Trichoderma reesei host cells expressing glucoamylase from aspergillus fumigatus and methods of use thereof

Abstract

The present invention provides a fungal glucoamylase (AfGATR) from Aspergillus fumigatus expressed in a Trichoderma reesei host cell. The Trichoderma reesei host cell expresses AfGATR at a higher or at least comparable level than naturally expressed AfGA Aspergillus fumigatus. According to the present invention, AfGATR (including AfGA1TR and AfGA2TR) exhibits high activity at a high temperature and a low pH, and thus the AfGATR can be effectively used in a saccharification process in the presence of an alpha-amylase such as Aspergillus flavus alpha-amylase (AkAA). Aspergillus niger glucoamylase (AnGA) expressed in Aspergillus fumigatus or a natural AfGA catalyzed saccharification product, the AfGATR advantageously catalyzes the saccharification of starch to an oligosaccharide composition that is significantly enriched in DP1 (i.e., glucose), as compared to the Aspergillus niger glucoamylase (AnGA) or the natural AfGA catalyzed saccharification product. In some embodiments, AfGATRs, such as AfGA1TR, AfGA2TR, or variants thereof, can be used at lower doses than AnGA and naturally expressed AfGA to produce considerable levels of glucose.

Description

[0001] The present application is a divisional application of Chinese patent application No. 201380064450.0 (PCT / US2013 / 071154), filed on November 21, 2013, and entitled “Trichoderma reesei host cells expressing glucoamylase from Aspergillus fumigatus and methods of use thereof”.

[0002] Cross-references to related patent applications

[0003] This patent application claims priority to international patent application no. PCT / CN2012 / 086349 filed on December 11, 2012, which is incorporated herein by reference in its entirety.

[0004] Attached is a sequence listing comprising SEQ ID NOs: 1-14, which are incorporated herein by reference in their entirety. Technical Field

[0005] The present invention provides Trichoderma reesei host cells expressing a glucoamylase from Aspergillus fumigatus (AfGATR) or a variant thereof, and methods of using the same. Background Art

[0006] Starch consists of a mixture of amylose (15-30% w / w) and amylopectin (70-85% w / w). Amylose consists of a linear chain of α-1,4-linked glucose units with a molecular weight (MW) of about 60,000 to about 800,000. Amylopectin is a branched polymer containing α-1,6 branch points every 24-30 glucose units; its MW may be as high as 100,000,000.

[0007] Sugars derived from starch in the form of concentrated dextrose syrup are currently produced by an enzymatic process involving: (1) liquefaction (or reduction of viscosity) of solid starch into dextrins having an average degree of polymerization of about 7-10 with α-amylase, and (2) saccharification of the resulting liquefied starch (i.e., starch hydrolysate) with glucoamylase (also known as amyloglucosidase or GA). The resulting syrup has a high glucose content. Most commercially produced glucose syrups are then enzymatically isomerized to a dextrose / fructose mixture known as isosyrup. The resulting syrup can also be fermented with microorganisms (e.g., yeast) to produce commercial products, including, for example, ethanol, citric acid, lactic acid, succinic acid, itaconic acid, monosodium glutamate, gluconate, lysine, other organic acids, other amino acids, and other biochemicals. Fermentation and saccharification can be performed simultaneously (i.e., SSF process) to achieve greater economy and efficiency.

[0008] Glucoamylase (glucan 1,4-α-glucohydrolase, EC 3.2.1.3) is an exo-acting carbohydrase that hydrolyzes starch, catalyzing the removal of consecutive glucose units from the non-reducing end of starch or related oligosaccharide and polysaccharide molecules. Glucoamylase can hydrolyze both linear and branched glycosidic bonds of starch (e.g., amylose and amylopectin). On the other hand, α-amylase hydrolyzes starch, glycogen and related polysaccharides by randomly cleaving internal α-1,4-glycosidic bonds. Glucoamylase has been used for a variety of different purposes, including starch saccharification, brewing, baking, production of syrups for the food industry, production of raw materials for fermentation processes, and in animal feed to increase digestibility.

[0009] Glucoamylase is produced by many strains of bacteria, fungi and plants. For example, glucoamylase is produced by a strain of Aspergillus fumigatus. Luo et al., (2008), "Production of acid proof raw starch-digesting glucoamylase from a newly isolated strain of Aspergillus fumigatus MS-09", Sci. Tech. Food Indus., 29(5): 151-154; Sellars et al., (1976), "Degradation of barley by Aspergillus fumigatus Fres", Proc. Int. Biodegradation Symp., 3rd edition, SJ Miles et al., eds., Appl. Sci., Barking, UK, pp. 635-43; Domingues et al., (1993), "Production of amylase by soil fungi and partial Biochemical characterization of amylase of a selected strain (Aspergillus fumigatus Fresenius)", Can. J. Microbiol., 39(7): 681-85; Cherry et al. (2004), "Extracellular glucoamylase from the isolate Aspergillus fumigatus", Pakistan J. Biol. Sci., 7(11): 1988-92. However, Aspergillus fumigatus is highly allergenic and pathogenic to humans and plants. Therefore, Aspergillus fumigatus is not a viable production host for glucoamylase used in industrial processes for manufacturing products for human consumption. It is necessary to produce Aspergillus fumigatus glucoamylase from a suitable host. Summary of the invention

[0010] Glucoamylase (AfGATR) from Aspergillus fumigatus expressed in Trichoderma reesei catalyzes saccharification at high temperature and acidic pH for a long time. Examples of known glucoamylases (SEQ ID NO: 1 and 2) from Aspergillus fumigatus, encoding nucleic acids, and Trichoderma reesei host cells expressing the polynucleotides are provided. Trichoderma reesei host cells express AfGATR at a higher or at least comparable level compared to naturally expressed AfGA Aspergillus fumigatus. AfGATR (including AfGA1TR and AfGA2TR) exhibits high activity at high temperature and low pH, so AfGATR can be effectively used in saccharification processes in the presence of α-amylases such as Aspergillus kawachii α-amylase (AkAA). Compared to saccharification products catalyzed by Aspergillus niger glucoamylase (AnGA) or natural AfGA expressed in Aspergillus fumigatus, AfGATR advantageously catalyzes starch saccharification into oligosaccharide compositions significantly enriched in DP1 (i.e., glucose). AfGATR, such as AfGA1TR, AfGA2TR or variants thereof can be used at lower doses than AnGA and naturally expressed AfGA to generate comparable levels of glucose. AfGATR or variants thereof can be used in combination with enzymes derived from plants (e.g., cereals and grains). AfGATR or variants thereof can also be used in combination with enzymes secreted by host cells or endogenous to host cells. For example, AfGATR or variants thereof can be added to a saccharification reaction or SSF process during which one or more amylases, other glucoamylases, proteases, lipases, phytases, esterases, oxidoreductases, transferases or other enzymes are secreted by the production host. AfGATR or variants thereof can also work in combination with endogenous non-secreted production host enzymes. In another example, AfGATR or variants thereof can be secreted by the production host cell along with other enzymes during saccharification or SSF. AfGATR glucoamylase or variants thereof can be used in processes involving direct hydrolysis of starch into syrups and / or biochemicals (e.g., alcohols, organic acids, amino acids, other biochemicals, and biological materials) wherein the reaction temperature is below the gelatinization temperature of the substrate. AfGATR or variants thereof can be secreted by the Trichoderma reesei host cell along with other enzymes during saccharification or SSF.

[0011] Thus, a recombinant Trichoderma reesei host cell expressing AfGATR or a variant thereof having at least 80% sequence identity to SEQ ID NO: 12 or 13 is provided, wherein the Trichoderma reesei host cell expresses AfGATR or a variant thereof at a comparable level as compared to an Aspergillus fumigatus host cell expressing AfGA or a variant thereof having the same amino acid sequence as AfGATR or a variant thereof under the same conditions.

[0012] Also provided is a recombinant Trichoderma reesei host cell expressing AfGATR or a variant thereof, wherein the AfGATR or a variant thereof has at least 80% sequence identity with SEQ ID NO: 12 or 13, wherein the AfGATR or a variant thereof is more thermostable than AfGA or a variant thereof having the same amino acid sequence as the AfGATR or a variant thereof, and wherein the AfGA or a variant thereof is expressed in an Aspergillus fumigatus host cell.

[0013] Also provided is a method for producing recombinant AfGATR or a variant thereof, comprising: (a) providing a Trichoderma reesei host cell expressing a recombinant AfGATR or a variant thereof, wherein the AfGATR or a variant thereof has at least 80% sequence identity to SEQ ID NO: 12 or 13; (b) culturing the host cell under conditions that allow production of the recombinant AfGATR or a variant thereof; and (c) isolating the recombinant AfGATR or a variant thereof, wherein the AfGATR or a variant thereof is more thermostable than AfGA or a variant thereof having the same amino acid sequence as the AfGATR or a variant thereof, and wherein the AfGA or a variant thereof is expressed in an Aspergillus fumigatus host cell.

[0014] Also provided is a recombinant AfGATR or variant thereof produced by the disclosed host cell. The recombinant AfGATR or variant thereof may have at least 70% activity at pH 5.0 for 10 minutes at 74°C. The recombinant AfGATR or variant thereof may be AfGA1TR. The AfGA1TR may have at least 70% activity at pH 5.0 for 10 minutes in a temperature range of 55°C-74°C. The AfGA1TR may have an optimum temperature of about 68°C. The recombinant AfGATR or variant thereof may also be AfGA2TR. The AfGA2TR may have at least 70% activity at pH 5.0 for 10 minutes in a temperature range of 61°C-74°C. The AfGA2TR may have an optimum temperature of about 69°C. The recombinant AfGATR or variant thereof may comprise an amino acid sequence having at least 90%, 95% or 99% amino acid sequence identity to SEQ ID NO: 12. The recombinant AfGATR or variant thereof may comprise SEQ ID NO: 12. The recombinant AfGATR or variant thereof may also consist of an amino acid sequence having at least 90%, 95% or 99% amino acid sequence identity with SEQ ID NO: 13. The recombinant AfGATR or variant thereof may consist of SEQ ID NO: 12. The recombinant AfGATR or variant thereof may also comprise an amino acid sequence having at least 90%, 95% or 99% amino acid sequence identity with SEQ ID NO: 13. The recombinant AfGATR or variant thereof may comprise SEQ ID NO: 13. The recombinant AfGATR or variant thereof may also consist of an amino acid sequence having at least 90%, 95% or 99% amino acid sequence identity with SEQ ID NO: 13. The recombinant AfGATR or variant thereof may consist of SEQ ID NO: 13.

[0015] Also provided is a method of saccharifying a composition comprising starch to produce a composition comprising glucose, wherein the method comprises: (i) contacting a starch composition with an isolated AfGATR or variant thereof according to any one of claims 4-15; and (ii) saccharifying the starch composition to produce the glucose composition; wherein the AfGA1TR or variant thereof catalyzes the saccharification of the composition comprising starch into a composition comprising glucose. The composition comprising glucose may be enriched in DP1 compared to a second composition comprising DP1 prepared by AnGA under the same conditions. The composition comprising glucose may also be enriched in DP1 compared to a second composition comprising DP1 prepared by wild-type AfGA under the same conditions. The AfGATR or variant thereof may be AfGATR2, and the composition comprising glucose may be enriched in DP1 compared to a second composition comprising DP1 prepared by AfGA1TR under the same conditions. The dosage of the AfGA1TR or variant thereof may be about 40%-50% of the dosage of AnGA to produce the same DP1 yield under the same conditions.

[0016] In addition, it is provided that the composition comprising starch comprises liquefied starch, gelatinized starch or granular starch. Saccharification can be carried out in a temperature range of about 30°C to about 65°C. The temperature range can be 47°C-60°C. Saccharification can be carried out in a pH range of pH 2.0-pH6.0. The pH range can be pH 3.5-pH 5.5. The pH range can also be pH 4.0-pH 5.0.

