Leu Gly Glu Trp Asp Thr Ser Asp Ala Val 50 55 60Lys Leu Ser Ala Asp Asp Tyr Thr Ser Ser Asn Pro Glu Trp Ser Val65 70 75 80Thr Val Ser Leu Pro Val Gly Thr Thr Phe Glu Tyr Lys Phe Ile Lys 85 90 95Val Asp Glu Gly Gly Ser Val Thr Trp Glu Ser Asp Pro Asn Arg Glu 100 105 110Tyr Thr Val Pro Glu Cys Gly Asn Gly Ser Gly Glu Thr Val Val Asp 115 120 125Thr Trp Arg 13014102PRTBacillus flavothermusMISC_FEATURE(1)..(102)CBM 14Ile Ser Thr Thr Ser Gln Ile Thr Phe Thr Val Asn Asn Ala Thr Thr1 5 10 15Val Trp Gly Gln Asn Val Tyr Val Val Gly Asn Ile Ser Gln Leu Gly 20 25 30Asn Trp Asp Pro Val His Ala Val Gln Met Thr Pro Ser Ser Tyr Pro 35 40 45Thr Trp Thr Val Thr Ile Pro Leu Leu Gln Gly Gln Asn Ile Gln Phe 50 55 60Lys Phe Ile Lys Lys Asp Ser Ala Gly Asn Val Ile Trp Glu Asp Ile65 70 75 80Ser Asn Arg Thr Tyr Thr Val Pro Thr Ala Ala Ser Gly Ala Tyr Thr 85 90 95Ala Ser Trp Asn Val Pro 1001599PRTBacillus sp.MISC_FEATURE(1)..(99)CBM 15Thr Ser Asn Val Thr Phe Thr Val Asn Asn Ala Thr Thr Val Tyr Gly1 5 10 15Gln Asn Val Tyr Val Val Gly Asn Ile Pro Glu Leu Gly Asn Trp Asn 20 25 30Ile Ala Asn Ala Ile Gln Met Thr Pro Ser Ser Tyr Pro Thr Trp Lys 35 40 45Thr Thr Val Ser Leu Pro Gln Gly Lys Ala Ile Glu Phe Lys Phe Ile 50 55 60Lys Lys Asp Ser Ala Gly Asn Val Ile Trp Glu Asn Ile Ala Asn Arg65 70 75 80Thr Tyr Thr Val Pro Phe Ser Ser Thr Gly Ser Tyr Thr Ala Asn Trp 85 90 95Asn Val Pro16102PRTAlcaliphilic BacillusMISC_FEATURE(1)..(102)CBM 16Thr Ser Thr Thr Ser Gln Ile Thr Phe Thr Val Asn Asn Ala Thr Thr1 5 10 15Val Trp Gly Gln Asn Val