[0017] In addition, the saccharification method may further include contacting the starch composition with an α-amylase. The α-amylase may be AkAA. The saccharification method may further include contacting the starch composition with a pullulanase.

[0018] In addition, the saccharification method may also include fermenting the glucose composition to produce a fermentation end (EOF) product. The fermentation may also be a simultaneous saccharification and fermentation (SSF) reaction. The fermentation may be carried out at pH 2-8 and at a temperature range of 25°C-70°C for 24-70 hours. The EOF product may contain ethanol. The EOF product may contain 8%-18% (v / v) ethanol. The method may also include contacting mash and / or wort with pullulanase, α-amylase and AfGA1TR or a variant thereof. The method may also include: (a) preparing mash; (b) filtering the mash to obtain wort, and (c) fermenting the wort to obtain a fermented beverage, wherein pullulanase, α-amylase and AfGA1TR or a variant thereof are added to: (i) the mash of step (a) and / or (ii) the wort of step (b) and / or (iii) the fermented wort of step (c). The EOF product may contain metabolites. The metabolite can be citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, sodium erythorbate, omega 3 fatty acids, butanol, amino acids, lysine, itaconic acid, 1,3-propanediol or isoprene.

[0019] In addition, the saccharification method may further include adding other glucoamylases, hexokinases, xylanases, glucose isomerases, xylose isomerases, phosphatases, phytases, proteases, pullulanases, β-amylases, other α-amylases, proteases, cellulases, hemicellulases, lipases, cutinases, trehalases, isoamylases, oxidoreductases, esterases, transferases, pectinases, α-glucosidases, β-glucosidases, lyases, hydrolases, or combinations thereof to the starch composition. The AfGATR or variant thereof may be added at a dose of 0.1 to 2 glucoamylase units (GAU) / g dry solids. The AfGATR or variant thereof may be added at a dose of about 49.5 μg protein / g solids. Pullulanase may also be added. The isolated AfGATR or variant thereof may be secreted by the Trichoderma reesei host cell. The host cell may also express and secrete α-amylase. The host cell may also express and secrete pullulanase.

[0020] In addition, the saccharification method may further include contacting the starch-containing composition with the host cell. The host cell is capable of fermenting the glucose composition.

[0021] Also contemplated is a composition comprising glucose produced by the disclosed saccharification method. Also contemplated is a liquefied starch produced by the disclosed saccharification method. Also contemplated is a fermented beverage produced by the disclosed saccharification method.

[0022] Also contemplated is the use of a composition comprising starch, comprising isolated AfGA1TR or a variant thereof. The composition may be a cultured cell material. The composition may further comprise a glucoamylase. The AfGA1TR or a variant thereof may be purified. The AfGA1TR or a variant thereof may be secreted by a host cell.

[0023] Also contemplated is the use of AfGA1TR or a variant thereof in the preparation of a composition comprising glucose. Also contemplated is the use of AfGA1TR or a variant thereof in the preparation of liquefied starch. Also contemplated is the use of AfGA1TR or a variant thereof in the preparation of a fermented beverage. Also contemplated is the disclosed saccharification method of a fermented beverage, or the disclosed use of a fermentation end product, wherein the fermented beverage or fermentation end product is selected from: i) beer, the beer selected from full malt beer, beer brewed according to the "Reinheitsgebot", ale, India pale ale (IPA), lager, bitter beer, low malt beer (Happoshu) (second beer), third beer, dry beer, thin beer, pale beer, low alcohol beer, low calorie beer, porter, bock, stout, malt liquor, non-alcoholic beer and non-alcoholic malt liquor; and ii) a cereal or malt beverage, the cereal or malt beverage selected from a fruit-flavored malt beverage, an alcohol-flavored malt beverage and a coffee-flavored malt beverage.

[0024] Also contemplated is a method of preparing a food composition comprising combining: (i) one or more food ingredients, and (ii) an isolated AfGA1TR or variant thereof according to claims 4-15, wherein the pullulanase and the isolated AfGA1TR or variant thereof catalyze the hydrolysis of starch components present in the food ingredients to produce glucose. The food composition may be selected from a food, a baking composition, a food additive, an animal food, a feed product, a feed additive, an oil, a meat, and a lard. The food ingredient may include a baking ingredient or additive. The one or more food ingredients may be selected from: flour; an anti-staling amylase; a phospholipase; a phospholipid; a malting alpha-amylase or a variant, homolog, or mutant thereof having malting alpha-amylase activity; a xylanase for baking (EC 3.2.1.8); and a lipase. The one or more food ingredients may be selected from: (i) a maltogenic alpha-amylase from Bacillus stearothermophilus; (ii) a xylanase for baked goods from Bacillus, Aspergillus, Thermomyces or Trichoderma; (iii) a glycolipidase from Fusarium heterosporum. The food composition may include dough or a dough product, preferably a processed dough product. The method may include baking the food composition to produce a baked product. The method may also include: (i) providing a starch medium; (ii) adding a pullulanase and AfGA1TR or a variant thereof to the starch medium; and (iii) heating the starch medium during or after step (b) to produce a baked product.

[0025] Also contemplated is a composition for preparing a food composition comprising AfGA1TR or a variant thereof. Also contemplated is the use of AfGA1TR or a variant thereof in preparing a food composition. The food composition may comprise dough or a dough product, preferably a processed dough product. The food composition may be a baked composition. Also contemplated is the use of AfGA1TR or a variant thereof in a dough product to delay or mitigate staling of the dough product, preferably to delay or mitigate harmful retrogradation of the dough product. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings are incorporated into and constitute a part of this specification and illustrate various methods and compositions disclosed herein. In the drawings:

[0027] Figure 1A to Figure 1BClustalW alignment of the AfGA1 catalytic core and carbohydrate binding domain (residues 27 to 476 and 524-631 of SEQ ID NO: 1, respectively) or full length with the corresponding residues from the following glucoamylases is shown: Aspergillus fumigatus A1163 (AfGA2) (residues 27-476 and 524-631 of SEQ ID NO: 2, respectively); Neosartorya fisheri NRRL 181 (residues 28-476 and 520-627 of SEQ ID NO: 3, respectively); Talaromyces stipitatus ATCC 10500 (residues 28-478 and 530-637 of SEQ ID NO: 4, respectively); Penicillium marneffei ATCC 18224 (residues 28-478 and 530-637 of SEQ ID NO: 5, respectively). NO:5, residues 31-481 and 534-641); and Aspergillus nidulans FGSC A4 (residues 55-493 and 544-661 of SEQ ID NO:6, respectively). The residues marked by asterisks in FIG1 are AfGA1 residues corresponding to the conserved residues in SEQ ID NO:1-6.

[0028] Figure 2 A map of the pJG222 expression vector pJG222 (Trex3gM-AfGA1) comprising a polynucleotide encoding an AfGA1 polypeptide is shown.

[0029] Figure 3 The dependence of glucoamylase activity (relative units) on pH is shown. The glucoamylases include (1) wild-type AfGA expressed in Aspergillus fumigatus, (2) AfGA1TR expressed in Trichoderma reesei, and (2) AnGA expressed in Aspergillus niger. Glucoamylase activity was determined by the release of glucose from soluble starch at 50°C.

[0030] Figure 4 The dependence of glucoamylase activity (relative units) on temperature is shown. The glucoamylases include (1) wild-type AfGA expressed in Aspergillus fumigatus, (2) AfGA1TR expressed in Trichoderma reesei, and (3) AnGA. ​​Glucoamylase activity was determined by releasing glucose from soluble starch at pH 5.0.

[0031] Figure 5 AB show AfGA1TR and AnGA glucoamylase activities measured by glucose release from 35% dry solids starch at pH 4.5 and 5.0.

[0032] Figure 6 AB shows the hydrolysis of 35% dry solids starch to DP1 and the reversal of DP1 to DP2 by a composition containing AfGATR1, pullulanase and AkAA.

[0033] Figure 7 AB shows the hydrolysis of 35% dry solids starch to DP1 and the reversal of DP1 to DP2 by a composition containing AfGATR1, pullulanase and variable doses of AkAA.

[0034] Figure 8 Shown is the amount of DP2 present in a high glucose composition containing 96% DP1 after the release of reducing sugars from 35% dry solids starch by a composition containing AfGA1TR or AnGA and in addition alpha-amylase (OPTIMAX L-100) and PU (GC636).

[0035] Fig. 9 A map of the pJG313 expression vector pJG313 (Trex3gM-AfGA2) comprising a polynucleotide encoding an AfGA2 polypeptide is shown.

[0036] Fig.10 Shown is the pH dependence of glucoamylase (relative units) of AfGA2TR expressed in T. reesei. Glucoamylase activity was determined by the release of glucose from a soluble starch substrate at 50°C.

[0037] Fig.11 Shown is the temperature dependence of glucoamylase (relative units) of AfGA2TR expressed in T. reesei. Glucoamylase activity was determined by the release of glucose from soluble starch substrates at pH 5.0.

[0038] Fig.12 The thermal stability of AfGA2TR in 50 mM sodium acetate buffer (pH 5.0) is shown. The enzyme was incubated in a thermocycler at the appropriate temperature for 2 hours and then added to the soluble starch substrate.

[0039] Fig.13 An SDS gel of AfGA1TR expressed in Trichoderma reesei is shown. Column M contains a protein molecular weight (MW) ladder in kDa. Columns 1-4 represent samples of AfGATR produced from Trichoderma reesei fermentations at 40.5 hours, 64.5 hours, 88.3 hours, and 112 hours of elapsed fermentation time, respectively. The 75 kDa band marked with an arrow is AfGA1TR. DETAILED DESCRIPTION

[0040] Provided are fungal glucoamylases (AfGA1TR or AfGA2TR) from Aspergillus fumigatus and variants thereof. AfGA1TR or variants thereof have an optimum pH of pH 5.0 and have at least 70% activity in the range of pH 3.5 to pH 7.5. When tested at pH 5.0, the enzyme has an optimum temperature of 68°C and has at least 70% activity in the temperature range of 55°C-74°C. AfGA2TR or variants thereof have an optimum pH of pH 5.3 and have at least 70% activity in the range of pH 3.3 to pH 7.3. When tested at pH 5.0, the enzyme has an optimum temperature of 69°C and has at least 70% activity in the temperature range of 61°C-74°C. These properties allow these enzymes to be used in combination with alpha amylases under the same reaction conditions. This eliminates the need to carry out saccharification reactions in a batch process, in which pH and temperature need to be adjusted in order to optimally use alpha amylases or glucoamylases.

[0041] Exemplary applications of glucoamylases such as AfGATR (including AfGA1TR and AfGA2TR) or variants thereof can be used in starch saccharification processes such as SSF; preparation of food compositions; preparation of cleaning compositions, such as detergent compositions for cleaning clothes, dishes and other surfaces; textile treatment (e.g., desizing). AfGATR advantageously catalyzes starch saccharification into an oligosaccharide composition significantly enriched in DP1 (i.e., glucose) compared to the saccharification product catalyzed by Aspergillus niger glucoamylase (AnGA). AfGATR can be secreted by the host cell along with other enzymes during fermentation or SSF. For example, AfGATR shows a greater saccharification rate than AnGA, producing more than 96% glucose within 24 hours. AfGATR can also be used at a lower dose than AnGA to generate comparable levels of DP1. Savings of at least 50% of the dose can be expected. During saccharification, AfGATR is also statistically significantly more thermostable than AnGA. ​​AfGATR can be used in combination with enzymes derived from plants (e.g., cereals and grains). AfGATR can also be used in combination with enzymes secreted by host cells such as T. reesei or endogenous to host cells such as T. reesei. For example, AfGATR can be added to a saccharification, fermentation or SSF process during which one or more amylases, glucoamylases, proteases, lipases, phytases, esterases, oxidoreductases, transferases or other enzymes are secreted by the production host. AfGATR can be combined with an auxiliary α-amylase to further improve the saccharification rate. For example, the addition of AkAA at 0.1 SSU / g dry solids can improve the saccharification rate. When combined with AkAA and pullulanase, it was found that AfGATR reduced DP3 by 0.1% compared to AnGA at the same glucose yield in a single pH process. AfGATR can also work in combination with endogenous non-secreted production host enzymes. In another example, AfGATR can be secreted by the production host cell with other enzymes during fermentation or SSF. AfGATR amylases can also effectively hydrolyze starch directly into syrups and / or biochemicals (e.g., alcohols, organic acids, amino acids, other biochemicals, and biological materials) where the reaction temperature is below the gelatinization temperature of the substrate.

[0042] 1. Definitions and Abbreviations

[0043] According to this specific embodiment, the following abbreviations and definitions apply. Note that the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes and reference to "a dosage" includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art. The following terms are provided below.

[0045] 1.1. Abbreviations and acronyms

[0046] Unless otherwise indicated, the following abbreviations / acronyms have the following meanings:

[0047] ABTS 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid

[0048] AcAmyl Aspergillus clavatus alpha-amylase

[0049] AE Alcohol Ethoxylate

[0050] AEO Alcohol Ethoxylate

[0051] AEOS Alcohol Ethoxysulfate

[0052] AES Alcohol Ethoxysulfate

[0053] AfGA Aspergillus fumigatus glucoamylase

[0054] AfGA1 Aspergillus fumigatus glucoamylase 1

[0055] AfGA2 Aspergillus fumigatus glucoamylase 2

[0056] AfGATR Aspergillus fumigatus glucoamylase expressed in Trichoderma reesei

[0057] AfGA1TR Aspergillus fumigatus glucoamylase 1 expressed in Trichoderma reesei

[0058] AfGA2TR Aspergillus fumigatus glucoamylase 2 expressed in Trichoderma reesei

[0059] AkAA Aspergillus kawachii α-amylase

[0060] AnGA Aspergillus niger glucoamylase

[0061] AOS α-olefin sulfonate

[0062] AS Alkyl Sulfate

[0063] cDNA complementary DNA

[0064] CMC Carboxymethyl Cellulose

[0065] DE Dextrose equivalent

[0066] DNA deoxyribonucleic acid

[0067] DPn degree of polymerization of polysaccharide with n subunits

[0068] ds or DS dry solids

[0069] DTMPA Diethylenetriaminepentaacetic acid

[0070] EC Enzyme Commission

[0071] EDTA Ethylenediaminetetraacetic acid

[0072] EO Ethylene oxide (polymer fragment)

[0073] EOF End of fermentation

[0074] FGSC Fungal Genetics Resource Center

[0075] GA Glucoamylase

[0076] GAU / g ds glucoamylase activity unit / g dry solids

[0077] HFCS High Fructose Corn Syrup

[0078] HgGA Humicola grisea glucoamylase

[0079] HS High Sugar

[0080] IPTG Isopropyl β-D-thiogalactopyranoside

[0081] IRS Insoluble residual starch

[0082] kDa kilodaltons

[0083] LAS Linear Alkyl Benzene Sulfonate

[0084] MW Molecular weight

[0085] MWU Modified Wohlgemuth Unit; 1.6x10 -5mg / MWU = Activity Unit

[0086] NCBI National Center for Biotechnology Information

[0087] NOBS Nonanoyloxybenzenesulfonate

[0088] NTA Nitriloacetic acid

[0089] OxAm Purastar HPAM 5000L (Danisco US Inc.)

[0090] PAHBAH Para-hydroxybenzoic acid hydrazide

[0091] PEG Polyethylene glycol

[0092] pI isoelectric point

[0093] ppm parts per million

[0094] PVA Poly(vinyl alcohol)

[0095] PVP Poly(vinyl pyrrolidone)

[0096] RNA Ribonucleic acid

[0097] SAS Alkane Sulfonate

[0098] SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

[0099] SSF simultaneous saccharification and fermentation

[0100] SSU / g solid soluble starch unit / g dry solids

[0101] sp. species

[0102] TAED Tetraacetylethylenediamine

[0103] TrGA Trichoderma reesei glucoamylase

[0104] w / v weight / volume

[0105] w / w weight / weight

[0106] v / v volume / volume

[0107] wt% weight%

[0108] ℃ Celsius

[0109] H 2 O Water

[0110] dH 2 O or DI deionized water

[0111] HkDJ 2 Milli-Q filtered deionized water

[0112] g or gm gram

[0113] μg microgram

[0114] mg milligram

[0115] kg kilogram

[0116] μL and μl microliter

[0117] mL and ml

[0118] mm Millimeters

[0119] μm micrometer

[0120] M Moore

[0121] mM millimole

[0122] μM micromolar

[0123] U Unit

[0124] sec seconds

[0125] min

[0126] hr hours

[0127] DO Dissolved oxygen

[0128] Ncm Newton centimeter

[0129] ETOH Ethanol

[0130] eq. equivalent

[0131] N equivalent concentration

[0132] 1.2. Definitions

[0133] The term "amylase" or "amylolytic enzyme" refers to an enzyme that is capable of, among other things, catalyzing the degradation of starch. α-Amylases are hydrolases that cleave α-D-(1→4) O-glycosidic bonds in starch. In general, α-amylases (EC 3.2.1.1; α-D-(1→4)-glucan glucanohydrolase) are described as endo-acting enzymes that cleave α-D-(1→4) O-glycosidic bonds within starch molecules in a random manner to generate polysaccharides containing three or more (1-4)-α-linked D-glucose units. In contrast, exo-amylases such as β-amylases (EC 3.2.1.2; α-D-(1→4)-glucan maltohydrolase) and some product-specific amylases such as maltogenic α-amylase (EC 3.2.1.133) cleave polysaccharide molecules from the non-reducing end of the substrate. β-Amylases, α-glucosidases (EC 3.2.1.20; α-D-glucoside glucohydrolase), glucoamylases (EC 3.2.1.3; α-D-(1→4)-glucan glucohydrolase) and product-specific amylases such as maltotetraosidase (EC 3.2.1.60) and maltohexaosidase (EC 3.2.1.98) can produce maltooligosaccharides of specific length or enriched syrups of specific maltooligosaccharides.

[0134] As used herein, the term "glucoamylase" (EC 3.2.1.3) (also known as glucan 1,4-α-glucosidase; glucoamylase; amyloglucosidase; γ-amylase; lysosomal α-glucosidase; acid maltase; exo-1,4-α-glucosidase; glucoamylase; γ-1,4-glucan glucohydrolase; acid maltase; 1,4-α-D-glucan glucohydrolase; or 4-α-D-glucan glucohydrolase) refers to a class of enzymes that catalyze the release of D-glucose from the non-reducing ends of starch and related oligosaccharides and polysaccharides. These enzymes are exo-acting enzymes that release glucosyl residues from the non-reducing ends of amylose and amylopectin molecules. The enzyme also hydrolyzes α-1,6 and α-1,3-bonds, but at a much slower rate than the hydrolysis of α-1,4-bonds. The term "hydrolysis of starch" refers to the cleavage of glycosidic bonds by the addition of water molecules.

[0135] The term "pullulanase" (EC 3.2.1.41, pullulan 6-glucanohydrolase) refers to a class of enzymes that are capable of hydrolyzing the α1-6 glucosidic bonds in the pullulan molecule.

[0136] "Enzyme unit" herein refers to the amount of product formed per time period under specified assay conditions. For example, a "glucoamylase activity unit" (GAU) is defined as the amount of enzyme that produces 1 g of glucose per hour from a soluble starch substrate (4% DS) at 60°C and pH 4.2. A "soluble starch unit" (SSU) is the amount of enzyme that produces 1 mg of glucose per minute from a soluble starch substrate (4% DS) at pH 4.5 and 50°C. DS refers to "dry solids".

[0137] As used herein, "dry solids" content refers to the total solids of the slurry as a percentage of dry weight. The term "slurry" refers to an aqueous mixture containing insoluble solids. The term "high ds" refers to an aqueous starch slurry containing greater than 38% dry solids.

[0138] The term "Brix" refers to the well-known hydrometer scale for measuring the sugar content of a solution at a given temperature. The Brix scale measures the grams of sucrose present per 100 grams of a sugar-water solution (total dissolved solids content). Brix measurements are often made using a hydrometer or refractometer.

[0139] The term "degree of polymerization" (DP) refers to the number (n) of anhydrous pyranose glucopyranose units in a given saccharide. Examples of DP1 are monosaccharides such as glucose and fructose. Examples of DP2 are disaccharides such as maltose and sucrose. HS or DP4+ (>DP3) represents polymers with a degree of polymerization greater than 3. The term "DE" or "dextrose equivalent" is defined as the percentage of reducing sugars, i.e., D-glucose, as a fraction of the total carbohydrates in a syrup. It is an industry standard for the concentration of total reducing sugars and is expressed as % D-glucose by dry weight. Non-hydrolyzed granular starch has a DE of almost 0, while the DE of D-glucose is 100.

[0140] As used herein, the term "starch" refers to any material composed of complex polysaccharide carbohydrates of plants having the formula (C 6 H 10 O 5 ) x (wherein X can be any number) of amylose and amylopectin. The term includes plant-based materials such as grains, grasses, tubers and roots, and more particularly materials obtained from wheat, barley, corn, rye, rice, sorghum, bran, cassava, millet, potato, sweet potato and tapioca. The term "starch" includes granular starch. The term "granular starch" refers to raw, i.e. uncooked, starch, e.g., starch that has not been subjected to gelatinization.

[0141] The term "glucose syrup" refers to an aqueous composition containing glucose solids. Glucose syrup will have a DE of at least 20. In some embodiments, glucose syrup will contain no more than 21% water and no less than 25% reducing sugars calculated as dextrose. Glucose syrup will include at least 90% D-glucose, or at least 95% D-glucose. In some embodiments, the terms glucose and glucose syrup are used interchangeably.

[0142] The term "total sugar content" refers to the total sugar content present in the starch composition.

[0143] The term "refractive index dry matter" (RIDS) is defined as measuring the refractive index of a starch solution at a known DE at controlled temperature and then converting the RI to dry matter using an appropriate relationship, such as the Critical Data Tables of the Corn Refiners Association.

[0144] The term "contacting" means placing the corresponding enzyme in sufficient proximity to the corresponding substrate so that the enzyme can convert the substrate into the final product. Those skilled in the art will recognize that mixing the enzyme solution with the corresponding substrate can achieve contacting.

[0145] The term "wild type", "parent" or "reference" about a polypeptide refers to a naturally occurring polypeptide that does not include artificially made displacements, insertions or deletions at one or more amino acid positions. Similarly, the term "wild type", "parent" or "reference" about a polynucleotide refers to a naturally occurring polynucleotide that does not include artificially made nucleoside changes. However, it should be noted that the polynucleotides encoding wild-type polypeptides, parent polypeptides or reference polypeptides are not limited to naturally occurring polynucleotides, but encompass any polynucleotides encoding wild-type polypeptides, parent polypeptides or reference polypeptides. In addition, as used herein and as will be apparent from the context, it should be understood that referring to a particular sequence as "wild type" does not mean that other sequences not appended with the prefix "wild type" in the example are not wild type.

[0146] As used herein, unless the context clearly indicates otherwise, the term "comparable" with respect to expression levels means that the variation between samples of interest does not exceed 20%.

[0147] Reference to wild-type protein should be understood to include the mature form of the protein. A "mature" polypeptide means a polypeptide or variant thereof lacking a signal sequence. For example, the signal sequence may be removed during expression of the polypeptide. The length of mature AfGA1 or AfGA2 is 612 amino acids, covering residues 1 to 612 of SEQ ID NO:1 and SEQ ID NO:2, respectively, wherein the positions are counted from the N-terminus. The signal sequence of wild-type AfGA1 or AfGA2 is 19 amino acids in length and has the sequence shown in SEQ ID NO:11. Mature AfGA1, AfGA2 or variants thereof may contain signal sequences taken from different proteins. The mature protein may be a fusion protein between a mature polypeptide and a signal sequence polypeptide.