Tyr Val Val Gly Asn Ile Ser Gln Leu Gly 20 25 30Asn Trp Asp Pro Val Asn Ala Val Gln Met Thr Pro Ser Ser Tyr Pro 35 40 45Thr Trp Val Val Thr Val Pro Leu Pro Gln Ser Gln Asn Ile Gln Phe 50 55 60Lys Phe Ile Lys Lys Asp Gly Ser Gly Asn Val Ile Trp Glu Asn Ile65 70 75 80Ser Asn Arg Thr Tyr Thr Val Pro Thr Ala Ala Ser Gly Ala Tyr Thr 85 90 95Ala Asn Trp Asn Val Pro 10017112PRTHormoconis resinaeMISC_FEATURE(1)..(112)CBM 17Cys Gln Val Ser Ile Thr Phe Asn Ile Asn Ala Thr Thr Tyr Tyr Gly1 5 10 15Glu Asn Leu Tyr Val Ile Gly Asn Ser Ser Asp Leu Gly Ala Trp Asn 20 25 30Ile Ala Asp Ala Tyr Pro Leu Ser Ala Ser Ala Tyr Thr Gln Asp Arg 35 40 45Pro Leu Trp Ser Ala Ala Ile Pro Leu Asn Ala Gly Glu Val Ile Ser 50 55 60Tyr Gln Tyr Val Arg Gln Glu Asp Cys Asp Gln Pro Tyr Ile Tyr Glu65 70 75 80Thr Val Asn Arg Thr Leu Thr Val Pro Ala Cys Gly Gly Ala Ala Val 85 90 95Thr Thr Asp Asp Ala Trp Met Gly Pro Val Gly Ser Ser Gly Asn Cys 100 105 1101895PRTLentinula edodesMISC_FEATURE(1)..(95)CBM 18Val Ser Val Thr Phe Asn Val Asp Ala Ser Thr Leu Glu Gly Gln Asn1 5 10 15Val Tyr Leu Thr Gly Ala Val Asp Ala Leu Glu Asp Trp Ser Thr Asp 20 25 30Asn Ala Ile Leu Leu Ser Ser Ala Asn Tyr Pro Thr Trp Ser Val Thr 35 40 45Val Asp Leu Pro Gly Ser Thr Asp Val Gln Tyr Lys Tyr Ile Lys Lys 50 55 60Asp Gly Ser Gly Thr Val Thr Trp Glu Ser Asp Pro Asn Met Glu Ile65 70 75 80Thr Thr Pro Ala Asn Gly Thr Tyr Ala Thr Asn Asp Thr Trp Arg 85 90 9519107PRTNeurospora crassaMISC_FEATURE(1)..(107)CBM 19Cys Ala Ala Asp His Glu Val Leu Val Thr Phe Asn Glu Lys Val Thr1 5 10 15Thr Ser Tyr Gly Gln Thr Val Lys Val Val Gly Ser Ile Ala Ala Leu 20 25 30Gly Asn Trp Ala Pro Ala Ser Gly Val Thr Leu Ser Ala Lys Gln Tyr 35 40 45Ser Ser Ser Asn Pro Leu Trp Ser Thr Thr Ile Ala Leu Pro Gln Gly 50 55 60Thr Ser Phe Lys Tyr Lys Tyr Val Val Val Asn Ser Asp Gly Ser Val65 70 75 80Lys Trp Glu Asn Asp Pro Asp Arg Ser Tyr Ala Val Gly Thr Asp Cys 85 90 95Ala Ser Thr Ala Thr Leu Asp Asp Thr Trp Arg 100 10520115PRTTalaromyces byssochlamydioidesMISC_FEATURE(1)..(115)CBM 20Thr Thr Thr Gly Ala Ala Pro Cys Thr Thr Pro Thr Thr Val Ala Val1 5 10 15Thr Phe Asp Glu Ile Val Thr Thr Thr Tyr Gly Glu Thr Val Tyr Leu 20 25 30Ser Gly Ser Ile Pro Ala Leu Gly Asn Trp Asp Thr Ser Ser Ala Ile 35 40 45Ala Leu Ser Ala Val Asp Tyr Thr Ser Ser Asn Pro Leu Trp Tyr Val 50 55 60Thr Val Asn Leu Pro Ala Gly Thr Ser Phe Glu Tyr Lys Phe Phe Val65 70 75 80Gln Gln Thr Asp Gly Thr Ile Val Trp Glu Asp Asp Pro Asn Arg Ser 85 90 95Tyr Thr Val Pro Ala Asn Cys Gly Gln Thr Thr Ala Ile Ile Asp Asp 100 105 110Ser Trp Gln 11521115PRTGeosmithia cylindrosporaMISC_FEATURE(1)..(115)CBM 21Thr Ser Thr Gly Ser Ala Pro Cys Thr Thr Pro Thr Thr Val Ala Val1 5 10 15Thr Phe Asp Glu Ile Val Thr Thr Ser Tyr Gly Glu Thr Val Tyr Leu 20 25 30Ala Gly Ser Ile Ala Ala Leu Gly Asn Trp Asp Thr Asn Ser Ala Ile 35 40 45Ala Leu Ser Ala Ala Asp Tyr Thr Ser Asn Asn Asn Leu Trp Tyr Val 50 55 60Thr Val Asn Leu Ala Ala Gly Thr Ser Phe Gln Tyr Lys Phe Phe Val65 70 75 80Lys Glu Thr Asp Ser Thr Ile Val Trp Glu Asp Asp Pro Asn Arg Ser 85 90 95Tyr Thr Val Pro Ala Asn Cys Gly Gln Thr Thr Ala Ile Ile Asp Asp 100 105 110Thr Trp Gln 11522139PRTScorias spongiosa CBMMISC_FEATURE(1)..(139)CBM 22Ala Lys Val Pro Ser Thr Cys Ser Ala Ser Ser Ala Thr Gly Thr Cys1 5 10 15Thr Thr Ala Thr Ser Thr Phe Gly Gly Ser Thr Pro Thr Thr Ser Cys 20 25 30Ala Thr Thr Pro Thr Leu Thr Thr Val Leu Phe Asn Glu Arg Ala Thr 35 40 45Thr Asn Phe Gly Gln Asn Val His Leu Thr Gly Ser Ile Ser Gln Leu 50 55 60Gly Ser Trp Asp Thr Asp Ser Ala Val Ala Leu Ser Ala Val Asn Tyr65 70 75 80Thr Ser Ser Asp Pro Leu Trp Phe Val Arg Val Gln Leu Pro Ala Gly 85 90 95Thr Ser Phe Gln Tyr Lys Tyr Phe Lys Lys Asp Ser Ser Asn Ala Val 100 105 110Ala Trp Glu Ser Asp Pro Asn Arg Ser Tyr Thr Val Pro Leu Asn Cys 115 120 125Ala Gly Thr Ala Thr Glu Asn Asp Thr Trp Arg 130 13523126PRTEupenicillium ludwigiiMISC_FEATURE(1)..(126)CBM 23Ser Thr Thr Thr Thr Ser Thr Thr Lys Thr Thr Thr Thr Ser Thr Thr1 5 10 15Thr Ser Cys Thr Thr Pro Thr Ala Val Ala Val Thr Phe Asp Leu Ile 20 25 30Ala Thr Thr Tyr Tyr Gly Glu Asn Ile Lys Ile Ala Gly Ser Ile Ser 35 40 45Gln Leu Gly Asp Trp Asp Thr Ser Asn Ala Val Ala Leu Ser Ala Ala 50 55 60Asp Tyr Thr Ser Ser Asp His Leu Trp Phe Val Asp Ile Asp Leu Pro65 70 75 80Ala Gly Thr Val Phe Glu Tyr Lys Tyr Ile Arg Ile Glu Ser Asp Gly 85 90 95Ser Ile Glu Trp Glu Ser Asp Pro Asn Arg Ser Tyr Thr Val Pro Ala 100 105 110Ala Cys Ala Thr Thr Ala Val Thr Glu Asn Asp Thr Trp Arg 115 120 12524116PRTAspergillus japonicusMISC_FEATURE(1)..(116)CBM 24Lys Thr Ser Thr Thr Thr Ser Ser Cys Ser Thr Pro Thr Ser Val Ala1 5 10 15Val Thr Phe Asp Val Ile Ala Thr Thr Thr Tyr Gly Glu Asn Val Tyr 20 25 30Ile Ser Gly Ser Ile Ser Gln Leu Gly Ser Trp Asp Thr Ser Ser Ala 35 40 45Ile Ala Leu Ser Ala Ser Gln Tyr Thr Ser Ser Asn Asn Leu Trp Tyr 50 55 60Ala Thr Val His Leu Pro Ala Gly Thr Thr Phe Gln Tyr Lys Tyr Ile65 70 75 80Arg Lys Glu Thr Asp Gly Ser Val Thr Trp Glu Ser Asp Pro Asn Arg 85 90 95Ser Tyr Thr Val Pro Ser Ser Cys Gly Val Ser Ser Ala Thr Glu Ser 100 105 110Asp Thr Trp Arg 11525133PRTPenicillium cf. miczynskiiMISC_FEATURE(1)..(133)CBM 25Thr Thr Thr Gly Gly Thr Thr Thr Ser Gln Gly Ser Thr Thr Thr Thr1 5 10 15Ser Lys Thr Ser Thr Thr Thr Ser Ser Cys Thr Ala Pro Thr Ser Val 20 25 30Ala Val Thr Phe Asp Leu Ile Ala Thr Thr Val Tyr Asp Glu Asn Val 35 40 45Gln Leu Ala Gly Ser Ile Ser Ala Leu Gly Ser Trp Asp Thr Ser Ser 50 55 60Ala Ile Arg Leu Ser Ala Ser Gln Tyr Thr Ser Ser Asn His Leu Trp65 70 75 80Tyr Val Ala Val Ser Leu Pro Ala Gly Gln Val Phe Gln Tyr Lys Tyr 85 90 95Ile Arg Val Ala Ser Ser Gly Thr Ile Thr Trp Glu Ser Asp Pro Asn 100 105 110Leu Ser Tyr Thr Val Pro Val Ala Cys Ala Ala Thr Ala Val Thr Ile 115 120 125Ser Asp Thr Trp Arg 13026116PRTMz1 Penicillium sp.MISC_FEATURE(1)..(116)CBM 26Thr Lys Thr Ser Thr Ser Thr Ser Cys Thr Thr Pro Thr Ala Val Ala1 5 10 15Val Thr Phe Asp Leu Ile Ala Thr Thr Thr Tyr Gly Glu Asn Ile Lys 20 25 30Ile Ala Gly Ser Ile Ala Ala Leu Gly Ala Trp Asp Thr Asp Asp Ala 35 40 45Val Ala Leu Ser Ala Ala Asp Tyr Thr Asp Ser Asp His Leu Trp Phe 50 55 60Val Thr Gln Ser Ile Pro Ala Gly Thr Val Phe Glu Tyr Lys Tyr Ile65 70 75 80Arg Val Glu Ser Asp Gly Thr Ile Glu Trp Glu Ser Asp Pro Asn Arg 85 90 95Ser Tyr Thr Val Pro Ala Ala Cys Ala Thr Thr Ala Val Thr Glu Ser 100 105 110Asp Thr Trp Arg 11527114PRTThysanophora sp.MISC_FEATURE(1)..(114)CBM 27Phe Thr Ser Thr Thr Lys Thr Ser Cys Thr Thr Pro Thr Ser Val Ala1 5 10 15Val Thr Phe Asp Leu Ile Ala Thr Thr Thr Tyr Gly Glu Ser Ile Arg 20 25 30Leu Val Gly Ser Ile Ser Glu Leu Gly Asp Trp Asp Thr Gly Ser Ala 35 40 45Ile Ala Leu His Ala Thr Asp Tyr Thr Asp Ser Asp His Leu Trp Phe 50 55 60Val Thr Val Gly Leu Pro Ala Gly Ala Ser Phe Glu Tyr Lys Tyr Ile65 70 75 80Arg Val Glu Ser Ser Gly Thr Ile Glu Trp Glu Ser Asp Pro Asn Arg 85 90 95Ser Tyr Thr Val Pro Ala Ala Cys Ala Thr Thr Ala Val Thr Glu Ser 100 105 110Asp Thr28111PRTHumicola grisea var. thermoideaMISC_FEATURE(1)..(111)CBM 28Ala Asp Ala Ser Glu Val Tyr Val Thr Phe Asn Glu Arg Val Ser Thr1 5 10 15Ala Trp Gly Glu Thr Ile Lys Val Val Gly Asn Val Pro Ala Leu Gly 20 25 30Asn Trp Asp Thr Ser Lys Ala Val Thr Leu Ser Ala Ser Gly Tyr Lys 35 40 45Ser Asn Asp Pro Leu Trp Ser Ile Thr Val Pro Ile Lys Ala Thr Gly 50 55 60Ser Ala Val Gln Tyr Lys Tyr Ile Lys Val Gly Thr Asn Gly Lys Ile65 70 75 80Thr Trp Glu Ser Asp Pro Asn Arg Ser Ile Thr Leu Gln Thr Ala Ser 85 90 95Ser Ala Gly Lys Cys Ala Ala Gln Thr Val Asn Asp Ser Trp Arg 100 105 11029108PRTAspergillus nigerMISC_FEATURE(1)..(108)CBM 29Cys Thr Thr Pro Thr Ala Val Ala Val Thr Phe Asp Leu Thr Ala Thr1 5 10 15Thr Thr Tyr Gly Glu Asn Ile Tyr Leu Val Gly Ser Ile Ser Gln Leu 20 25 30Gly Asp Trp Glu Thr Ser Asp Gly Ile Ala Leu Ser Ala Asp Lys Tyr 35 40 45Thr Ser Ser Asp Pro Leu Trp Tyr Val Thr Val Thr Leu Pro Ala Gly 50 55 60Glu Ser Phe Glu Tyr Lys Phe Ile Arg Ile Glu Ser Asp Asp Ser Val65 70 75 80Glu Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val Pro Gln Ala Cys 85 90 95Gly Thr Ser Thr Ala Thr Val Thr Asp Thr Trp Arg 100 1053097PRTAthelia rolfsiiMISC_FEATURE(1)..(97)CBM 30Val Glu Val Thr Phe Asp Val Tyr Ala Thr Thr Val Tyr Gly Gln Asn1 5 10 15Ile Tyr Ile Thr Gly Asp Val Ser Glu Leu Gly Asn Trp Thr Pro Ala 20 25 30Asn Gly Val Ala Leu Ser Ser Ala Asn Tyr Pro Thr Trp Ser Ala Thr 35 40 45Ile Ala Leu Pro Ala Asp Thr Thr Ile Gln Tyr Lys Tyr Val Asn Ile 50 55 60Asp Gly Ser Thr Val Ile Trp Glu Asp Ala Ile Ser Asn Arg Glu Ile65 70 75 80Thr Thr Pro Ala Ser Gly Thr Tyr Thr Glu Lys Asp Thr Trp Asp Glu 85 90 95Ser31640PRTAspergillus kawachi alpha-amylasemat_peptide(22)..(640) 31Met Arg Val Ser Thr Ser Ser Ile Ala Leu Ala Val Ser Leu Phe Gly -20 -15 -10Lys Leu Ala Leu Gly Leu Ser Ala Ala Glu Trp Arg Thr Gln Ser Ile-5 -1 1 5 10Tyr Phe Leu Leu Thr Asp Arg Phe Gly Arg Thr Asp Asn Ser Thr Thr 15 20 25Ala Thr Cys Asn Thr Gly Asp Gln Ile Tyr Cys Gly Gly Ser Trp Gln 30 35 40Gly Ile Ile Asn His Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala 45 50 55Ile Trp Ile Ser Pro Ile Thr Glu Gln Leu Pro Gln Asp Thr Ser Asp60 65 70 75Gly Glu Ala Tyr His Gly Tyr Trp Gln Gln Lys Ile Tyr Tyr Val Asn 80 85 90Ser Asn Phe Gly Thr Ala Asp Asp Leu Lys Ser Leu Ser Asp Ala Leu 95 100 105His Ala Arg Gly Met Tyr Leu Met Val Asp Val Val Pro Asn His Met 110 115 120Gly Tyr Ala Gly Asn Gly Asn Asp Val Asp Tyr Ser Val Phe Asp Pro 125 130 135Phe Asp Ser Ser Ser Tyr Phe His Pro Tyr Cys Leu Ile Thr Asp Trp140 145 150 155Asp Asn Leu Thr Met Val Gln Asp Cys Trp Glu Gly Asp Thr Ile Val 160 165 170Ser Leu Pro Asp Leu Asn Thr Thr Glu Thr Ala Val Arg Thr Ile Trp 175 180 185Tyr Asp Trp Val Ala Asp Leu Val Ser Asn Tyr Ser Val Asp Gly Leu 190 195 200Arg Ile Asp Ser Val Glu Glu Val Glu Pro Asp Phe Phe Pro Gly Tyr 205 210 215Gln Glu Ala Ala Gly Val Tyr Cys Val Gly Glu Val Asp Asn Gly Asn220 225 230 235Pro Ala Leu Asp Cys Pro Tyr Gln Lys Tyr Leu Asp Gly Val Leu Asn 240 245 250Tyr Pro Ile Tyr Trp Gln Leu Leu Tyr Ala Phe Glu Ser Ser Ser Gly 255 260 265Ser Ile Ser Asn Leu Tyr Asn Met Ile Lys Ser Val Ala Ser Asp Cys 270 275 280Ser Asp Pro Thr Leu Leu Gly Asn Phe Ile Glu Asn His Asp Asn Pro 285 290 295Arg Phe Ala Ser Tyr Thr Ser Asp Tyr Ser Gln Ala Lys Asn Val Leu300 305 310 315Ser Tyr Ile Phe Leu Ser Asp Gly Ile Pro Ile Val Tyr Ala Gly Glu 320 325 330Glu Gln His Tyr Ser Gly Gly Asp Val Pro Tyr Asn Arg Glu Ala Thr 335 340 345Trp Leu Ser Gly Tyr Asp Thr Ser Ala Glu Leu Tyr Thr Trp Ile Ala 350 355 360Thr Thr Asn Ala Ile Arg Lys Leu Ala Ile Ser Ala Asp Ser Asp Tyr 365 370 375Ile Thr Tyr Lys Asn Asp Pro Ile Tyr Thr Asp Ser Asn Thr Ile Ala380 385 390 395Met Arg Lys Gly Thr Ser Gly Ser Gln Ile Ile Thr Val Leu Ser Asn 400 405 410Lys Gly Ser Ser Gly Ser Ser Tyr Thr Leu Thr Leu Ser Gly Ser Gly 415 420 425Tyr Thr Ser Gly Thr Lys Leu Ile Glu Ala Tyr Thr Cys Thr Ser Val 430 435 440Thr Val Asp Ser Asn Gly Asp Ile Pro Val Pro Met Ala Ser Gly Leu 445 450 455Pro Arg Val Leu Leu Pro Ala Ser Val Val Asp Ser Ser Ser Leu Cys460 465 470 475Gly Gly Ser Gly Asn Thr Thr Thr Thr Thr Thr Ala Ala Thr Ser Thr 480 485 490Ser Lys Ala Thr Thr Ser Ser Ser Ser Ser Ser Ala Ala Ala Thr Thr 495 500 505Ser Ser Ser Cys Thr Ala Thr Ser Thr Thr Leu Pro Ile Thr Phe Glu 510 515 520Glu Leu Val Thr Thr Thr Tyr Gly Glu Glu Val Tyr Leu Ser Gly Ser 525 530 535Ile Ser Gln Leu Gly Glu Trp His Thr Ser Asp Ala Val Lys Leu Ser540 545 550 555Ala Asp Asp Tyr Thr Ser Ser Asn Pro Glu Trp Ser Val Thr Val Ser 560 565 570Leu Pro Val Gly Thr Thr Phe Glu Tyr Lys Phe Ile Lys Val Asp Glu 575 580 585Gly Gly Ser Val Thr Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val 590 595 600Pro Glu Cys Gly Ser Gly Ser Gly Glu Thr Val Val Asp Thr Trp Arg 605 610 615321860DNAArtificialhybrid consisting of Aspergillus niger acid alpha-amylase catalytic domain-Aspergillus kawachii alpha-amylase linker-Aspergillus niger glucoamylase CBM 32ctg tcg gct gca gaa tgg cgc act cag tcg att tac ttc