[0148] The putative "catalytic core" of AfGA1, AfGA2 or variants thereof spans residues 41 to 453 of SEQ ID NO: 1. Amino acid residues 534-630 constitute the putative "carbohydrate binding domain" of AfGA1, AfGA2 or variants thereof. The "linker" or "linker region" of AfGA1, AfGA2 or variants thereof spans the region between the "catalytic core" and the "carbohydrate binding domain".

[0149] The term "variant" with respect to a polypeptide refers to a polypeptide that is different from a specified wild-type polypeptide, a parent polypeptide, or a reference polypeptide because it includes one or more naturally occurring or artificially made amino acid substitutions, insertions, or deletions. Similarly, the term "variant" with respect to a polynucleotide refers to a polynucleotide whose nucleotide sequence is different from a specified wild-type polynucleotide, a parent polynucleotide, or a reference polynucleotide. The identity of the wild-type, parent, or reference polypeptide or polynucleotide will be apparent from the context.

[0150] With respect to enzymes of the invention, such as glucoamylases, "activity" refers to enzyme activity, which can be measured as described herein.

[0151] The term "recombinant" when applied to a subject cell, nucleic acid, protein or vector means that the subject has been modified from its native state. Thus, for example, a recombinant cell expresses genes that are not present in the native (non-recombinant) form of the cell, or expresses native genes at levels or conditions different from those found in nature. A recombinant nucleic acid differs from a native sequence by one or more nucleotides and / or is operably linked to a heterologous sequence, such as a heterologous promoter in an expression vector. A recombinant protein may differ from a native sequence by one or more amino acids and / or be fused to a heterologous sequence. A vector comprising a nucleic acid encoding AfGA1, AfGA2 or a variant thereof is a recombinant vector.

[0152] The terms "recovered", "isolated" and "separated" refer to a compound, protein (polypeptide), cell, nucleic acid, amino acid or other specified substance or component that is removed from at least one other substance or component with which it is naturally associated as it exists in nature, such as AfGATR isolated from a recombinant host cell. "Isolated" AfGATR or variants thereof include, but are not limited to, a culture broth containing secreted AfGATR expressed in a heterologous host cell (i.e., a host cell other than Aspergillus fumigatus).

[0153] As used herein, the term "purified" refers to a material (e.g., an isolated polypeptide or polynucleotide) that is in a relatively pure state, such as at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.

[0154] The terms "thermostable" and "thermostability" with respect to enzymes refer to the ability of an enzyme to maintain activity after exposure to elevated temperatures. The thermostability of an enzyme (e.g., amylase) can be measured by its T m Measurement during which the enzyme activity is lost by half under defined conditions. m It can be calculated by measuring the residual glucoamylase activity after exposure to (ie, subjecting to) elevated temperature.

[0155] "pH range" with respect to an enzyme refers to the range of pH values ​​over which the enzyme exhibits catalytic activity.

[0156] As used herein, the terms "pH stable" and "pH stability" with respect to an enzyme relate to the ability of the enzyme to remain active over a wide range of pH values ​​over a predetermined period of time (eg, 15 minutes, 30 minutes, and 1 hour).

[0157] As used herein, the term "amino acid sequence" is synonymous with the terms "polypeptide", "protein" and "peptide", and is used interchangeably. When these amino acid sequences exhibit activity, they may be referred to as "enzymes". Conventional single-letter or three-letter codes for amino acid residues are used, with amino acid sequences given in the standard amino to carboxyl terminal orientation (i.e., N→C).

[0158] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding polypeptides. Nucleic acids may be single-stranded or double-stranded, and may be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Due to the degeneracy of the genetic code, more than one codon may be used to encode a specific amino acid, and the compositions and methods of the present invention encompass nucleotide sequences encoding a specific amino acid sequence. Unless otherwise specified, nucleic acid sequences are given in a 5' to 3' orientation.

[0159] As used herein, "hybridization" refers to the process in which one strand of nucleic acid forms a duplex with a complementary strand, i.e., base pairs with the complementary strand, during the blot hybridization technique and the PCR technique. Stringent hybridization conditions are exemplified by hybridization under the following conditions: 65°C and 0.1X SSC (where 1X SSC = 0.15M NaCl, 0.015M trisodium citrate, pH 7.0). The hybridized double-stranded nucleic acid is determined by the melting temperature (T m ) characterizes that half of the hybridized nucleic acids are not paired with the complementary strand at the melting temperature. Mismatched nucleotides within the duplex reduce T m The nucleic acid encoding the variant glucoamylase may have a T value lowered by 1°C to 3°C or more than that formed by the duplex between the nucleotides of SEQ ID NO: 8 and its identical complementary chain. m .

[0160] As used herein, a "synthetic" molecule is produced by in vitro chemical or enzymatic synthesis rather than by an organism.

[0161] As used herein, the terms "transformed," "stably transformed," and "transgenic" used with respect to cells refer to cells that contain a non-native (e.g., heterologous) nucleic acid sequence integrated into their genome or carried as an episome that is maintained through generations.

[0162] The term "introducing" in the context of inserting a nucleic acid sequence into a cell means "transfection," "transformation," or "transduction" as known in the art.

[0163] A "host strain" or "host cell" is an organism into which an expression vector, phage, virus or other DNA construct (including a polynucleotide encoding a polypeptide of interest (e.g., AfGATR or a variant thereof)) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi and yeast, such as Trichoderma reesei) that are capable of expressing the polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced by a cell.

[0164] The term "heterologous" with respect to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in the host cell.

[0165] The term "endogenous" with respect to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.

[0166] As used herein, the term "expression" refers to the process of producing a polypeptide based on a nucleic acid sequence. The process includes both transcription and translation.

[0167] "Selectable marker" or "selectable marker" refers to a gene that can be expressed in a host to facilitate selection of host cells carrying the gene. Examples of selectable markers include, but are not limited to, antimicrobial agents (e.g., hygromycin, bleomycin, or chloramphenicol) and / or genes that confer metabolic benefits (e.g., nutritional benefits) to the host cell.

[0168] "Vector" refers to a polynucleotide sequence designed to introduce a nucleic acid into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, expression cassettes, and the like.

[0169] "Expression vector" refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, the coding sequence being operably linked to a suitable control sequence capable of affecting expression of the DNA in a suitable host. Such control sequences may include a promoter that affects transcription, an optional operator sequence that controls transcription, a sequence encoding a suitable ribosome binding site on mRNA, an enhancer, and sequences that control transcription and translation termination.

[0170] The term "operably linked" means that the specified components are in a relationship (including but not limited to juxtaposition) that allows them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence so that the expression of the coding sequence is controlled by the regulatory sequence.

[0171] A "signal sequence" is a sequence of amino acids attached to the N-terminal portion of a protein that promotes secretion of the protein outside the cell. The mature form of the extracellular protein does not have a signal sequence, which is cleaved off during the secretion process.

[0172] As used herein, "biologically active" refers to a sequence that has a particular biological activity (eg, enzymatic activity).

[0173] As used herein, a "swatch" is a piece of material, such as fabric, onto which a stain is applied. The material may be, for example, a fabric made of cotton, polyester, or a mixture of natural and synthetic fibers. The swatch may also be paper, such as filter paper or nitrocellulose, or a piece of hard material, such as ceramic, metal, or glass. For amylases, the stain is starch-based, but may also include blood, milk, ink, grass, tea, wine, spinach, gravy, chocolate, eggs, cheese, clay, pigments, oils, or mixtures of these compounds.

[0174] As used herein, "small swatches" are portions cut from a swatch with a single-hole punch, or portions cut from a custom 96-hole punch (wherein the multi-hole punch pattern matches a standard 96-hole microtiter plate), or portions otherwise removed from a swatch. The swatch can be a textile, paper, metal, or other suitable material. The small swatch can have a fixed stain before or after it is placed in a 24-hole, 48-hole, or 96-hole microtiter plate well. "Small swatches" can also be made by applying stains to small pieces of material. For example, a small swatch can be a fabric with a diameter of 5 / 8 inch or 0.25 inch on which stains are applied. The custom punch is designed to deliver 96 swatches to all holes of a 96-hole plate simultaneously. The device can allow more than one swatch to be delivered to each hole by simply loading the same 96-hole plate multiple times. It is contemplated that the multi-hole punch device can be used to deliver multiple samples simultaneously to any format of plate (including but not limited to 24-well, 48-well and 96-well plates). In another contemplated method, the dirty test platform can be a stain-coated bead made of metal, plastic, glass, ceramic or another suitable material. One or more coated beads are then placed in the wells of a 96-well, 48-well or 24-well plate or larger format containing a suitable buffer and enzyme.

[0175] As used herein, "cultured cell material comprising AfGATR or a variant thereof" or similar terms refers to a cell lysate or supernatant (including culture medium) containing AfGATR or a variant thereof as a component. The cell material may be from a heterologous host grown in culture for the purpose of preparing AfGATR or a variant thereof.

[0176] "Percentage of sequence identity" means that when the variant is aligned using the CLUSTAL W algorithm with default parameters, it has at least a certain percentage of amino acid residues that are identical to the wild-type AfGA1 or AfGA2. See Thompson et al., (1994) Nucleic Acids Res., 22:4673-4680. The default parameters of the CLUSTAL W algorithm are:

[0177]

[0178]

[0179] Deletions are counted as non-identical residues compared to the reference sequence. Deletions occurring at either end are included. For example, a variant having six amino acid deletions at the C-terminus of the mature AfGA1 polypeptide of SEQ ID NO: 12 would have a percent sequence identity of 99% relative to the mature polypeptide (606 / 612 identical residues x 100, rounded to the nearest integer). Such a variant would be encompassed by a variant having "at least 99% sequence identity" to the mature AfGA1 polypeptide.

[0180] "Fused" polypeptide sequences are joined, ie, operably linked, via a peptide bond between the two polypeptide sequences.

[0181] The term "filamentous fungi" refers to all filamentous forms of the subdivision Eumycotina.

[0182] The phrase "simultaneous saccharification and fermentation (SSF)" refers to a process in the production of biochemicals in which a microbial organism, such as an ethanologenic microorganism, and at least one enzyme, such as AfGA or a variant thereof, are present during the same process step. SSF involves the simultaneous hydrolysis of a starch substrate (granular, liquefied, or solubilized) into sugars (including glucose) and fermentation of the sugars into alcohol or other biochemicals or biological materials in the same reactor vessel.

[0183] As used herein, "ethanologenic microorganism" refers to a microorganism that is capable of converting sugars or oligosaccharides into ethanol.

[0184] The term "fermented beverage" refers to any beverage prepared by a process involving a fermentation process such as microbial fermentation, for example bacterial and / or yeast fermentation.

[0185] "Beer" is an example of such a fermented beverage, and the term "beer" is intended to include any fermented wort produced by fermentation / brewing of starch-containing plant materials. Typically, beer is prepared specifically by malt or auxiliary materials or any combination of malt and auxiliary materials. The example of beer includes: full malt beer, beer brewed under the "purity method", ale, India pale ale (IPA), lager, bitter beer, low malt beer (second beer), third beer, dry beer, thin beer, light beer, low alcohol beer, low calorie beer, porter beer, bock beer, stout beer, malt liquor, non-alcoholic beer, non-alcoholic malt liquor, etc., but there are also alternative forms of grain and malt beverages, such as fruit-flavored malt beverages, such as citrus flavors such as lemon, sweet orange, lime or berry flavored malt beverages; alcohol-flavored malt beverages, such as vodka, rum or tequila-flavored malt liquors; or coffee-flavored malt beverages, such as caffeine-flavored malt liquors, etc.

[0186] The term "malt" refers to any malted grain, such as malted barley or wheat.

[0187] The term "adjunct material" refers to any starch and / or sugar-containing plant material other than malt, such as barley or wheat malt. Examples of adjunct materials include coarse ground corn meal, refined coarse ground corn meal, brewery mill yeast, rice, sorghum, refined corn starch, barley, barley starch, hulled barley, wheat, wheat starch, baked cereals, cereal flakes, rye, oats, potatoes, tapioca flour, cassava, and syrups, such as corn syrup, cane syrup, invert syrup, barley and / or wheat syrup, etc.

[0188] The term "mash" refers to an aqueous slurry of any plant material containing starch and / or sugars, such as grist (e.g., including crushed malt, crushed barley) and / or other adjunct materials, or combinations thereof, which is subsequently mixed with water to separate into wort and spent grains.

[0189] The term "wort" refers to the unfermented liquid outflow after the milling grains are extracted during the mashing process.

[0190] "Iodine-positive starch" or "IPS" refers to (1) unhydrolyzed amylose or (2) retrograded starch polymers after liquefaction and saccharification. When saccharified starch or sugar solution is tested with iodine, high DPn amylose or retrograded starch polymers will bind iodine and produce a characteristic blue color. The sugar solution is thus called "iodine-positive sugar", "blue sugar" or "blue sugar".

[0191] The terms "retrograded starch" or "starch retrogradation" refer to the changes that occur spontaneously in starch pastes or gels as they age.

[0192] The term "about" refers to a reference value ± 15%.

[0193] 2. Aspergillus fumigatus glucoamylase (AfGA1 and AfGA2)

[0194] Provided is an isolated and / or purified AfGA1 or variant thereof from Aspergillus fumigatus species having glucoamylase activity. Glucoamylase consists of three different domains, including a catalytic domain, followed by a linker region, which in turn is connected to a starch binding domain. The AfGA1 polypeptide can be the mature AfGA1 polypeptide shown in SEQ ID NO:12. The polypeptide can be fused to additional amino acid sequences at the N-terminal and / or C-terminal ends. Additional N-terminal sequences can be signal peptides, which can have, for example, the sequence shown in SEQ ID NO:11. Other amino acid sequences fused at either end include fusion partner polypeptides that can be used for marking or purifying proteins.

[0195] For example, the AfGA1 precursor includes the following sequence (SEQ ID NO: 1):

[0196]

[0197] Provided is an AfGA2 or variant thereof separated and / or purified from Aspergillus fumigatus species having glucoamylase activity. Glucoamylase consists of three different domains, including a catalytic domain, followed by a linker region, which is in turn connected to a starch binding domain. The AfGA2 polypeptide can be the mature AfGA2 polypeptide shown in SEQ ID NO:13. The polypeptide can be fused to additional amino acid sequences at the N-terminal and / or C-terminal ends. Additional N-terminal sequences can be signal peptides, which can have, for example, the sequence shown in SEQ ID NO:11. Other amino acid sequences fused at either end include fusion protein polypeptides that can be used for marking or purifying proteins.

[0198] For example, the AfGA2 precursor includes the following sequence (SEQ ID NO: 2):

[0199]

[0200] For AfGA1 and AfGA2, the amino acids in bold above constitute the C-terminal carbohydrate binding (CBM) domain (SEQ ID NO: 7). The glycosylation linker region connects the N-terminal catalytic core to the CBM domain. The CBM domains in AfGA1 and AfGA2 are conserved, with the CBM20 domain being present in a large number of starch-degrading enzymes, including α-amylases, β-amylases, glucoamylases, and cyclodextrin glucanotransferases. CBM20 folds into an antiparallel β-barrel structure with two starch-binding sites 1 and 2. The two sites are thought to be functionally different: site 1 may serve as an initial starch recognition site, while site 2 may be involved in specific recognition of the appropriate region of starch. See Sorimachi et al., (1997), "Solution structure of the granular starch binding domain of Aspergillus niger glucoamylase bound to beta-cyclodextrin", Structure, 5(5):647-61.

[0201] The conserved residues in starch binding sites 1 and 2 in the AfGA1 and AfGA2 CBM domains are indicated by numbers 1 and 2, respectively, in the following sequences:

[0202]

[0203] The variant AfGA1 or AfGA2 may comprise some or zero amino acid residues of the CBM domain of SEQ ID NO: 7. Alternatively, the variant may comprise a CBM domain having at least 80%, 85%, 90%, 95% or 98% sequence identity to the CBM domain of SEQ ID NO: 7. The variant may comprise a heterologous or engineered CBM20 domain.

[0204] AfGA or a variant thereof can be expressed in a eukaryotic host cell, such as a filamentous fungal cell, which allows, for example, proper glycosylation of the linker sequence.

[0205] A representative polynucleotide encoding AfGA1 is the polynucleotide sequence shown in SEQ ID NO: 8. A representative polynucleotide encoding AfGA2 is the polynucleotide sequence shown in SEQ ID NO: 14. (NCBI reference sequence NC_007195, Aspergillus fumigatus genome.) The polypeptide sequence MPRLSYALCALSLGHAAIA (SEQ ID NO: 11) shown in italics above in the AfGA1 and AfGA2 precursor sequences is an N-terminal signal peptide that is cleaved when the protein is expressed in an appropriate host cell.

[0206] The polypeptide sequence of AfGA1 is similar to other fungal glucoamylases, including AfGA2. For example, AfGA1 has a high degree of sequence identity with the following fungal glucoamylases:

[0207] 99% sequence identity to glycosyl hydrolase (AfGA2) from Aspergillus fumigatus A1163 (SEQ ID NO: 2);

[0208] has 92% sequence identity to a glycosyl hydrolase from Neosartorya fischeri NRRL 181 (SEQ ID NO: 3); and

[0209] 82% sequence identity to a putative glucoamylase from Talaromyces stylosus ATCC 10500 (SEQ ID NO: 4);

[0210] 81% sequence identity to a putative glucoamylase from Penicillium marneffei ATCC 18224 (SEQ ID NO: 5);

[0211] 81% sequence identity to a putative glucoamylase from Aspergillus nidulans FGSC A4 (SEQ ID NO: 6);

[0212] Sequence identity is determined by BLAST alignment using the precursor form of AfGA1 of SEQ ID NO: 1 as the query sequence. See Altschul et al., (1990), J. Mol. Biol. 215: 403-410. Sequence identity may also optionally be based on the mature form of the enzyme.

[0213] Variants of AfGA1 polypeptides are provided. The variants may consist of or include a polypeptide having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity with a polypeptide of residues 1-631 of SEQ ID NO:1, wherein the variant comprises one or more amino acid modifications selected from the group consisting of substitutions, insertions or deletions of one or more corresponding amino acids in SEQ ID NOs:2-6. Variants of AfGA2 polypeptides are provided. The variants may consist of or include a polypeptide having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity with a polypeptide of residues 1-631 of SEQ ID NO:2, wherein the variant comprises one or more amino acid modifications selected from the group consisting of substitutions, insertions or deletions of one or more corresponding amino acids in SEQ ID NOs:1 and / or 3-6. For example, a variant consisting of a polypeptide having at least 99% sequence identity with a polypeptide having residues 1 to 612 of SEQ ID NO: 1 may have one to six amino acid substitutions, insertions or deletions compared to AfGA1 of SEQ ID NO: 1. Insertions or deletions may occur, for example, at either end of the polypeptide. Alternatively, a variant may "comprise" a polypeptide consisting of a polypeptide having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity with a polypeptide having residues 1 to 631 of SEQ ID NO: 1 or 2. In a variant, additional amino acid residues may be fused to either end of the polypeptide. A variant may be glycosylated, regardless of whether the variant "comprises" or "consists of" a given amino acid sequence.

[0214] A ClustalW alignment between AfGA1 (SEQ ID NO: 1); AfGA2 (SEQ ID NO: 2); glucoamylase from Neosartorya fischeri NRRL 181 (SEQ ID NO: 3); glucoamylase from Talaromyces stalk ATCC 10500 (SEQ ID NO: 4); glucoamylase from Penicillium marneffei ATCC 18224 (SEQ ID NO: 5); glucoamylase from Aspergillus nidulans FGSC A4 (SEQ ID NO: 6) is shown in Figure 1. See Thompson et al., (1994) Nucleic Acids Res., 22: 4673-4680. In general, the degree of conservation of amino acids in an alignment of related protein sequences is proportional to the importance of the amino acid position relative to the function of the protein. That is, amino acids that are common in all related sequences may play an important functional role and cannot be easily replaced. Likewise, positions that vary between sequences are likely to be substituted with other amino acids or otherwise modified while maintaining protein activity.

[0215] The alignment shown in FIG. 1 can, for example, guide the construction of variant AfGA polypeptides having glucoamylase activity. Variants of the AfGA1 polypeptide of SEQ ID NO: 1 can include, but are not limited to, those having amino acid modifications selected from the group consisting of substitutions, insertions or deletions of corresponding amino acids in polypeptides selected from SEQ ID NO: 2 (AfGA2), 3, 4, 5 and 6. The correspondence between the positions in the AfGA1 of SEQ ID NO: 1 and the glucoamylases of SEQ ID NO: 2, 3, 4, 5 and 6 is determined with reference to the alignment shown in FIG. 1 . For example, referring to the alignment in FIG. 1 , a variant AfGA1 polypeptide can have a D23N substitution, wherein Asn is the corresponding amino acid in SEQ ID NO: 6. Variant AfGA1 polypeptides also include, but are not limited to, those having 1, 2, 3 or 4 randomly selected amino acid modifications. Amino acid modifications can be performed using well-known methods, such as oligonucleotide-directed mutagenesis. Similarly, variants of the AfGA2 polypeptide of SEQ ID NO: 2 may include, but are not limited to, those having amino acid modifications selected from: substitution, insertion or deletion of the corresponding amino acid in the polypeptides selected from SEQ ID NO: 1 (AfGA1), 3, 4, 5 and 6.

[0216] Also provided are nucleic acids encoding AfGA1 polypeptides or variants thereof. The nucleic acid encoding AfGA1 may be genomic DNA. Alternatively, the nucleic acid may be a cDNA comprising SEQ ID NO:8. Similarly, nucleic acids encoding AfGA2 polypeptides or variants thereof are provided. The nucleic acid encoding AfGA2 may also be genomic DNA. Alternatively, the nucleic acid may be a cDNA comprising SEQ ID NO:14. As is well known to those skilled in the art, the genetic code is degenerate, meaning that in some cases multiple codons may encode the same amino acid. Nucleic acids include all genomic DNA, mRNA, and cDNA sequences encoding AfGA1, AfGA2, or variants thereof.

[0217] AfGA1, AfGA2 or variants thereof may be "precursor", "immature" or "full length", in which case they include a signal sequence; or may be "mature", in which case they lack a signal sequence. Variant glucoamylases may also be truncated at the N-terminus or C-terminus, as long as the resulting polypeptide retains glucoamylase activity.

[0218] 2.1. Characterization of AfGA variants

[0219] Variant AfGA polypeptides retain glucoamylase activity. They may have a specific activity higher or lower than that of wild-type AfGA polypeptides. Additional features of AfGA variants include, for example, stability, pH range, temperature profile, oxidative stability, and thermostability. For example, the variants may be pH stable for 24-60 hours at pH 3 to about pH 8, such as pH 3.0-7.8, such as pH 3.0-7.5, pH 3.5-7.0, pH 4.0-6.7, or pH 5.0. AfGA variants can be expressed at a level higher than that of wild-type AfGA while maintaining the performance characteristics of wild-type AfGA. Compared to the parent glucoamylase, AfGA variants may also have altered oxidative stability. For example, reduced oxidative stability may be advantageous in compositions for starch liquefaction. Variant AfGA has an altered temperature profile compared to wild-type glucoamylase. Such AfGA variants are advantageously used in baking or other processes requiring high temperatures. Expression levels and enzyme activity can be evaluated using standard assays known to those skilled in the art, including those disclosed below.