cta ttg acg 48Leu Ser Ala Ala Glu Trp Arg Thr Gln Ser Ile Tyr Phe Leu Leu Thr1 5 10 15gat cgg ttc ggt agg acg gac aat tcg acg aca gct aca tgc gat acg 96Asp Arg Phe Gly Arg Thr Asp Asn Ser Thr Thr Ala Thr Cys Asp Thr 20 25 30ggt gac caa atc tat tgt ggt ggc agt tgg caa gga atc atc aac cat 144Gly Asp Gln Ile Tyr Cys Gly Gly Ser Trp Gln Gly Ile Ile Asn His 35 40 45ctg gat tat atc cag ggc atg gga ttc acg gcc atc tgg atc tcg cct 192Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Ser Pro 50 55 60atc act gaa cag ctg ccc cag gat act gct gat ggt gaa gct tac cat 240Ile Thr Glu Gln Leu Pro Gln Asp Thr Ala Asp Gly Glu Ala Tyr His65 70 75 80gga tat tgg cag cag aag ata tac gac gtg aac tcc aac ttc ggc act 288Gly Tyr Trp Gln Gln Lys Ile Tyr Asp Val Asn Ser Asn Phe Gly Thr 85 90 95gca gat gac ctc aag tcc ctc tca gat gcg ctt cat gcc cgc gga atg 336Ala Asp Asp Leu Lys Ser Leu Ser Asp Ala Leu His Ala Arg Gly Met 100 105 110tac ctc atg gtg gac gtc gtc cct aac cac atg ggc tac gcc ggc aac 384Tyr Leu Met Val Asp Val Val Pro Asn His Met Gly Tyr Ala Gly Asn 115 120 125ggc aac gat gta gac tac agc gtc ttc gac ccc ttc gat tcc tcc tcc 432Gly Asn Asp Val Asp Tyr Ser Val Phe Asp Pro Phe Asp Ser Ser Ser 130 135 140tac ttc cac cca tac tgc ctg atc aca gat tgg gac aac ttg acc atg 480Tyr Phe His Pro Tyr Cys Leu Ile Thr Asp Trp Asp Asn Leu Thr Met145 150 155 160gtc caa gat tgt tgg gag ggt gac acc atc gta tct ctg cca gac cta 528Val Gln Asp Cys Trp Glu Gly Asp Thr Ile Val Ser Leu Pro Asp Leu 165 170 175aac acc acc gaa act gcc gtg aga aca atc tgg tat gac tgg gta gcc 576Asn Thr Thr Glu Thr Ala Val Arg Thr Ile Trp Tyr Asp Trp Val Ala 180 185 190gac ctg gta tcc aat tat tca gtc gac gga ctc cgc atc gac agt gtc 624Asp Leu Val Ser Asn Tyr Ser Val Asp Gly Leu Arg Ile Asp Ser Val 195 200 205ctc gaa gtc gaa cca gac ttc ttc ccg ggc tac cag gaa gca gca ggt 672Leu Glu Val Glu Pro Asp Phe Phe Pro Gly Tyr Gln Glu Ala Ala Gly 210 215 220gtc tac tgc gtc ggc gaa gtc gac aac ggc aac cct gcc ctc gac tgc 720Val Tyr Cys Val Gly Glu Val Asp Asn Gly Asn Pro Ala Leu Asp Cys225 230 235 240cca tac cag aag gtc ctg gac ggc gtc ctc aac tat ccg atc tac tgg 768Pro Tyr Gln Lys Val Leu Asp Gly Val Leu Asn Tyr Pro Ile Tyr Trp 245 250 255caa ctc ctc tac gcc ttc gaa tcc tcc agc ggc agc atc agc aat ctc 816Gln Leu Leu Tyr Ala Phe Glu Ser Ser Ser Gly Ser Ile Ser Asn Leu 260 265 270tac aac atg atc aaa tcc gtc gca agc gac tgc tcc gat ccg aca cta 864Tyr Asn Met Ile Lys Ser Val Ala Ser Asp Cys Ser Asp Pro Thr Leu 275 280 285ctc ggc aac ttc atc gaa aac cac gac aat ccc cgt ttc gcc tcc tac 912Leu Gly Asn Phe Ile Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300acc tcc gac tac tcg caa gcc aaa aac gtc ctc agc tac atc ttc ctc 960Thr Ser Asp Tyr Ser Gln Ala Lys Asn Val Leu Ser Tyr Ile Phe Leu305 310 315 320tcc gac ggc atc ccc atc gtc tac gcc ggc gaa gaa cag cac tac tcc 1008Ser Asp Gly Ile Pro Ile Val Tyr Ala Gly Glu Glu Gln His Tyr Ser 325 330 335ggc ggc aag gtg ccc tac aac cgc gaa gcg acc tgg ctt tca ggc tac 1056Gly Gly Lys Val Pro Tyr Asn Arg Glu Ala Thr Trp Leu Ser Gly Tyr 340 345 350gac acc tcc gca gag ctg tac acc tgg ata gcc acc acg aac gcg atc 1104Asp Thr Ser Ala Glu Leu Tyr Thr Trp Ile Ala Thr Thr Asn Ala Ile 355 360 365cgc aaa cta gcc atc tca gct gac tcg gcc tac att acc tac gcg aat 1152Arg Lys Leu Ala Ile Ser Ala Asp Ser Ala Tyr Ile Thr Tyr Ala Asn 370 375 380gat gca ttc tac act gac agc aac acc atc gca atg cgc aaa ggc acc 1200Asp Ala Phe Tyr Thr Asp Ser Asn Thr Ile Ala Met Arg Lys Gly Thr385 390 395 400tca ggg agc caa gtc atc acc gtc ctc tcc aac aaa ggc tcc tca gga 1248Ser Gly Ser Gln Val Ile Thr Val Leu Ser Asn Lys Gly Ser Ser Gly 405 410 415agc agc tac acc ctg acc ctc agc gga agc ggc tac aca tcc ggc acg 1296Ser Ser Tyr Thr Leu Thr Leu Ser Gly Ser Gly Tyr Thr Ser Gly Thr 420 425 430aag ctg atc gaa gcg tac aca tgc aca tcc gtg acc gtg gac tcg agc 1344Lys Leu Ile Glu Ala Tyr Thr Cys Thr Ser Val Thr Val Asp Ser Ser 435 440 445ggc gat att ccc gtg ccg atg gcg tcg gga tta ccg aga gtt ctt ctg 1392Gly Asp Ile Pro Val Pro Met Ala Ser Gly Leu Pro Arg Val Leu Leu 450 455 460ccc gcg tcc gtc gtc gat agc tct tcg ctc tgt ggc ggg agc gga aga 1440Pro Ala Ser Val Val Asp Ser Ser Ser Leu Cys Gly Gly Ser Gly Arg465 470 475 480aca acc acg acc aca act gct gct gct act agt aca tcc aaa gcc acc 1488Thr Thr Thr Thr Thr Thr Ala Ala Ala Thr Ser Thr Ser Lys Ala Thr 485 490 495acc tcc tct tct tct tct tct gct gct gct act act tct tca tca tgt 1536Thr Ser Ser Ser Ser Ser Ser Ala Ala Ala Thr Thr Ser Ser Ser Cys 500 505 510acc act ccc acc gcc gtg gct gtg act ttc gat ctg aca gct acc acc 1584Thr Thr Pro Thr Ala Val Ala Val Thr Phe Asp Leu Thr Ala Thr Thr 515 520 525acc tac ggc gag aac atc tac ctg gtc gga tcg atc tct cag ctg ggt 1632Thr Tyr Gly Glu Asn Ile Tyr Leu Val Gly Ser Ile Ser Gln Leu Gly 530 535 540gac tgg gaa acc agc gac ggc ata gct ctg agt gct gac aag tac act 1680Asp Trp Glu Thr Ser Asp Gly Ile Ala Leu Ser Ala Asp Lys Tyr Thr545 550 555 560tcc agc gac ccg ctc tgg tat gtc act gtg act ctg ccg gct ggt gag 1728Ser Ser Asp Pro Leu Trp Tyr Val Thr Val Thr Leu Pro Ala Gly Glu 565 570 575tcg ttt gag tac aag ttt atc cgc att gag agc gat gac tcc gtg gag 1776Ser Phe Glu Tyr Lys Phe Ile Arg Ile Glu Ser Asp Asp Ser Val Glu 580 585 590tgg gag agt gat ccc aac cga gaa tac acc gtt cct cag gcg tgc gga 1824Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val Pro Gln Ala Cys Gly 595 600 605acg tcg acc gcg acg gtg act gac acc tgg cgg tag 1860Thr Ser Thr Ala Thr Val Thr Asp Thr Trp Arg 610 61533619PRTArtificialSynthetic Construct 33Leu Ser Ala Ala Glu Trp Arg Thr Gln Ser Ile Tyr Phe Leu Leu Thr1 5 10 15Asp Arg Phe Gly Arg Thr Asp Asn Ser Thr Thr Ala Thr Cys Asp Thr 20 25 30Gly Asp Gln Ile Tyr Cys Gly Gly Ser Trp Gln Gly Ile Ile Asn His 35 40 45Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Ser Pro 50 55 60Ile Thr Glu Gln Leu Pro Gln Asp Thr Ala Asp Gly Glu Ala Tyr His65 70 75 80Gly Tyr Trp Gln Gln Lys Ile Tyr Asp Val Asn Ser Asn Phe Gly Thr 85 90 95Ala Asp Asp Leu Lys Ser Leu Ser Asp Ala Leu His Ala Arg Gly Met 100 105 110Tyr Leu Met Val Asp Val Val Pro Asn His Met Gly Tyr Ala Gly Asn 115 120 125Gly Asn Asp Val Asp Tyr Ser Val Phe Asp Pro Phe Asp Ser Ser Ser 130 135 140Tyr Phe His Pro Tyr Cys Leu Ile Thr Asp Trp Asp Asn Leu Thr Met145 150 155 160Val Gln Asp Cys Trp Glu Gly Asp Thr Ile Val Ser Leu Pro Asp Leu 165 170 175Asn Thr Thr Glu Thr Ala Val Arg Thr Ile Trp Tyr Asp Trp Val Ala 180 185 190Asp Leu Val Ser Asn Tyr Ser Val Asp Gly Leu Arg Ile Asp Ser Val 195 200 205Leu Glu Val Glu Pro Asp Phe Phe Pro Gly Tyr Gln Glu Ala Ala Gly 210 215 220Val Tyr Cys Val Gly Glu Val Asp Asn Gly Asn Pro Ala Leu Asp Cys225 230 235 240Pro Tyr Gln