[0220] 3. Preparation of AfGA and its variants

[0221] AfGA or its variant can be isolated from host cells, for example, by secretion of AfGA or variant from host cells. Cultured cell material containing AfGA or its variant can be obtained after AfGA or variant is secreted from host cells. AfGA or its variant is optionally purified before use. The AfGA gene can be cloned and expressed according to methods well known in the art. Suitable host cells include bacteria, plants or yeast cells, such as filamentous fungal cells. Particularly useful host cells include Trichoderma reesei. Trichoderma reesei host cells express AfGATR at a higher or at least comparable level compared to naturally expressed AfGA Aspergillus fumigatus.

[0222] In some embodiments, AfGA is heterologously expressed in a host at least 10 g / L. In some embodiments, AfGA is heterologously expressed at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, or 110 g / L. In some embodiments, AfGA is heterologously expressed in a Trichoderma reesei host, wherein the expression is at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, or 110 g / L. In some embodiments, AfGA is heterologously expressed in an Aspergillus host, wherein the expression is at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, or 110 g / L.

[0223] The host cell may also express a homologous or heterologous amylase (i.e., an amylase of a species other than the host cell) or one or more other enzymes. The amylase may be a variant amylase. In addition, the host may express one or more auxiliary enzymes, proteins, peptides. These may be beneficial to processes such as liquefaction, saccharification, fermentation, SSF, etc. In addition, the host cell may produce biochemical substances in addition to enzymes for digesting one or more carbon raw materials. Such host cells may be used in fermentation processes or simultaneous saccharification and fermentation processes to reduce or eliminate the need for adding enzymes.

[0224] 3.1. Carrier

[0225] A DNA construct comprising a nucleic acid encoding AfGA or a variant thereof can be constructed for expression in a host cell. Representative nucleic acids encoding AfGA1 include SEQ ID NO:8. Representative nucleic acids encoding AfGA2 include SEQ ID NO:14. Due to the well-known degeneracy of the genetic code, variant polynucleotides encoding the same amino acid sequence can be designed and prepared with routine skills. It is also well known in the art to optimize codon usage for specific host cells. Nucleic acids encoding AfGA or a variant thereof can be incorporated into a vector. The vector can be transferred to a host cell using well-known transformation techniques, such as those disclosed below.

[0226] The vector can be any vector that can be transformed into a host cell and replicated in the host cell. For example, as a means of propagating and amplifying the vector, a vector comprising a nucleic acid encoding AfGA or its variant can be transformed into a bacterial host cell and replicated in the bacterial host cell. The vector can also be transformed into an expression host so that the encoding nucleic acid can be expressed as a functional AfGA or its variant. The host cell serving as an expression host can include, for example, filamentous fungi. The Fungal Genetics Stock Center (Fungal Genetics Stock Center, FGSC) strain catalog lists vectors suitable for expression in fungal host cells. See the FGSC, Catalogue of Strains, University of Missouri (University of Missouri strain catalog) at the website www.fgsc.net (last modified on January 17, 2007). Representative vectors include pJG222 (Trex3gM-AfGA1) ( Figure 2 ) and pJG313(Trex3gM-AfGA2)( Fig.10 ), each of which contains the pTrex3gM expression vector (published U.S. Application No. 2011 / 0136197A1) and allows the expression of nucleic acids encoding AfGA in fungal hosts under the control of the cbhl promoter. pJG222 and pJG313 can be modified using routine techniques to contain nucleic acids encoding AfGA variants and express the nucleic acids.

[0227] The nucleic acid encoding AfGA or its variant can be operably linked to a suitable promoter so that it can be transcribed in a host cell. The promoter can be any DNA sequence that shows transcriptional activity in a selected host cell, and can be derived from a gene encoding a protein that is homologous or heterologous to the host cell. For transcription in a fungal host, examples of available promoters are those derived from genes encoding the following enzymes: Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid stable α-amylase, Aspergillus niger glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, or Aspergillus nidulans acetamidase. When expressing a gene encoding AfGA or its variant in a bacterial species such as Escherichia coli, a suitable promoter can be selected, for example, from a phage promoter (including a T7 promoter and a lambda phage promoter). Examples of promoters suitable for expression in yeast species include, but are not limited to, the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the AOX1 or AOX2 promoters of Pichia pastoris. Figure 2 The pJG222 vector shown in , for example, contains the cbhl promoter operably linked to AfGA1. Fig.10 The pJG313 vector shown in, for example, contains the cbhl promoter operably linked to AfGA2. cbhl is an endogenous inducible promoter from Trichoderma reesei. See Liu et al., (2008), "Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbhl) promoter optimization", Acta Biochim. Biophys. Sin (Shanghai) 40 (2): 158-65.

[0228] The coding sequence may be operably linked to the signal sequence. The DNA encoding the signal sequence may be a DNA sequence naturally associated with the AfGA gene to be expressed. For example, the DNA may encode the AfGA1 and AfGA2 signal sequences of SEQ ID NO: 11 operably linked to a nucleic acid encoding AfGA or a variant thereof. The DNA encodes a signal sequence from a species other than Aspergillus fumigatus. The signal sequence or promoter sequence comprising the DNA construct or vector may be introduced into a fungal host cell and may be derived from the same source. For example, the signal sequence is the cbhl signal sequence operably linked to the cbhl promoter.

[0229] The expression vector may also contain a suitable transcription terminator and (in eukaryotes) a polyadenylation sequence operably linked to the DNA sequence encoding AfGA or its variant. The termination sequence and polyadenylation sequence may suitably be derived from the same source as the promoter.

[0230] The vector may also comprise a DNA sequence which enables the vector to replicate in the host cell. Examples of such sequences are the replication origins of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.

[0231] The vector may also contain a selectable marker, such as a gene whose product can complement a defect in an isolated host cell, such as the dal gene from Bacillus subtilis or Bacillus licheniformis, or a gene that confers antibiotic resistance (such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance). In addition, the vector may contain Aspergillus selection markers, such as amdS, argB, niaD and sC (a marker for hygromycin resistance), or selection may be achieved by co-transformation, such as known in the art. See, for example, PCT International Patent Application WO 91 / 17243.

[0232] Intracellular expression may be advantageous in some aspects, for example, when certain bacteria or fungi are used as host cells to produce large amounts of AfGA or its variants for subsequent purification. Extracellular secretion of AfGA or its variants into the culture medium can also be used to prepare cultured cell materials containing isolated AfGA or its variants.

[0233] The expression vector generally comprises the components of a cloning vector, for example, an element that allows autonomous replication of the vector in a selected host organism and one or more phenotypic detectable markers for selection purposes. The expression vector generally comprises a control nucleotide sequence, such as a promoter, an operator, a ribosome binding site, a translation initiation signal, and optionally a repressor gene or one or more activator genes. In addition, the expression vector may comprise a coding sequence for an amino acid sequence that is capable of targeting AfGA or its variants to a host cell organelle (e.g., a peroxisome), or to a specific host cell compartment. Such targeting sequences include, but are not limited to, serine-lysine-leucine (SKL) sequences, which are known peroxisomal target signals. To be expressed under the guidance of a control sequence, the nucleic acid sequence of AfGA or its variants is operably linked to the control sequence in a manner suitable for expression.

[0234] The operations for ligating a DNA construct encoding AfGA or its variants, promoters, terminators and other elements, respectively, and for inserting them into a suitable vector containing the information necessary for replication are well known to those skilled in the art (see, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition, Cold Spring Harbor, 1989, and 3rd edition, 2001).

[0235] 3.2. Transformation and culture of host cells

[0236] Trichoderma reesei host cells containing DNA constructs or expression vectors are advantageously used as host cells in the process of recombinant production of AfGATR or its variants. The cell can be transformed conveniently by integrating the DNA construct (in one or more copies) into the host chromosome with a DNA construct encoding the enzyme. It is generally believed that such integration is advantageous because the DNA sequence is more likely to be stably maintained in the cell. The DNA construct can be integrated into the host chromosome according to conventional methods, for example by homologous or heterologous recombination. Alternatively, the cell can be transformed using the above-mentioned expression vectors for different types of host cells.

[0237] It is advantageous to remove genes from the expression host, where the gene defect can be remedied by the transformed expression vector. Known methods can be used to obtain fungal host cells with one or more inactivated genes. Gene inactivation can be achieved by complete or partial deletion, by insertional inactivation, or by any other means that renders the gene non-functional for its intended purpose, thereby preventing the gene from expressing a functional protein. Any gene from a cloned Trichoderma species or other filamentous fungal host, such as the cbhl gene, cbh2 gene, egll gene, and egl2 gene, can be removed. Gene deletion can be accomplished by inserting a form of the desired gene to be inactivated into a plasmid using methods known in the art.

[0238] The introduction of a DNA construct or vector into a host cell includes the following techniques: for example, transformation; electroporation; nuclear microinjection; transduction; transfection, such as lipofection-mediated and DEAE-dextran-mediated transfection; incubation with calcium phosphate DNA precipitates; high-speed bombardment with DNA-coated microprojectiles; and protoplast fusion. General transformation techniques are known in the art. See, for example, Sambrook et al., (2001), supra. Expression of heterologous proteins in Trichoderma is described, for example, in U.S. Patent No. 6,022,725. For transformation of Aspergillus strains, also refer to Cao et al., (2000) Science 9:991-1001. Genetically stable transformants can be constructed using vector systems, whereby nucleic acids encoding AfGA or its variants are stably integrated into the host cell chromosome. Transformants are then selected and purified by known techniques.

[0239] Preparation of Trichoderma species for transformation may involve, for example, preparation of protoplasts from fungal mycelium. See Campbell et al., (1989) Curr. Genet, 16:53-56. Mycelium may be obtained from germinated vegetative spores. Mycelium may be treated with enzymes that digest the cell wall to obtain protoplasts. Protoplasts are protected by the presence of osmotic stabilizers in the suspension medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like. Typically, the concentration of these stabilizers varies between 0.8 M and 1.2 M, for example, a 1.2 M sorbitol solution may be used in the suspension medium.

[0240] DNA uptake by the host Trichoderma sp. strain depends on the calcium ion concentration. Generally, about 10-50 mM CaCl is used in the uptake solution. 2Other suitable compounds include buffer systems such as TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 and polyethylene glycol. Polyethylene glycol is believed to fuse cell membranes, thereby allowing the contents of the medium to be delivered into the cytoplasm of the Trichoderma species strain. Such fusion often results in multiple copies of the plasmid DNA being integrated into the host chromosome.

[0241] Typically, transformation of Trichoderma species is usually carried out at a rate of 10 5 Up to 10 7 / mL, especially 2×10 6 Protoplasts or cells that have undergone osmotic treatment are used at a density of / mL. A volume of 100 μL of a suitable solution (e.g., 1.2 M sorbitol and 50 mM CaCl) can be added. 2 ) are mixed with the desired DNA. Generally, a high concentration of PEG is added to the uptake solution. 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension; however, it is useful to add about 0.25 volume to the protoplast suspension. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, etc. can also be added to the uptake solution to aid transformation. Similar procedures exist for other fungal host cells. See, for example, U.S. Patent No. 6,022,725.

[0242] 3.3. Expression

[0243] A method of producing AfGATR or a variant thereof may comprise culturing a Trichoderma reesei host cell as described above under conditions conducive to production of the enzyme and recovering the enzyme from the cells and / or culture medium. The Trichoderma reesei host cell expresses AfGATR at a higher or at least comparable level compared to the naturally expressed AfGA in Aspergillus fumigatus.

[0244] The medium used to culture the cells may be any conventional medium suitable for culturing the host cells in question and obtaining expression of AfGATR or its variants. Suitable culture media and medium components are available from commercial suppliers or may be prepared according to published recipes (e.g., recipes as described in catalogs of the American Type Culture Collection).