Lys Val Leu Asp Gly Val Leu Asn Tyr Pro Ile Tyr Trp 245 250 255Gln Leu Leu Tyr Ala Phe Glu Ser Ser Ser Gly Ser Ile Ser Asn Leu 260 265 270Tyr Asn Met Ile Lys Ser Val Ala Ser Asp Cys Ser Asp Pro Thr Leu 275 280 285Leu Gly Asn Phe Ile Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300Thr Ser Asp Tyr Ser Gln Ala Lys Asn Val Leu Ser Tyr Ile Phe Leu305 310 315 320Ser Asp Gly Ile Pro Ile Val Tyr Ala Gly Glu Glu Gln His Tyr Ser 325 330 335Gly Gly Lys Val Pro Tyr Asn Arg Glu Ala Thr Trp Leu Ser Gly Tyr 340 345 350Asp Thr Ser Ala Glu Leu Tyr Thr Trp Ile Ala Thr Thr Asn Ala Ile 355 360 365Arg Lys Leu Ala Ile Ser Ala Asp Ser Ala Tyr Ile Thr Tyr Ala Asn 370 375 380Asp Ala Phe Tyr Thr Asp Ser Asn Thr Ile Ala Met Arg Lys Gly Thr385 390 395 400Ser Gly Ser Gln Val Ile Thr Val Leu Ser Asn Lys Gly Ser Ser Gly 405 410 415Ser Ser Tyr Thr Leu Thr Leu Ser Gly Ser Gly Tyr Thr Ser Gly Thr 420 425 430Lys Leu Ile Glu Ala Tyr Thr Cys Thr Ser Val Thr Val Asp Ser Ser 435 440 445Gly Asp Ile Pro Val Pro Met Ala Ser Gly Leu Pro Arg Val Leu Leu 450 455 460Pro Ala Ser Val Val Asp Ser Ser Ser Leu Cys Gly Gly Ser Gly Arg465 470 475 480Thr Thr Thr Thr Thr Thr Ala Ala Ala Thr Ser Thr Ser Lys Ala Thr 485 490 495Thr Ser Ser Ser Ser Ser Ser Ala Ala Ala Thr Thr Ser Ser Ser Cys 500 505 510Thr Thr Pro Thr Ala Val Ala Val Thr Phe Asp Leu Thr Ala Thr Thr 515 520 525Thr Tyr Gly Glu Asn Ile Tyr Leu Val Gly Ser Ile Ser Gln Leu Gly 530 535 540Asp Trp Glu Thr Ser Asp Gly Ile Ala Leu Ser Ala Asp Lys Tyr Thr545 550 555 560Ser Ser Asp Pro Leu Trp Tyr Val Thr Val Thr Leu Pro Ala Gly Glu 565 570 575Ser Phe Glu Tyr Lys Phe Ile Arg Ile Glu Ser Asp Asp Ser Val Glu 580 585 590Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val Pro Gln Ala Cys Gly 595 600 605Thr Ser Thr Ala Thr Val Thr Asp Thr Trp Arg 610 615341827DNAArtificialHybrid containing Aspergillus niger acid alpha- amylase catalytic domain-Aspergillus kawachii alpha-amylase linker-Athelia rolfsii glucoamylase CBD 34ctg tcg gct gca gaa tgg cgc act cag tcg att tac ttc cta ttg acg 48Leu Ser Ala Ala Glu Trp Arg Thr Gln Ser Ile Tyr Phe Leu Leu Thr1 5 10 15gat cgg ttc ggt agg acg gac aat tcg acg aca gct aca tgc gat acg 96Asp Arg Phe Gly Arg Thr Asp Asn Ser Thr Thr Ala Thr Cys Asp Thr 20 25 30ggt gac caa atc tat tgt ggt ggc agt tgg caa gga atc atc aac cat 144Gly Asp Gln Ile Tyr Cys Gly Gly Ser Trp Gln Gly Ile Ile Asn His 35 40 45ctg gat tat atc cag ggc atg gga ttc acg gcc atc tgg atc tcg cct 192Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Ser Pro 50 55 60atc act gaa cag ctg ccc cag gat act gct gat ggt gaa gct tac cat 240Ile Thr Glu Gln Leu Pro Gln Asp Thr Ala Asp Gly Glu Ala Tyr His65 70 75 80gga tat tgg cag cag aag ata tac gac gtg aac tcc aac ttc ggc act 288Gly Tyr Trp Gln Gln Lys Ile Tyr Asp Val Asn Ser Asn Phe Gly Thr 85 90 95gca gat gac ctc aag tcc ctc tca gat gcg ctt cat gcc cgc gga atg 336Ala Asp Asp Leu Lys Ser Leu Ser Asp Ala Leu His Ala Arg Gly Met 100 105 110tac ctc atg gtg gac gtc gtc cct aac cac atg ggc tac gcc ggc aac 384Tyr Leu Met Val Asp Val Val Pro Asn His Met Gly Tyr Ala Gly Asn 115 120 125ggc aac gat gta gac tac agc gtc ttc gac ccc ttc gat tcc tcc tcc 432Gly Asn Asp Val Asp Tyr Ser Val Phe Asp Pro Phe Asp Ser Ser Ser 130 135 140tac ttc cac cca tac tgc ctg atc aca gat tgg gac aac ttg acc atg 480Tyr Phe His Pro Tyr Cys Leu Ile Thr Asp Trp Asp Asn Leu Thr Met145 150 155 160gtc caa gat tgt tgg gag ggt gac acc atc gta tct ctg cca gac cta 528Val Gln Asp Cys Trp Glu Gly Asp Thr Ile Val Ser Leu Pro Asp Leu 165 170 175aac acc acc gaa act gcc gtg aga aca atc tgg tat gac tgg gta gcc 576Asn Thr Thr Glu Thr Ala Val Arg Thr Ile Trp Tyr Asp Trp Val Ala 180 185 190gac ctg gta tcc aat tat tca gtc gac gga ctc cgc atc gac agt gtc 624Asp Leu Val Ser Asn Tyr Ser Val Asp Gly Leu Arg Ile Asp Ser Val 195 200 205ctc gaa gtc gaa cca gac ttc ttc ccg ggc tac cag gaa gca gca ggt 672Leu Glu Val Glu Pro Asp Phe Phe Pro Gly Tyr Gln Glu Ala Ala Gly 210 215 220gtc tac tgc gtc ggc gaa gtc gac aac ggc aac cct gcc ctc gac tgc 720Val Tyr Cys Val Gly Glu Val Asp Asn Gly Asn Pro Ala Leu Asp Cys225 230 235 240cca tac cag aag gtc ctg gac ggc gtc ctc aac tat ccg atc tac tgg 768Pro Tyr Gln Lys Val Leu Asp Gly Val Leu Asn Tyr Pro Ile Tyr Trp 245 250 255caa ctc ctc tac gcc ttc gaa tcc tcc agc ggc agc atc agc aat ctc 816Gln Leu Leu Tyr Ala Phe Glu Ser Ser Ser Gly Ser Ile Ser Asn Leu 260 265 270tac aac atg atc aaa tcc gtc gca agc gac tgc tcc gat ccg aca cta 864Tyr Asn Met Ile Lys Ser Val Ala Ser Asp Cys Ser Asp Pro Thr Leu 275 280 285ctc ggc aac ttc atc gaa aac cac gac aat ccc cgt ttc gcc tcc tac 912Leu Gly Asn Phe Ile Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300acc tcc gac tac tcg caa gcc aaa aac gtc ctc agc tac atc ttc ctc 960Thr Ser Asp Tyr Ser Gln Ala Lys Asn Val Leu Ser Tyr Ile Phe Leu305 310 315 320tcc gac ggc atc ccc atc gtc tac gcc ggc gaa gaa cag cac tac tcc 1008Ser Asp Gly Ile Pro Ile Val Tyr Ala Gly Glu Glu Gln His Tyr Ser 325 330 335ggc ggc aag gtg ccc tac aac cgc gaa gcg acc tgg ctt tca ggc tac 1056Gly Gly Lys Val Pro Tyr Asn Arg Glu Ala Thr Trp Leu Ser Gly Tyr 340 345 350gac acc tcc gca gag ctg tac acc tgg ata gcc acc acg aac gcg atc 1104Asp Thr Ser Ala Glu Leu Tyr Thr Trp Ile Ala Thr Thr Asn Ala Ile 355 360 365cgc aaa cta gcc atc tca gct gac tcg gcc tac att acc tac gcg aat 1152Arg Lys Leu Ala Ile Ser Ala Asp Ser Ala Tyr Ile Thr Tyr Ala Asn 370 375 380gat gca ttc tac act gac agc aac acc atc gca atg cgc aaa ggc acc 1200Asp Ala Phe Tyr Thr Asp Ser Asn Thr Ile Ala Met Arg Lys Gly Thr385 390 395 400tca ggg agc caa gtc atc acc gtc ctc tcc aac aaa ggc tcc tca gga 1248Ser Gly Ser Gln Val Ile Thr Val Leu Ser Asn Lys Gly Ser Ser Gly 405 410 415agc agc tac acc ctg acc ctc agc gga agc ggc tac aca tcc ggc acg 1296Ser Ser Tyr Thr Leu Thr Leu Ser Gly Ser Gly Tyr Thr Ser Gly Thr 420 425 430aag ctg atc gaa gcg tac aca tgc aca tcc gtg acc gtg gac tcg agc 1344Lys Leu Ile Glu Ala Tyr Thr Cys Thr Ser Val Thr Val Asp Ser Ser 435 440 445ggc gat att ccc gtg ccg atg gcg tcg gga tta ccg aga gtt ctt ctg 1392Gly Asp Ile Pro Val Pro Met Ala Ser Gly Leu Pro Arg Val Leu Leu 450 455 460ccc gcg tcc gtc gtc gat agc tct tcg ctc tgt ggc ggg agc gga aga 1440Pro Ala Ser Val Val Asp Ser Ser Ser Leu Cys Gly Gly Ser Gly Arg465 470 475 480aca acc acg acc aca act gct gct gct act agt aca tcc aaa gcc acc 1488Thr Thr Thr Thr Thr Thr Ala Ala Ala Thr Ser Thr Ser Lys Ala Thr 485 490 495acc tcc tct tct tct tct tct gct gct gct act act tct tca tca gtc 1536Thr Ser Ser Ser Ser Ser Ser Ala Ala Ala Thr Thr Ser Ser Ser Val 500 505 510gag gtc act ttc gac gtt tac gct acc aca gta tat ggc cag aac atc 1584Glu Val Thr Phe Asp Val Tyr Ala Thr Thr Val Tyr Gly Gln Asn Ile

515 520 525tat atc acc ggt gat gtg agt gag ctc ggc aac tgg aca ccc gcc aat 1632Tyr Ile Thr Gly Asp Val Ser Glu Leu Gly Asn Trp Thr Pro Ala Asn 530 535 540ggt gtt gca ctc tct tct gct aac tac ccc acc tgg agt gcc acg atc 1680Gly Val Ala Leu Ser Ser Ala Asn Tyr Pro Thr Trp Ser Ala Thr Ile545 550 555 560gct ctc ccc gct gac acg aca atc cag tac aag tat gtc aac att gac 1728Ala Leu Pro Ala Asp Thr Thr Ile Gln Tyr Lys Tyr Val Asn Ile Asp 565 570 575ggc agc acc gtc atc tgg gag gat gct atc agc aat cgc gag atc acg 1776Gly Ser Thr Val Ile Trp Glu Asp Ala Ile Ser Asn Arg Glu Ile Thr 580 585 590acg ccc gcc agc ggc aca tac acc gaa aaa gac act tgg gat gaa tct 1824Thr Pro Ala Ser Gly Thr Tyr Thr Glu Lys Asp Thr Trp Asp Glu Ser 595 600 605tag 182735608PRTArtificialSynthetic Construct 35Leu Ser Ala Ala Glu Trp Arg Thr Gln Ser Ile Tyr Phe Leu Leu Thr1 5 10 15Asp Arg Phe Gly Arg Thr Asp Asn Ser Thr Thr Ala Thr Cys Asp Thr 20 25 30Gly Asp Gln Ile Tyr Cys Gly Gly Ser Trp Gln Gly Ile Ile Asn His 35 40 45Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Ser Pro 50 55 60Ile Thr Glu Gln Leu Pro Gln Asp Thr Ala Asp Gly Glu Ala Tyr His65 70 75 80Gly Tyr Trp Gln Gln Lys Ile Tyr Asp Val Asn Ser Asn Phe Gly Thr 85 90 95Ala Asp Asp Leu Lys Ser Leu Ser Asp Ala Leu His Ala Arg Gly Met 100 105 110Tyr Leu Met Val Asp Val Val Pro Asn His Met Gly Tyr Ala Gly Asn 115 120 125Gly Asn Asp Val Asp Tyr Ser Val Phe Asp Pro Phe Asp Ser Ser Ser 130 135 140Tyr Phe His Pro Tyr Cys Leu Ile Thr Asp Trp Asp Asn Leu Thr Met145 150 155 160Val Gln Asp Cys Trp Glu Gly Asp Thr Ile Val Ser Leu Pro Asp Leu 165 170 175Asn Thr Thr Glu Thr Ala Val Arg Thr Ile Trp Tyr Asp Trp Val Ala 180 185 190Asp Leu Val Ser Asn Tyr Ser Val Asp Gly Leu Arg Ile Asp Ser Val 195 200 205Leu Glu Val Glu Pro Asp Phe Phe Pro Gly Tyr Gln Glu Ala Ala Gly 210 215 220Val Tyr Cys Val Gly Glu Val Asp Asn Gly Asn Pro Ala Leu Asp Cys225 230 235 240Pro Tyr Gln Lys Val Leu Asp Gly Val Leu Asn Tyr Pro Ile Tyr Trp 245 250 255Gln Leu Leu Tyr Ala Phe Glu Ser Ser Ser Gly Ser Ile Ser Asn Leu 260 265 270Tyr Asn Met Ile Lys Ser Val Ala Ser Asp Cys Ser Asp Pro Thr Leu 275 280 285Leu Gly Asn Phe Ile Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300Thr Ser Asp Tyr Ser Gln Ala Lys Asn Val Leu Ser Tyr Ile Phe Leu305 310 315 320Ser Asp Gly Ile Pro Ile Val Tyr Ala Gly Glu Glu Gln His Tyr Ser 325 330 335Gly Gly Lys Val Pro Tyr Asn Arg Glu Ala Thr Trp Leu Ser Gly Tyr 340 345 350Asp Thr Ser Ala Glu Leu Tyr Thr Trp Ile Ala Thr Thr Asn Ala Ile 355 360 365Arg Lys Leu Ala Ile Ser Ala Asp Ser Ala Tyr Ile Thr Tyr Ala Asn 370 375 380Asp Ala Phe Tyr Thr Asp Ser Asn Thr Ile Ala Met Arg Lys Gly Thr385 390 395 400Ser Gly Ser Gln Val Ile Thr Val Leu Ser Asn Lys Gly Ser Ser Gly 405 410 415Ser Ser Tyr Thr Leu Thr Leu Ser Gly Ser Gly Tyr Thr Ser Gly Thr 420 425 430Lys Leu Ile Glu Ala Tyr Thr Cys Thr Ser Val Thr Val Asp Ser Ser 435 440 445Gly Asp Ile Pro Val Pro Met Ala Ser Gly Leu Pro Arg Val Leu Leu 450 455 460Pro Ala Ser Val Val Asp Ser Ser Ser Leu Cys Gly Gly Ser Gly Arg465 470 475 480Thr Thr Thr Thr Thr Thr Ala Ala Ala Thr Ser Thr Ser Lys Ala Thr 485 490 495Thr Ser Ser Ser Ser Ser Ser Ala Ala Ala Thr Thr Ser Ser Ser Val 500 505 510Glu Val Thr Phe Asp Val Tyr Ala Thr Thr Val Tyr Gly Gln Asn Ile 515 520 525Tyr Ile Thr Gly Asp Val Ser Glu Leu Gly Asn Trp Thr Pro Ala Asn 530 535 540Gly Val Ala Leu Ser Ser Ala Asn Tyr Pro Thr Trp Ser Ala Thr Ile545 550 555 560Ala Leu Pro Ala Asp Thr Thr Ile Gln Tyr Lys Tyr Val Asn Ile Asp 565 570 575Gly Ser Thr Val Ile Trp Glu Asp Ala Ile Ser Asn Arg Glu Ile Thr 580 585 590Thr Pro Ala Ser Gly Thr Tyr Thr Glu Lys Asp Thr Trp Asp Glu Ser 595 600 605361863DNAArtificialHybrid consisting of A.oryzae alpha-amylase catalytic domain-A. kawachii alpha-amylase linker-A. kawachi alpha-amylase CBD 36gca acg cct gcg gac tgg cga tcg caa tcc att tat ttc ctt ctc acg 48Ala Thr Pro Ala Asp Trp Arg Ser Gln Ser Ile Tyr Phe Leu Leu Thr1 5 10 15gat cga ttt gca agg acg gat ggg tcg acg act gcg act tgt aat act 96Asp Arg Phe Ala Arg Thr Asp Gly Ser Thr Thr Ala Thr Cys Asn Thr 20 25 30gcg gat cag aaa tac tgt ggt gga aca tgg cag ggc atc atc gac aag 144Ala Asp Gln Lys Tyr Cys Gly Gly Thr Trp Gln Gly Ile Ile Asp Lys 35 40 45ttg gac tat atc cag gga atg ggc ttc aca gcc atc tgg atc acc ccc 192Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Thr Pro 50 55 60gtt aca gcc cag ctg ccc cag acc acc gca tat gga gat gcc tac cat 240Val Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly Asp Ala Tyr His65 70 75 80ggc tac tgg cag cag gat ata tac tct ctg aac gaa aac tac ggc act 288Gly Tyr Trp Gln Gln Asp Ile Tyr Ser Leu Asn Glu Asn Tyr Gly Thr 85 90 95gca gat gac ttg aag gcg ctc tct tcg gcc ctt cat gag agg ggg atg 336Ala Asp Asp Leu Lys Ala Leu Ser Ser Ala Leu His Glu Arg Gly Met 100 105 110tat ctt atg gtc gat gtg gtt gct aac cat atg ggc tat gat gga gcg 384Tyr Leu Met Val Asp Val Val Ala Asn His Met Gly Tyr Asp Gly Ala 115 120 125ggt agc tca gtc gat tac agt gtg ttt aaa ccg ttc agt tcc caa gac 432Gly Ser Ser Val Asp Tyr Ser Val Phe Lys Pro Phe Ser Ser Gln Asp 130 135 140tac ttc cac ccg ttc tgt ttc att caa aac tat gaa gat cag act cag 480Tyr Phe His Pro Phe Cys Phe Ile Gln Asn Tyr Glu Asp Gln Thr Gln145 150 155 160gtt gag gat tgc tgg cta gga gat aac act gtc tcc ttg cct gat ctc 528Val Glu Asp Cys Trp Leu Gly Asp Asn Thr Val Ser Leu Pro Asp Leu 165 170 175gat acc acc aag gat gtg gtc aag aat gaa tgg tac gac tgg gtg gga 576Asp Thr Thr Lys Asp Val Val Lys Asn Glu Trp Tyr Asp Trp Val Gly 180 185 190tca ttg gta tcg aac tac tcc att gac ggc ctc cgt atc gac aca gta 624Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly Leu Arg Ile Asp Thr Val 195 200 205aaa cac gtc cag aag gac ttc tgg ccc ggg tac aac aaa gcc gca ggc 672Lys His Val Gln Lys Asp Phe Trp Pro Gly Tyr Asn Lys Ala Ala Gly 210 215 220gtg tac tgt atc ggc gag gtg ctc gac ggt gat ccg gcc tac act tgt 720Val Tyr Cys Ile Gly Glu Val Leu Asp Gly Asp Pro Ala Tyr Thr Cys225 230 235 240ccc tac cag aac gtc atg gac ggc gta ctg aac tat ccc att tac tat 768Pro Tyr Gln Asn Val Met Asp Gly Val Leu Asn Tyr Pro Ile Tyr Tyr 245 250 255cca ctc ctc aac gcc ttc aag tca acc tcc ggc agc atg gac gac ctc 816Pro Leu Leu Asn Ala Phe Lys Ser Thr Ser Gly Ser Met Asp Asp Leu 260 265 270tac aac atg atc aac acc gtc aaa tcc gac tgt cca gac tca aca ctc 864Tyr Asn Met Ile Asn Thr Val Lys Ser Asp Cys Pro Asp Ser Thr Leu 275 280 285ctg ggc aca ttc gtc gag aac cac gac aac cca cgg ttc gct tct tac 912Leu Gly Thr Phe Val Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300acc aac gac ata gcc ctc gcc aag aac gtc gca gca ttc atc atc ctc 960Thr Asn Asp Ile Ala Leu Ala Lys Asn Val Ala Ala Phe Ile Ile Leu305 310 315 320aac gac gga atc ccc atc atc tac gcc ggc caa gaa cag cac tac gcc 1008Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly Gln Glu Gln His Tyr Ala 325 330 335ggc gga aac gac ccc gcg aac cgc gaa gca acc tgg ctc tcg ggc tac 1056Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala Thr Trp Leu Ser Gly Tyr 340 345 350ccg acc gac agc gag ctg tac aag tta att gcc tcc gcg aac gca atc 1104Pro Thr Asp Ser Glu Leu Tyr Lys Leu Ile Ala Ser Ala Asn Ala Ile 355 360 365cgg aac tat gcc att agc aaa gat aca gga ttc gtg acc tac aag aac 1152Arg Asn Tyr Ala Ile Ser Lys Asp Thr Gly Phe Val Thr Tyr Lys Asn 370 375 380tgg ccc atc tac aaa gac gac aca acg atc gcc atg cgc aag ggc aca 1200Trp Pro Ile Tyr Lys Asp Asp Thr Thr Ile Ala Met Arg Lys Gly Thr385 390 395 400gat ggg tcg cag atc gtg act atc ttg tcc aac aag ggt gct tcg ggt 1248Asp Gly Ser Gln Ile Val Thr Ile Leu Ser Asn Lys Gly Ala Ser Gly 405 410 415gat tcg tat acc ctc tcc ttg agt ggt gcg ggt tac aca gcc ggc cag 1296Asp Ser Tyr Thr Leu Ser Leu Ser Gly Ala Gly Tyr Thr Ala Gly Gln 420 425 430caa ttg acg gag gtc att ggc tgc acg acc gtg acg gtt ggt tcg gat 1344Gln Leu Thr Glu Val Ile Gly Cys Thr Thr Val Thr Val Gly Ser Asp 435 440 445gga aat gtg cct gtt cct atg gca ggt ggg cta cct agg gta ttg tat 1392Gly Asn Val Pro Val Pro Met Ala Gly Gly Leu Pro Arg Val Leu Tyr 450 455 460ccg act gag aag ttg gca ggt agc aag atc tgt agt agc tcg gga aga 1440Pro Thr Glu Lys Leu Ala Gly Ser Lys Ile Cys Ser Ser Ser Gly Arg465 470 475 480aca acc acg acc aca act gct gct gct act agt aca tcc aaa gcc acc 1488Thr Thr Thr Thr Thr Thr Ala Ala Ala Thr Ser Thr Ser Lys Ala Thr 485 490 495acc tcc tct tct tct tct tct gct gct gct act act tct tca tca tgc 1536Thr Ser Ser Ser Ser Ser Ser Ala Ala Ala Thr Thr Ser Ser Ser Cys 500 505 510acc gca aca agc acc acc ctc ccc atc acc ttc gaa gaa ctc gtc acc 1584Thr Ala Thr Ser Thr Thr Leu Pro Ile Thr Phe Glu Glu Leu Val Thr 515 520 525act acc tac ggg gaa gaa gtc tac ctc agc gga tct atc tcc cag ctc 1632Thr Thr Tyr Gly Glu Glu Val Tyr Leu Ser Gly Ser Ile Ser Gln Leu 530 535 540gga gag tgg gat acg agt gac gcg gtg aag ttg tcc gcg gat gat tat 1680Gly Glu Trp Asp Thr Ser Asp Ala Val Lys Leu Ser Ala Asp Asp Tyr545 550 555 560acc tcg agt aac ccc gag tgg tct gtt act gtg tcg ttg ccg gtg ggg 1728Thr Ser Ser Asn Pro Glu Trp Ser Val Thr Val Ser Leu Pro Val Gly 565 570 575acg acc ttc gag tat aag ttt att aag gtc gat gag ggt gga agt gtg 1776Thr Thr Phe Glu Tyr Lys Phe Ile Lys Val Asp Glu Gly Gly Ser Val 580 585 590act tgg gaa agt gat ccg aat agg gag tat act gtg cct gaa tgt ggg 1824Thr Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val Pro Glu Cys Gly 595 600 605aat ggg agt ggg gag acg gtg gtt gat acg tgg agg tag 1863Asn Gly Ser Gly Glu Thr Val Val Asp Thr Trp Arg 610 615 62037620PRTArtificialSynthetic Construct 37Ala Thr Pro Ala Asp Trp Arg Ser Gln Ser Ile Tyr Phe Leu Leu Thr1 5 10 15Asp Arg Phe Ala Arg Thr Asp Gly Ser Thr Thr Ala Thr Cys Asn Thr 20 25 30Ala Asp Gln Lys Tyr Cys Gly Gly Thr Trp Gln Gly Ile Ile Asp Lys 35 40 45Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Thr Pro 50 55 60Val Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly Asp Ala Tyr His65 70 75 80Gly Tyr Trp Gln Gln Asp Ile Tyr Ser Leu Asn Glu Asn Tyr Gly Thr 85 90 95Ala Asp Asp Leu Lys Ala Leu Ser Ser Ala Leu His Glu Arg Gly Met 100 105 110Tyr Leu Met Val Asp Val Val Ala Asn His Met Gly Tyr Asp Gly Ala 115 120 125Gly Ser Ser Val Asp Tyr Ser Val Phe Lys Pro Phe Ser Ser Gln Asp 130 135 140Tyr Phe His Pro Phe Cys Phe Ile Gln Asn Tyr Glu Asp Gln Thr Gln145 150 155 160Val Glu Asp Cys Trp Leu Gly Asp Asn Thr Val Ser Leu Pro Asp Leu 165 170 175Asp Thr Thr Lys Asp Val Val Lys Asn Glu Trp Tyr Asp Trp Val Gly 180 185 190Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly Leu Arg Ile Asp Thr Val 195 200 205Lys His Val Gln Lys Asp Phe Trp Pro Gly Tyr Asn Lys Ala Ala Gly 210 215 220Val Tyr Cys Ile Gly Glu Val Leu Asp Gly Asp Pro Ala Tyr Thr Cys225 230 235 240Pro Tyr Gln Asn Val Met Asp Gly Val Leu Asn Tyr Pro Ile Tyr Tyr 245 250 255Pro Leu Leu Asn Ala Phe Lys Ser Thr Ser Gly Ser Met Asp Asp Leu 260 265 270Tyr Asn Met Ile Asn Thr Val Lys Ser Asp Cys Pro Asp Ser Thr Leu 275 280 285Leu Gly Thr Phe Val Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300Thr Asn Asp Ile Ala Leu Ala Lys Asn Val Ala Ala Phe Ile Ile Leu305 310 315 320Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly Gln Glu Gln His Tyr Ala 325 330 335Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala Thr Trp Leu Ser Gly Tyr 340 345 350Pro Thr Asp Ser Glu Leu Tyr Lys Leu Ile Ala Ser Ala Asn Ala Ile 355 360 365Arg Asn Tyr Ala Ile Ser Lys Asp Thr Gly Phe Val Thr Tyr Lys Asn 370 375 380Trp Pro Ile Tyr Lys Asp Asp Thr Thr Ile Ala Met Arg Lys Gly Thr385 390 395 400Asp Gly Ser Gln Ile Val Thr Ile Leu Ser Asn Lys Gly Ala Ser Gly 405 410 415Asp Ser Tyr Thr Leu Ser Leu Ser Gly Ala Gly Tyr Thr Ala Gly Gln 420 425 430Gln Leu Thr Glu Val Ile Gly Cys Thr Thr Val Thr Val Gly Ser Asp 435 440 445Gly Asn Val Pro Val Pro Met Ala Gly Gly Leu Pro Arg Val Leu Tyr 450 455 460Pro Thr Glu Lys Leu Ala Gly Ser Lys Ile Cys Ser Ser Ser Gly Arg465 470 475 480Thr Thr Thr Thr Thr Thr Ala Ala Ala Thr Ser Thr Ser Lys Ala Thr 485 490 495Thr Ser Ser Ser Ser Ser Ser Ala Ala Ala Thr Thr Ser Ser Ser Cys 500 505 510Thr Ala Thr Ser Thr Thr Leu Pro Ile Thr Phe Glu Glu Leu Val Thr 515 520 525Thr Thr Tyr Gly Glu Glu Val Tyr Leu Ser Gly Ser Ile Ser Gln Leu 530 535 540Gly Glu Trp Asp Thr Ser Asp Ala Val Lys Leu Ser Ala Asp Asp Tyr545 550 555 560Thr Ser Ser Asn Pro Glu Trp Ser Val Thr Val Ser Leu Pro Val Gly 565 570 575Thr Thr Phe Glu Tyr Lys Phe Ile Lys Val Asp Glu Gly Gly Ser Val 580 585 590Thr Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val Pro Glu Cys Gly 595 600 605Asn Gly Ser Gly Glu Thr Val Val Asp Thr Trp Arg 610 615 620381767DNAArtificialHybrid consisting of A. niger acid alpha- amylase catalytic domain- A.rolfsii glucoamylase linker- A. rolfsii glucoamylase CBM 38ctg tcg gct gca gaa tgg cgc act cag tcg att tac ttc cta ttg acg 48Leu Ser Ala Ala Glu Trp Arg Thr Gln Ser Ile Tyr Phe Leu Leu Thr1 5 10 15gat cgg ttc ggt agg acg gac aat tcg acg aca gct aca tgc gat acg 96Asp Arg Phe Gly Arg Thr Asp Asn Ser Thr Thr Ala Thr Cys Asp Thr 20