[0245] Enzymes secreted from host cells can be used for full fermentation broth preparation. In the method of the present invention, using any culture method known in the art that causes glucoamylase expression, the preparation of the full fermentation broth exhausted by recombinant microorganisms can be achieved. Therefore, fermentation can be understood as being included in a suitable medium and under conditions that allow glucoamylase expression or separation, shake flask culture in the laboratory, or small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, batch fed fermentation or solid-state fermentation) in an industrial fermentor. The term "exhausted full fermentation broth" is defined herein as the unfractionated content of fermentation material, including culture medium, extracellular protein (e.g., enzyme) and cell biomass. It should be understood that the term "exhausted full fermentation broth" also encompasses cell biomass that has been cracked using methods well known in the art or that has been permeabilized.

[0246] Enzymes secreted from the host cells can be conveniently recovered from the culture medium by well-known methods, including separation of the cells from the culture medium by centrifugation or filtration, and precipitation of protein components in the culture medium with the aid of salts such as ammonium sulfate, followed by chromatographic methods such as ion exchange chromatography, affinity chromatography, and the like.

[0247] The polynucleotide encoding AfGA or its variant in the vector may be operably linked to a control sequence that enables the host cell to express the coding sequence, i.e., the vector is an expression vector. The control sequence may be modified, for example, by adding other transcriptional regulatory elements, so that the transcription level directed by the control sequence is more responsive to transcriptional regulatory factors. The control sequence may particularly comprise a promoter.

[0248] The host cells can be cultured under suitable conditions that allow expression of AfGATR or its variants. The expression of enzymes can be constitutive, so that they can be produced continuously; or can be inducible, so that a stimulus is required to initiate expression. In the case of inducible expression, the production of the protein can be initiated when necessary by, for example, adding an inducing substance (such as dexamethasone or IPTG or Sepharose) to the culture medium. The polypeptide can also be expressed in an in vitro cell-free system (such as TNT TM Produced recombinantly in rabbit reticulocyte system (Promega).

[0249] The expression host can also be cultured in a culture medium suitable for the host under aerobic conditions. Shaking or a combination of stirring and aeration can be provided, and production can be carried out at a temperature suitable for the host, such as about 25°C to about 78°C (e.g., 30°C to 45°C), depending on the needs of the host and the needs of preparing the desired AfGATR or variant thereof. The culture can be about 12 to about 100 hours or longer (and any hour value therebetween, such as 24 to 72 hours). Typically, the pH of the culture broth is about 4.0 to about 8.0, which also depends on the culture conditions required by the host related to the preparation of AfGATR or its variant.

[0250] 3.4. Identification of AfGATR activity

[0251] To evaluate the expression of AfGATR or a variant thereof in a host cell, an assay can be used to measure the expressed protein, the corresponding mRNA, or the glucoamylase activity. For example, suitable assays include Northern blots, reverse transcriptase polymerase chain reaction, and in situ hybridization using appropriately labeled hybridization probes. Suitable assays also include measuring AfGATR activity in a sample, for example, by an assay that directly measures reducing sugars such as glucose in the culture medium. For example, the glucose concentration can be measured using a glucose kit No. 15-UV (Sigma Chemical Co.) or an instrument such as a Technicon autoanalyzer. Glucoamylase activity can also be measured by any known method, such as the PAHBAH assay or the ABTS assay described below.

[0252] 3.5. Methods for purifying AfGATR and its variants

[0253] Fermentation, separation and concentration techniques are well known in the art, and conventional methods can be used to prepare concentrated AfGATR or variant glucoamylase polypeptide-containing solutions.

[0254] After fermentation, the fermentation broth is obtained, and the microbial cells and various suspended solids (including residual fermentation raw materials) are removed by conventional separation techniques to obtain an amylase solution. Typically, filtration, centrifugation, microfiltration, drum vacuum filtration, ultrafiltration, centrifugation followed by ultrafiltration, extraction or chromatography, etc. are used.

[0255] It may be desirable to concentrate the solution containing the AfGATR or variant glucoamylase polypeptide to optimize recovery. Use of unconcentrated solutions may require increased incubation times in order to collect the purified enzyme precipitate.

[0256] The enzyme solution is concentrated using conventional concentration techniques until the desired enzyme content is obtained. Concentration of the enzyme solution can be achieved by any of the techniques discussed herein. Exemplary methods of purification include, but are not limited to, rotary vacuum filtration and / or ultrafiltration.

[0257] The enzyme solution is concentrated into a concentrated enzyme solution until the enzyme activity of the concentrated AfGATR or variant glucoamylase polypeptide-containing solution is at a desired level.

[0258] Concentration can be performed using, for example, a precipitating agent such as a metal halide precipitating agent. Metal halide precipitating agents include, but are not limited to, alkali metal chlorides, alkali metal bromides, and blends of two or more of these metal halides. Exemplary metal halides include sodium chloride, potassium chloride, sodium bromide, potassium bromide, and blends of two or more of these metal halides. The metal halide precipitating agent sodium chloride can also be used as a preservative.

[0259] The metal halide precipitation agent is used in an amount effective to precipitate AfGATR or a variant thereof. It will be apparent to one of ordinary skill in the art to select at least an effective amount and an optimal amount of a metal halide effective to cause precipitation of the enzyme, as well as precipitation conditions (including incubation time, pH, temperature, and enzyme concentration) for maximum recovery after routine testing.

[0260] Generally, at least about 5% w / v (weight / volume) to about 25% w / v of the metal halide is added to the concentrated enzyme solution, typically at least 8% w / v. Generally, no more than about 25% w / v of the metal halide is added to the concentrated enzyme solution, typically no more than about 20% w / v. The optimal concentration of the metal halide precipitation agent will depend, among other things, on the properties of the specific AfGATR or variant glucoamylase polypeptide and its concentration in the concentrated enzyme solution.

[0261] Another alternative approach to precipitating the enzyme is to use an organic compound. Exemplary organic compound precipitation agents include: 4-hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid, alkyl esters of 4-hydroxybenzoic acid, and blends of two or more of these organic compounds. The addition of the organic compound precipitation agent can occur before, simultaneously with, or after the addition of the metal halide precipitation agent, and the addition of the two precipitation agents (organic compound and metal halide) can be performed sequentially or simultaneously.

[0262] Typically, the organic precipitation agent is selected from alkali metal salts (such as sodium or potassium salts) of 4-hydroxybenzoic acid, and straight or branched alkyl esters of 4-hydroxybenzoic acid (wherein the alkyl group contains 1 to 12 carbon atoms), and blends of two or more of these organic compounds. The organic compound precipitation agent can be, for example, a straight or branched alkyl ester of 4-hydroxybenzoic acid (wherein the alkyl group contains 1 to 10 carbon atoms) and a blend of two or more of these organic compounds. Exemplary organic compounds are straight chain alkyl esters of 4-hydroxybenzoic acid (wherein the alkyl group contains 1 to 6 carbon atoms) and a blend of two or more of these organic compounds. Methyl esters of 4-hydroxybenzoic acid, propyl esters of 4-hydroxybenzoic acid, butyl esters of 4-hydroxybenzoic acid, ethyl esters of 4-hydroxybenzoic acid, and blends of two or more of these organic compounds can also be used. Additional organic compounds include, but are not limited to, methyl 4-hydroxybenzoate (known as methylparaben) and propyl 4-hydroxybenzoate (known as propylparaben), which are also amylase preservatives. For further description, see, for example, U.S. Pat. No. 5,281,526.

[0263] The addition of an organic compound precipitation agent offers the advantage of high flexibility in the precipitation conditions with respect to pH, temperature, AfGATR or variant glucoamylase polypeptide concentration, precipitation agent concentration, and incubation time.

[0264] The organic compound precipitation agent is used in an amount effective to improve enzyme precipitation by the metal halide precipitation agent. In light of the present disclosure, it will be apparent to one of ordinary skill in the art to select at least an effective amount and an optimal amount of the organic compound precipitation agent, as well as precipitation conditions (including incubation time, pH, temperature, and enzyme concentration) for maximum recovery after routine testing.

[0265] Generally, at least about 0.01% w / v of the organic compound precipitation agent is added to the concentrated enzyme solution, and usually at least about 0.02% w / v. Generally, no more than about 0.3% w / v of the organic compound precipitation agent is added to the concentrated enzyme solution, and usually no more than about 0.2% w / v.

[0266] The pH of the concentrated polypeptide solution containing the metal halide precipitation agent and the organic compound precipitation agent can be adjusted, and the pH will necessarily depend on the enzyme to be purified. Typically, the pH is adjusted to a level near the isoelectric point of the glucoamylase. The pH can be adjusted to a pH within the pH range of about 2.5 pH units below the isoelectric point (pi) to about 2.5 pH units above the isoelectric point.

[0267] The incubation time required to obtain a purified enzyme precipitate depends on the nature of the specific enzyme, the enzyme concentration, and the specific one or more precipitation agents and their concentrations. Generally, the time effective for precipitating the enzyme is between about 1 and about 30 hours; usually not more than about 25 hours. In the presence of an organic compound precipitation agent, the incubation time can also be reduced to less than about 10 hours, and in most cases even to about 6 hours.

[0268] Typically, the temperature during incubation is between about 4° C. and about 50° C. Typically, the method is performed at a temperature between about 10° C. and about 45° C., such as between about 20° C. and about 40° C. The optimal temperature for inducing precipitation varies depending on the solution conditions and the enzyme or one or more precipitation agents used.

[0269] The total recovery of the purified enzyme precipitate and the efficiency of the method are improved by stirring a solution comprising the enzyme, the added metal halide and the added organic compound. During the addition of the metal halide and the organic compound, and during the subsequent incubation period, a stirring step is performed. Suitable stirring methods include mechanical stirring or vibration, forced ventilation or any similar technology.

[0270] After the incubation period, the purified enzyme can then be separated from the dissociated pigment and other impurities and collected by conventional separation techniques such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, filter press, transmembrane microfiltration, cross-flow membrane microfiltration, etc. Further purification of the purified enzyme precipitate can be obtained by washing the precipitate with water. For example, the purified enzyme precipitate is washed with water containing a metal halide precipitation agent, or with water containing a metal halide and an organic compound precipitation agent.

[0271] During the fermentation process, the AfGATR or variant glucoamylase polypeptide accumulates in the culture broth. In order to isolate and purify the desired AfGATR or variant glucoamylase, the culture broth is centrifuged or filtered to remove cells, and the resulting cell-free broth is used for enzyme purification. In one embodiment, the cell-free broth is salted out using ammonium sulfate at about 70% saturation; the 70% saturation precipitation fraction is then dissolved in a buffer and applied to a column such as a Sephadex G-100 column, and eluted to recover the enzyme activity fraction. For further purification, conventional methods such as ion exchange chromatography can be used.

[0272] The purified enzymes can be used in laundry and cleaning applications. For example, they can be used in laundry detergents and stain removers. They can be made into liquid (solutions, slurries) or solid (granules, powder) forms of the final product.

[0273] More specific examples of purification are described in Sumitani et al., (2000) "New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp. 195 glucoamylase contributes to starch binding and raw starch degrading", Biochem. J. 350: 477-484 and are briefly summarized here. 4 ) 2 SO 4 The enzyme obtained from 4 liters of culture supernatant of Streptomyces lividans TK24 was processed. The precipitate was recovered by centrifugation at 10,000 × g (20 min and 4° C.) and redissolved in 5 mM CaCl 2 The dissolved precipitate was then dialyzed against the same buffer. The dialyzed sample was then applied to a 20 mM Tris / HCl buffer (pH 7.0) that had been previously diluted with 5 mM CaCl 2 The protein was purified by eluting on a Sephacryl S-200 column equilibrated with 20 mM Tris / HCl buffer (pH 7.0) and then eluting with the same buffer at a linear flow rate of 7 mL / h. Fractions from the column were collected and evaluated for activity as determined by enzyme assay and SDS-PAGE. The protein was further purified as follows. A Toyopearl HW55 column (Tosoh Bioscience, Montgomeryville, PA; Catalog No. 19812) was used with 5 mM CaCl 2 and 1.5M (NH 4 ) 2 SO 4 The samples were equilibrated in 20 mM Tris / HCl buffer (pH 7.0). 2 The linear gradient was from 1.5 to 0 M (NH 4 ) 2 SO 4 The enzyme was eluted. The active fractions were collected and washed with 80% saturation (NH 4 ) 2SO 4 The enzyme was precipitated. The precipitate was recovered, redissolved and dialyzed as described above. The dialyzed sample was then applied to a MonoQ HR5 / 5 column (Amersham Pharmacia; Catalog No. 17-5167-01) that had been previously treated with 5 mM CaCl 2 The active fractions were collected and added to 1.5 M (NH 4 ) 2 SO 4 The active enzyme fraction was rechromatographed on a Toyopearl HW55 column as described above to give a homogeneous enzyme as determined by SDS-PAGE. See Sumitani et al., (2000) Biochem. J. 350:477-484 for a general discussion of its methods and variations.