25 30ggt gac caa atc tat tgt ggt ggc agt tgg caa gga atc atc aac cat 144Gly Asp Gln Ile Tyr Cys Gly Gly Ser Trp Gln Gly Ile Ile Asn His 35 40 45ctg gat tat atc cag ggc atg gga ttc acg gcc atc tgg atc tcg cct 192Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Ser Pro 50 55 60atc act gaa cag ctg ccc cag gat act gct gat ggt gaa gct tac cat 240Ile Thr Glu Gln Leu Pro Gln Asp Thr Ala Asp Gly Glu Ala Tyr His65 70 75 80gga tat tgg cag cag aag ata tac gac gtg aac tcc aac ttc ggc act 288Gly Tyr Trp Gln Gln Lys Ile Tyr Asp Val Asn Ser Asn Phe Gly Thr 85 90 95gca gat gac ctc aag tcc ctc tca gat gcg ctt cat gcc cgc gga atg 336Ala Asp Asp Leu Lys Ser Leu Ser Asp Ala Leu His Ala Arg Gly Met 100 105 110tac ctc atg gtg gac gtc gtc cct aac cac atg ggc tac gcc ggc aac 384Tyr Leu Met Val Asp Val Val Pro Asn His Met Gly Tyr Ala Gly Asn 115 120 125ggc aac gat gta gac tac agc gtc ttc gac ccc ttc gat tcc tcc tcc 432Gly Asn Asp Val Asp Tyr Ser Val Phe Asp Pro Phe Asp Ser Ser Ser 130 135 140tac ttc cac cca tac tgc ctg atc aca gat tgg gac aac ttg acc atg 480Tyr Phe His Pro Tyr Cys Leu Ile Thr Asp Trp Asp Asn Leu Thr Met145 150 155 160gtc caa gat tgt tgg gag ggt gac acc atc gta tct ctg cca gac cta 528Val Gln Asp Cys Trp Glu Gly Asp Thr Ile Val Ser Leu Pro Asp Leu 165 170 175aac acc acc gaa act gcc gtg aga aca atc tgg tat gac tgg gta gcc 576Asn Thr Thr Glu Thr Ala Val Arg Thr Ile Trp Tyr Asp Trp Val Ala 180 185 190gac ctg gta tcc aat tat tca gtc gac gga ctc cgc atc gac agt gtc 624Asp Leu Val Ser Asn Tyr Ser Val Asp Gly Leu Arg Ile Asp Ser Val 195 200 205ctc gaa gtc gaa cca gac ttc ttc ccg ggc tac cag gaa gca gca ggt 672Leu Glu Val Glu Pro Asp Phe Phe Pro Gly Tyr Gln Glu Ala Ala Gly 210 215 220gtc tac tgc gtc ggc gaa gtc gac aac ggc aac cct gcc ctc gac tgc 720Val Tyr Cys Val Gly Glu Val Asp Asn Gly Asn Pro Ala Leu Asp Cys225 230 235 240cca tac cag aag gtc ctg gac ggc gtc ctc aac tat ccg atc tac tgg 768Pro Tyr Gln Lys Val Leu Asp Gly Val Leu Asn Tyr Pro Ile Tyr Trp 245 250 255caa ctc ctc tac gcc ttc gaa tcc tcc agc ggc agc atc agc aat ctc 816Gln Leu Leu Tyr Ala Phe Glu Ser Ser Ser Gly Ser Ile Ser Asn Leu 260 265 270tac aac atg atc aaa tcc gtc gca agc gac tgc tcc gat ccg aca cta 864Tyr Asn Met Ile Lys Ser Val Ala Ser Asp Cys Ser Asp Pro Thr Leu 275 280 285ctc ggc aac ttc atc gaa aac cac gac aat ccc cgt ttc gcc tcc tac 912Leu Gly Asn Phe Ile Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300acc tcc gac tac tcg caa gcc aaa aac gtc ctc agc tac atc ttc ctc 960Thr Ser Asp Tyr Ser Gln Ala Lys Asn Val Leu Ser Tyr Ile Phe Leu305 310 315 320tcc gac ggc atc ccc atc gtc tac gcc ggc gaa gaa cag cac tac tcc 1008Ser Asp Gly Ile Pro Ile Val Tyr Ala Gly Glu Glu Gln His Tyr Ser 325 330 335ggc ggc aag gtg ccc tac aac cgc gaa gcg acc tgg ctt tca ggc tac 1056Gly Gly Lys Val Pro Tyr Asn Arg Glu Ala Thr Trp Leu Ser Gly Tyr 340 345 350gac acc tcc gca gag ctg tac acc tgg ata gcc acc acg aac gcg atc 1104Asp Thr Ser Ala Glu Leu Tyr Thr Trp Ile Ala Thr Thr Asn Ala Ile 355 360 365cgc aaa cta gcc atc tca gct gac tcg gcc tac att acc tac gcg aat 1152Arg Lys Leu Ala Ile Ser Ala Asp Ser Ala Tyr Ile Thr Tyr Ala Asn 370 375 380gat gca ttc tac act gac agc aac acc atc gca atg cgc aaa ggc acc 1200Asp Ala Phe Tyr Thr Asp Ser Asn Thr Ile Ala Met Arg Lys Gly Thr385 390 395 400tca ggg agc caa gtc atc acc gtc ctc tcc aac aaa ggc tcc tca gga 1248Ser Gly Ser Gln Val Ile Thr Val Leu Ser Asn Lys Gly Ser Ser Gly 405 410 415agc agc tac acc ctg acc ctc agc gga agc ggc tac aca tcc ggc acg 1296Ser Ser Tyr Thr Leu Thr Leu Ser Gly Ser Gly Tyr Thr Ser Gly Thr 420 425 430aag ctg atc gaa gcg tac aca tgc aca tcc gtg acc gtg gac tcg agc 1344Lys Leu Ile Glu Ala Tyr Thr Cys Thr Ser Val Thr Val Asp Ser Ser 435 440 445ggc gat att ccc gtg ccg atg gcg tcg gga tta ccg aga gtt ctt ctg 1392Gly Asp Ile Pro Val Pro Met Ala Ser Gly Leu Pro Arg Val Leu Leu 450 455 460ccc gcg tcc gtc gtc gat agc tct tcg ctc tgt ggc ggg agc gga aga 1440Pro Ala Ser Val Val Asp Ser Ser Ser Leu Cys Gly Gly Ser Gly Arg465 470 475 480ggt gct aca agc ccg ggt ggc tcc tcg ggt agt gtc gag gtc act ttc 1488Gly Ala Thr Ser Pro Gly Gly Ser Ser Gly Ser Val Glu Val Thr Phe 485 490 495gac gtt tac gct acc aca gta tat ggc cag aac atc tat atc acc ggt 1536Asp Val Tyr Ala Thr Thr Val Tyr Gly Gln Asn Ile Tyr Ile Thr Gly 500 505 510gat gtg agt gag ctc ggc aac tgg aca ccc gcc aat ggt gtt gca ctc 1584Asp Val Ser Glu Leu Gly Asn Trp Thr Pro Ala Asn Gly Val Ala Leu 515 520 525tct tct gct aac tac ccc acc tgg agt gcc acg atc gct ctc ccc gct 1632Ser Ser Ala Asn Tyr Pro Thr Trp Ser Ala Thr Ile Ala Leu Pro Ala 530 535 540gac acg aca atc cag tac aag tat gtc aac att gac ggc agc acc gtc 1680Asp Thr Thr Ile Gln Tyr Lys Tyr Val Asn Ile Asp Gly Ser Thr Val545 550 555 560atc tgg gag gat gct atc agc aat cgc gag atc acg acg ccc gcc agc 1728Ile Trp Glu Asp Ala Ile Ser Asn Arg Glu Ile Thr Thr Pro Ala Ser 565 570 575ggc aca tac acc gaa aaa gac act tgg gat gaa tct tag 1767Gly Thr Tyr Thr Glu Lys Asp Thr Trp Asp Glu Ser 580 58539588PRTArtificialSynthetic Construct 39Leu Ser Ala Ala Glu Trp Arg Thr Gln Ser Ile Tyr Phe Leu Leu Thr1 5 10 15Asp Arg Phe Gly Arg Thr Asp Asn Ser Thr Thr Ala Thr Cys Asp Thr 20 25 30Gly Asp Gln Ile Tyr Cys Gly Gly Ser Trp Gln Gly Ile Ile Asn His 35 40 45Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Ser Pro 50 55 60Ile Thr Glu Gln Leu Pro Gln Asp Thr Ala Asp Gly Glu Ala Tyr His65 70 75 80Gly Tyr Trp Gln Gln Lys Ile Tyr Asp Val Asn Ser Asn Phe Gly Thr 85 90 95Ala Asp Asp Leu Lys Ser Leu Ser Asp Ala Leu His Ala Arg Gly Met 100 105 110Tyr Leu Met Val Asp Val Val Pro Asn His Met Gly Tyr Ala Gly Asn 115 120 125Gly Asn Asp Val Asp Tyr Ser Val Phe Asp Pro Phe Asp Ser Ser Ser 130 135 140Tyr Phe His Pro Tyr Cys Leu Ile Thr Asp Trp Asp Asn Leu Thr Met145 150 155 160Val Gln Asp Cys Trp Glu Gly Asp Thr Ile Val Ser Leu Pro Asp Leu 165 170 175Asn Thr Thr Glu Thr Ala Val Arg Thr Ile Trp Tyr Asp Trp Val Ala 180 185 190Asp Leu Val Ser Asn Tyr Ser Val Asp Gly Leu Arg Ile Asp Ser Val 195 200 205Leu Glu Val Glu Pro Asp Phe Phe Pro Gly Tyr Gln Glu Ala Ala Gly 210 215 220Val Tyr Cys Val Gly Glu Val Asp Asn Gly Asn Pro Ala Leu Asp Cys225 230 235 240Pro Tyr Gln Lys Val Leu Asp Gly Val Leu Asn Tyr Pro Ile Tyr Trp 245 250 255Gln Leu Leu Tyr Ala Phe Glu Ser Ser Ser Gly Ser Ile Ser Asn Leu 260 265 270Tyr Asn Met Ile Lys Ser Val Ala Ser Asp Cys Ser Asp Pro Thr Leu 275 280 285Leu Gly Asn Phe Ile Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300Thr Ser Asp Tyr Ser Gln Ala Lys Asn Val Leu Ser Tyr Ile Phe Leu305 310 315 320Ser Asp Gly Ile Pro Ile Val Tyr Ala Gly Glu Glu Gln His Tyr Ser 325 330 335Gly Gly Lys Val Pro Tyr Asn Arg Glu Ala Thr Trp Leu Ser Gly Tyr 340 345 350Asp Thr Ser Ala Glu Leu Tyr Thr Trp Ile Ala Thr Thr Asn Ala Ile 355 360 365Arg Lys Leu Ala Ile Ser Ala Asp Ser Ala Tyr Ile Thr Tyr Ala Asn 370 375 380Asp Ala Phe Tyr Thr Asp Ser Asn Thr Ile Ala Met Arg Lys Gly Thr385 390 395 400Ser Gly Ser Gln Val Ile Thr Val Leu Ser Asn Lys Gly Ser Ser Gly 405 410 415Ser Ser Tyr Thr Leu Thr Leu Ser Gly Ser Gly Tyr Thr Ser Gly Thr 420 425 430Lys Leu Ile Glu Ala Tyr Thr Cys Thr Ser Val Thr Val Asp Ser Ser 435 440 445Gly Asp Ile Pro Val Pro Met Ala Ser Gly Leu Pro Arg Val Leu Leu 450 455 460Pro Ala Ser Val Val Asp Ser Ser Ser Leu Cys Gly Gly Ser Gly Arg465 470 475 480Gly Ala Thr Ser Pro Gly Gly Ser Ser Gly Ser Val Glu Val Thr Phe 485 490 495Asp Val Tyr Ala Thr Thr Val Tyr Gly Gln Asn Ile Tyr Ile Thr Gly 500 505 510Asp Val Ser Glu Leu Gly Asn Trp Thr Pro Ala Asn Gly Val Ala Leu 515 520 525Ser Ser Ala Asn Tyr Pro Thr Trp Ser Ala Thr Ile Ala Leu Pro Ala 530 535 540Asp Thr Thr Ile Gln Tyr Lys Tyr Val Asn Ile Asp Gly Ser Thr Val545 550 555 560Ile Trp Glu Asp Ala Ile Ser Asn Arg Glu Ile Thr Thr Pro Ala Ser 565 570 575Gly Thr Tyr Thr Glu Lys Asp Thr Trp Asp Glu Ser 580 585401767DNAArtificialHybrid containing A. oryzae alpha-amylase catalytic domain- A. rolfsii glucoamylase linker- A. rolfsii glucoamylase CBM 40gca acg cct gcg gac tgg cga tcg caa tcc att tat ttc ctt ctc acg 48Ala Thr Pro Ala Asp Trp Arg Ser Gln Ser Ile Tyr Phe Leu Leu Thr1 5 10 15gat cga ttt gca agg acg gat ggg tcg acg act gcg act tgt aat act 96Asp Arg Phe Ala Arg Thr Asp Gly Ser Thr Thr Ala Thr Cys Asn Thr 20 25 30gcg gat cag aaa tac tgt ggt gga aca tgg cag ggc atc atc gac aag 144Ala Asp Gln Lys Tyr Cys Gly Gly Thr Trp Gln Gly Ile Ile Asp Lys 35 40 45ttg gac tat atc cag gga atg ggc ttc aca gcc atc tgg atc acc ccc 192Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Thr Pro 50 55 60gtt aca gcc cag ctg ccc cag acc acc gca tat gga gat gcc tac cat 240Val Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly Asp Ala Tyr His65 70 75 80ggc tac tgg cag cag gat ata tac tct ctg aac gaa aac tac ggc act 288Gly Tyr Trp Gln Gln Asp Ile Tyr Ser Leu Asn Glu Asn Tyr Gly Thr 85 90 95gca gat gac ttg aag gcg ctc tct tcg gcc ctt cat gag agg ggg atg 336Ala Asp Asp Leu Lys Ala Leu Ser Ser Ala Leu His Glu Arg Gly Met 100 105 110tat ctt atg gtc gat gtg gtt gct aac cat atg ggc tat gat gga gcg 384Tyr Leu Met Val Asp Val Val Ala Asn His Met Gly Tyr Asp Gly Ala 115 120 125ggt agc tca gtc gat tac agt gtg ttt aaa ccg ttc agt tcc caa gac 432Gly Ser Ser Val Asp Tyr Ser Val Phe Lys Pro Phe Ser Ser Gln Asp 130 135 140tac ttc cac ccg ttc tgt ttc att caa aac tat gaa gat cag act cag 480Tyr Phe His Pro Phe Cys Phe Ile Gln Asn Tyr Glu Asp Gln Thr Gln145 150 155 160gtt gag gat tgc tgg cta gga gat aac act gtc tcc ttg cct gat ctc 528Val Glu Asp Cys Trp Leu Gly Asp Asn Thr Val Ser Leu Pro Asp Leu 165 170 175gat acc acc aag gat gtg gtc aag aat gaa tgg tac gac tgg gtg gga 576Asp Thr Thr Lys Asp Val Val Lys Asn Glu Trp Tyr Asp Trp Val Gly 180 185 190tca ttg gta tcg aac tac tcc att gac ggc ctc cgt atc gac aca gta 624Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly Leu Arg Ile Asp Thr Val 195 200 205aaa cac gtc cag aag gac ttc tgg ccc ggg tac aac aaa gcc gca ggc 672Lys His Val Gln Lys Asp Phe Trp Pro Gly Tyr Asn Lys Ala Ala Gly 210 215 220gtg tac tgt atc ggc gag gtg ctc gac ggt gat ccg gcc tac act tgt 720Val Tyr Cys Ile Gly Glu Val Leu Asp Gly Asp Pro Ala Tyr Thr Cys225 230 235 240ccc tac cag aac gtc atg gac ggc gta ctg aac tat ccc att tac tat 768Pro Tyr Gln Asn Val Met Asp Gly Val Leu Asn Tyr Pro Ile Tyr Tyr 245 250 255cca ctc ctc aac gcc ttc aag tca acc tcc ggc agc atg gac gac ctc 816Pro Leu Leu Asn Ala Phe Lys Ser Thr Ser Gly Ser Met Asp Asp Leu 260 265 270tac aac atg atc aac acc gtc aaa tcc gac tgt cca gac tca aca ctc 864Tyr Asn Met Ile Asn Thr Val Lys Ser Asp Cys Pro Asp Ser Thr Leu 275 280 285ctg ggc aca ttc gtc gag aac cac gac aac cca cgg ttc gct tct tac 912Leu Gly Thr Phe Val Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300acc aac gac ata gcc ctc gcc aag aac gtc gca gca ttc atc atc ctc 960Thr Asn Asp Ile Ala Leu Ala Lys Asn Val Ala Ala Phe Ile Ile Leu305 310 315 320aac gac gga atc ccc atc atc tac gcc ggc caa gaa cag cac tac gcc 1008Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly Gln Glu Gln His Tyr Ala 325 330 335ggc gga aac gac ccc gcg aac cgc gaa gca acc tgg ctc tcg ggc tac 1056Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala Thr Trp Leu Ser Gly Tyr 340 345 350ccg acc gac agc gag ctg tac aag tta att gcc tcc gcg aac gca atc 1104Pro Thr Asp Ser Glu Leu Tyr Lys Leu Ile Ala Ser Ala Asn Ala Ile 355 360 365cgg aac tat gcc att agc aaa gat aca gga ttc gtg acc tac aag aac 1152Arg Asn Tyr Ala Ile Ser Lys Asp Thr Gly Phe Val Thr Tyr Lys Asn 370 375 380tgg ccc atc tac aaa gac gac aca acg atc gcc atg cgc aag ggc aca 1200Trp Pro Ile Tyr Lys Asp Asp Thr Thr Ile Ala Met Arg Lys Gly Thr385 390 395 400gat ggg tcg cag atc gtg act atc ttg tcc aac aag ggt gct tcg ggt 1248Asp Gly Ser Gln Ile Val Thr Ile Leu Ser Asn Lys Gly Ala Ser Gly 405 410 415gat tcg tat acc ctc tcc ttg agt ggt gcg ggt tac aca gcc ggc cag 1296Asp Ser Tyr Thr Leu Ser Leu Ser Gly Ala Gly Tyr Thr Ala Gly Gln 420 425 430caa ttg acg gag gtc att ggc tgc acg acc gtg acg gtt ggt tcg gat 1344Gln Leu Thr Glu Val Ile Gly Cys Thr Thr Val Thr Val Gly Ser Asp 435 440 445gga aat gtg cct gtt cct atg gca ggt ggg cta cct agg gta ttg tat 1392Gly Asn Val Pro Val Pro Met Ala Gly Gly Leu Pro Arg Val Leu Tyr 450 455 460ccg act gag aag ttg gca ggt agc aag atc tgt agt agc tcg gga aga 1440Pro Thr Glu Lys Leu Ala Gly Ser Lys Ile Cys Ser Ser Ser Gly Arg465 470 475 480ggt gct aca agc ccg ggt ggc tcc tcg ggt agt gtc gag gtc act ttc 1488Gly Ala Thr Ser Pro Gly Gly Ser Ser Gly Ser Val Glu Val Thr Phe 485 490 495gac gtt tac gct acc aca gta tat ggc cag aac atc tat atc acc ggt 1536Asp Val Tyr Ala Thr Thr Val Tyr Gly Gln Asn Ile Tyr Ile Thr Gly 500 505 510gat gtg agt gag ctc ggc aac tgg aca ccc gcc aat ggt gtt gca ctc 1584Asp Val Ser Glu Leu Gly Asn Trp Thr Pro Ala Asn Gly Val Ala Leu 515 520 525tct tct gct aac tac ccc acc tgg agt gcc acg atc gct ctc ccc gct 1632Ser Ser Ala Asn Tyr Pro Thr Trp Ser Ala Thr Ile Ala Leu Pro Ala 530 535 540gac acg aca atc cag tac aag tat gtc aac att gac ggc agc acc gtc 1680Asp Thr Thr Ile Gln Tyr Lys Tyr Val Asn Ile Asp Gly Ser Thr Val545 550 555 560atc tgg gag gat gct atc agc aat cgc gag atc acg acg ccc gcc agc 1728Ile Trp Glu Asp Ala Ile Ser Asn Arg Glu Ile Thr Thr Pro Ala Ser 565 570 575ggc aca tac acc gaa aaa gac act tgg gat gaa tct tag 1767Gly Thr Tyr Thr Glu Lys Asp Thr Trp Asp Glu Ser 580 58541588PRTArtificialSynthetic Construct 41Ala Thr Pro Ala Asp Trp Arg Ser Gln Ser Ile Tyr Phe Leu Leu Thr1 5

10 15Asp Arg Phe Ala Arg Thr Asp Gly Ser Thr Thr Ala Thr Cys Asn Thr 20 25 30Ala Asp Gln Lys Tyr Cys Gly Gly Thr Trp Gln Gly Ile Ile Asp Lys 35 40 45Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Thr Pro 50 55 60Val Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly Asp Ala Tyr His65 70 75 80Gly Tyr Trp Gln Gln Asp Ile Tyr Ser Leu Asn Glu Asn Tyr Gly Thr 85 90 95Ala Asp Asp Leu Lys Ala Leu Ser Ser Ala Leu His Glu Arg Gly Met 100 105 110Tyr Leu Met Val Asp Val Val Ala Asn His Met Gly Tyr Asp Gly Ala 115 120 125Gly Ser Ser Val Asp Tyr Ser Val Phe Lys Pro Phe Ser Ser Gln Asp 130 135 140Tyr Phe His Pro Phe Cys Phe Ile Gln Asn Tyr Glu Asp Gln Thr Gln145 150 155 160Val Glu Asp Cys Trp Leu Gly Asp Asn Thr Val Ser Leu Pro Asp Leu 165 170 175Asp Thr Thr Lys Asp Val Val Lys Asn Glu Trp Tyr Asp Trp Val Gly 180 185 190Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly Leu Arg Ile Asp Thr Val 195 200 205Lys His Val Gln Lys Asp Phe Trp Pro Gly Tyr Asn Lys Ala Ala Gly 210 215 220Val Tyr Cys Ile Gly Glu Val Leu Asp Gly Asp Pro Ala Tyr Thr Cys225 230 235 240Pro Tyr Gln Asn Val Met Asp Gly Val Leu Asn Tyr Pro Ile Tyr Tyr 245 250 255Pro Leu Leu Asn Ala Phe Lys Ser Thr Ser Gly Ser Met Asp Asp Leu 260 265 270Tyr Asn Met Ile Asn Thr Val Lys Ser Asp Cys Pro Asp Ser Thr Leu 275 280 285Leu Gly Thr Phe Val Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300Thr Asn Asp Ile Ala Leu Ala Lys Asn Val Ala Ala Phe Ile Ile Leu305 310 315 320Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly Gln Glu Gln His Tyr Ala 325 330 335Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala Thr Trp Leu Ser Gly Tyr 340 345 350Pro Thr Asp Ser Glu Leu Tyr Lys Leu Ile Ala Ser Ala Asn Ala Ile 355 360 365Arg Asn Tyr Ala Ile Ser Lys Asp Thr Gly Phe Val Thr Tyr Lys Asn 370 375 380Trp Pro Ile Tyr Lys Asp Asp Thr Thr Ile Ala Met Arg Lys Gly Thr385 390 395 400Asp Gly Ser Gln Ile Val Thr Ile Leu Ser Asn Lys Gly Ala Ser Gly 405 410 415Asp Ser Tyr Thr Leu Ser Leu Ser Gly Ala Gly Tyr Thr Ala Gly Gln 420 425 430Gln Leu Thr Glu Val Ile Gly Cys Thr Thr Val Thr Val Gly Ser Asp 435 440 445Gly Asn Val Pro Val Pro Met Ala Gly Gly Leu Pro Arg Val Leu Tyr 450 455 460Pro Thr Glu Lys Leu Ala Gly Ser Lys Ile Cys Ser Ser Ser Gly Arg465 470 475 480Gly Ala Thr Ser Pro Gly Gly Ser Ser Gly Ser Val Glu Val Thr Phe 485 490 495Asp Val Tyr Ala Thr Thr Val Tyr Gly Gln Asn Ile Tyr Ile Thr Gly 500 505 510Asp Val Ser Glu Leu Gly Asn Trp Thr Pro Ala Asn Gly Val Ala Leu 515 520 525Ser Ser Ala Asn Tyr Pro Thr Trp Ser Ala Thr Ile Ala Leu Pro Ala 530 535 540Asp Thr Thr Ile Gln Tyr Lys Tyr Val Asn Ile Asp Gly Ser Thr Val545 550 555 560Ile Trp Glu Asp Ala Ile Ser Asn Arg Glu Ile Thr Thr Pro Ala Ser 565 570 575Gly Thr Tyr Thr Glu Lys Asp Thr Trp Asp Glu Ser 580 585

Claims

1. A process for producing a fermentation product from milled starch-containing material comprising: (a) saccharifying milled starch-containing material with a glucoamylase having an amino acid sequence shown in SEQ ID NO: 2, or a glucoamylase being at least 70% identical thereto, at a temperature below the initial gelatinization temperature of said starch-containing material, (b) fermenting using a fermenting organism.

2. The process of claim 1, wherein the process is carried out for a period of 1 to 250 hours.

3. The process of claim 1, wherein the process is carried out at a pH in the range between 3 and 7.

4. The process of claim 1, wherein the dry solid content (DS) lies in the range from 20-55 wt.-%.

5. The process of claim 1, wherein the sugar concentration is kept at a level below about 3 wt. % during saccharification and fermentation.

6. The process of claim 1, wherein a slurry comprising water and milled starch-containing material is prepared before step (a).

7. The process of claim 1, wherein the milled starch-containing material is prepared by milling starch-containing material to a particle size of 0.1-0.5 mm.

8. The process of claim 1, wherein the milled starch-containing material is granular starch obtained by dry or wet milling.

9. The process of claim 1, wherein the milled starch containing material is whole grains.

10. The process of claim 1, wherein the saccharification is carried out simultaneously.

11. The process of claim 10, wherein the temperature during fermentation is between 28.degree. C. and 36.degree. C.

12. The process of claim 1, wherein the glucoamylase is derived from Athelia rolfsii.

13. The process of claim 1, wherein the glucoamylase is present in an amount of 0.001 to 10 AGU/g DS

14. The process of claim 1, wherein an acid alpha-amylase is present.

15. The process of claim 14, wherein the acid alpha-amylase is a fungal alpha-amylase.

16. (canceled)

17. The process of claim 14, wherein the fungal acid alpha-amylase is a hybrid enzyme comprising an alpha-amylase catalytic domain (CD) and a carbohydrate-binding module (CBM) and optionally linker or a wild-type fungal acid alpha-amylase catalytic domain (CD) and a carbohydrate-binding module (CBM) and optionally a linker.

18. The process of claim 17, wherein the CBM is derived from Aspergillus kawachii alpha-amylase, Athelia rolfsii glucoamylase, or Aspergillus niger glucoamylase.

19. The process of claim 17, wherein the CBM is derived from Athelia rolfsii glucoamylase, A. niger glucoamylase or A. kawachii alpha-amylase.

20-33. (canceled)

24. The process of claim 14, wherein the acid alpha-amylase is derived from a strain of Aspergillus.