[0274] For production scale recovery, the AfGATR or variant glucoamylase polypeptide can be partially purified by removing cells by flocculation with a polymer as generally described above. Alternatively, the enzyme can be purified by microfiltration using available membranes and equipment followed by concentration by ultrafiltration. However, for certain applications, the enzyme does not need to be purified and the whole fermentation broth culture can be dissolved and used without further processing. The enzyme can then be processed into, for example, granules.

[0275] 4. Compositions and uses of AfGATR and its variants

[0276] AfGATR and its variants can be used in a variety of industrial applications. For example, AfGATR and its variants can be used in starch conversion processes, especially in saccharification processes for starch that has been liquefied. The desired end product can be any product that can be produced by enzymatic conversion of a starch substrate. For example, the desired product can be a glucose-rich syrup that can be used in other processes, such as the preparation of HFCS, or it can be converted into a variety of other useful products, such as ascorbic acid intermediates (e.g., gluconate; 2-keto-L-gulonic acid; 5-keto-gluconate; and 2,5-diketo-gluconate); 1,3-propanediol; aromatic amino acids (e.g., tyrosine, phenylalanine, and tryptophan); organic acids (e.g., lactate, pyruvate, succinate, isocitrate, and oxaloacetate); amino acids (e.g., serine and glycine); antibiotics; antimicrobials; enzymes; microorganisms; and hormones.

[0277] The starch conversion process can be a precursor to a fermentation process designed to produce fuel alcohol or drinking alcohol (i.e., alcohol suitable for drinking) or can be carried out simultaneously with the fermentation process. Those skilled in the art are aware of various fermentation conditions that can be used to produce these end products. AfGATR and its variants can also be used in compositions and methods for food preparation. These various uses of AfGATR and its variants are described in more detail below.

[0278] 4.1. Preparation of starch substrate

[0279] Those of ordinary skill in the art are familiar with available methods for preparing starch substrates used in the processes disclosed herein. For example, available starch substrates can be obtained from tubers, roots, stems, legumes, cereals, or whole grains. More specifically, granular starch can be obtained from corn, corn cobs, wheat, barley, rye, triticale, milo, sago, millet, cassava, tapioca, sorghum, rice, peas, beans, bananas, or potatoes. Corn contains about 60-68% starch; barley contains about 55-65% starch; millet contains about 75-80% starch; wheat contains about 60-65% starch; and polished rice contains 70-72% starch. Specifically contemplated starch substrates are corn starch and wheat starch. Starch from grains can be ground or whole, including corn solids, such as corn kernels, bran, and / or cobs. Starch can be highly refined raw starch or raw material from a starch refining process. Various starches are also commercially available. For example, corn starch is available from Cerestar, Sigma, and Katayama Chemical Industry Co. (Japan); wheat starch is available from Sigma; sweet potato starch is available from Wako Pure Chemical Industry Co. (Japan); and potato starch is available from Nakaari Chemical Pharmaceutical Co. (Japan).

[0280] The starch substrate can be a crude starch from milled whole grains containing non-starch fractions such as germ residues and fiber. Milling includes wet milling or dry milling or grinding. In wet milling, whole grains can be soaked in water or dilute acid to separate grains into their components, such as starch, protein, germ, oil, grain fiber. Wet milling can effectively separate germ and meal (i.e. starch granules and protein), and can be particularly suitable for preparing syrups. In dry milling or grinding, whole grains are ground into fine powder and are usually processed without grading the grains into their components. In some cases, oil from grains is recovered. Dry milled grains will usually contain a large amount of non-starch carbohydrates in addition to starch. Dry milled starch substrates can be used to produce ethanol and other biochemical substances. The starch to be processed can be a highly refined starch quality, such as at least 90%, at least 95%, at least 97% or at least 99.5% purity.

[0281] 4.2. Starch gelatinization and liquefaction

[0282] As used herein, the term "liquefaction" means the process of converting starch into dextrins with less viscosity and shorter chain length. Generally speaking, the process involves the gelatinization of starch, while adding alpha-amylase or adding alpha-amylase after gelatinization, but optionally another enzyme that induces liquefaction can be added. In some embodiments, the starch substrate prepared as described above is adjusted to a slurry with water. The starch slurry may contain starch having a dry solid weight percentage of about 10-55%, about 20-45%, about 30-45%, about 30-40% or about 30-35%. Alpha amylase (EC 3.2.1.1) can be added to the slurry with, for example, a metering pump. Alpha amylases commonly used in this application are bacterial alpha amylases of heat stability, such as Bacillus stearothermophilus alpha amylases. Alpha-amylases are typically supplied, for example, at about 1500 units / kg of starch dry matter. To optimize the stability and activity of the alpha amylase, the pH of the slurry is typically adjusted to about pH 5.5-6.5, and about 1 mM calcium (about 40 ppm of free calcium ions) is typically added. Geobacillus stearothermophilus variants or other alpha-amylases may require different conditions. Bacterial alpha-amylases that remain in the slurry after liquefaction can be inactivated by a variety of methods, including lowering the pH in subsequent reaction steps or by removing calcium in cases where the enzyme is calcium-dependent.

[0283] The starch plus alpha-amylase slurry can be continuously pumped through a jet cooker heated to 105°C by steam. Gelatinization occurs rapidly under these conditions, and the enzymatic activity combined with the shear force begins the hydrolysis of the starch substrate. The residence time in the jet cooker is short. The partially gelatinized starch can be passed through a series of holding tubes maintained at 105-110°C and maintained for 5-8 minutes to complete the gelatinization process ("primary liquefaction"). Hydrolysis to the desired DE ("secondary liquefaction") is completed in about 1 to 2 hours in a holding tank at 85-95°C or higher. These tanks may have baffles to prevent back mixing. As used herein, the term "secondary liquefaction minutes" refers to the time elapsed from the start of secondary liquefaction to the measurement of dextrose equivalent (DE). The slurry is then allowed to cool to room temperature. The cooling step may be 30 minutes to 180 minutes, such as 90 minutes to 120 minutes.

[0284] The liquefied starch obtained by the above process generally comprises about 98% oligosaccharides and about 2% maltose and 0.3% D-glucose. The liquefied starch is generally in the form of a slurry having a dry solids content (w / w) of about 10-50%, about 10-45%, about 15-40%, about 20-40%, about 25-40% or about 25-35%.

[0285] AkAA, AtAmy1, AfAmy1 and AcAmy1 and variants thereof can be used in place of bacterial alpha amylase in liquefaction processes. Liquefaction with these alpha-amylases and variants thereof can advantageously be performed at low pH, thereby eliminating the need to adjust the pH to about pH 5.5-6.5. These alpha-amylases and variants thereof can be used for liquefaction in a pH range of 2 to 7, such as pH 3.0-7.5, pH 4.0-6.0, or pH 4.5-5.8. They can maintain liquefaction activity at a temperature range of about 85°C-95°C, such as 85°C, 90°C, or 95°C. For example, 800 μg AcAmy1 or variants thereof can be used in a 25% DS corn starch solution, such as at pH 5.8 and 85°C or pH 4.5 and 95°C for 10 minutes. The liquefaction activity can be measured by any of a variety of viscometric methods known in the art.

[0286] 4.3. Saccharification

[0287] AfGATR and variants thereof can be used, optionally in the presence of additional enzymes, to saccharify liquefied starch into a syrup rich in lower DP, especially DP1 sugars. The exact composition of the saccharification product depends on the combination of enzymes used and the type of granular starch treated. Advantageously, the syrups obtainable using the provided AfGATR and variants thereof can contain DP1 as a weight percentage of more than about 65% of the total oligosaccharides in the saccharified starch, such as 70%, 80%, 85%, 90%, 95% or 96%.

[0288] Although liquefaction is usually run as a continuous process, saccharification is usually carried out in a batch process. Saccharification is usually most effective at a temperature of about 55-75°C and a pH of about 4.0-6.7, such as pH 5.0, and the pH of the liquefied starch must be cooled and adjusted. Saccharification can be carried out, for example, at a temperature between about 40°C, about 55°C or about 65°C to about 70°C, about 75°C or about 80°C. Saccharification is usually carried out in a stirred tank, which may take several hours to fill or empty. Enzymes are usually added in a fixed ratio with dry solids when filling the tank, or in a single dose at the beginning of the filling stage. The saccharification reaction for preparing syrup usually runs for about 24-72 hours, such as 24-48 hours. When the maximum DE or the required DE has been obtained, the reaction is terminated by, for example, heating to 85°C for 5 minutes. Further incubation will result in lower DE, ultimately to about 90 DE, as the accumulated glucose is repolymerized to isomaltose and / or other reversion products via enzymatic reversion reactions and / or using thermodynamic equilibrium methods. When using an AfGATR polypeptide or variant thereof, saccharification is optimally performed at a temperature range of about 40°C to about 80°C, such as about 55°C to about 75°C or about 65°C to about 70°C. Saccharification can be performed at a pH range of about pH 3.0 to about pH 7.5, such as pH 3.5-pH 7.0, pH 4.0-pH 6.7, or pH 5.0.

[0289] AfGATR or a variant thereof can be added to a slurry in the form of a composition. AfGATR or a variant thereof can be added to a slurry of a granular starch substrate in an amount of about 0.6-10 ppm dry solids, for example, 2 ppm dry solids. AfGATR or a variant thereof can be added as a whole fermentation broth, a clarified enzyme, a partially purified enzyme, or a purified enzyme. The specific activity of the purified AfGATR or a variant thereof can be about 187.7 U / mg, for example, as measured using an ABTS assay. The specific activity of the purified AfGA2TR or a variant thereof can be about 213.7 U / mg, for example, as measured using an ABTS assay. AfGATR or a variant thereof can also be added as a whole fermentation broth product.

[0290] AfGATR or a variant thereof can be added to the slurry as a separate enzyme solution. For example, AfGATR or a variant thereof can be added in the form of a cultured cell material produced by a host cell expressing AfGATR or a variant thereof. AfGATR or a variant thereof can also be secreted by the host cell into the reaction medium during the fermentation or SSF process so that the enzyme is continuously provided to the reactants. The host cell that produces and secretes AfGATR or a variant thereof can also express additional enzymes, such as glucoamylase. For example, U.S. Patent No. 5,422,267 discloses the use of glucoamylase in yeast in the preparation of alcoholic beverages. For example, during saccharification, a host cell such as Trichoderma reesei can be engineered to co-express AfGATR or a variant thereof and an α-amylase, including but not limited to AkAA, AcAmy1, a native Trichoderma reesei α-amylase or a variant thereof. The host cell can be genetically modified so as not to express its endogenous glucoamylase and / or other enzymes, proteins or other substances. The host cell can be engineered to express a variety of different saccharolytic enzymes. For example, the recombinant yeast host cell can comprise a nucleic acid encoding a glucoamylase, an alpha-glucosidase, a pentose utilizing enzyme, an alpha amylase, a pullulanase, an isoamylase and / or an isopullulanase. See, for example, WO 2011 / 153516 A2.

[0291] 4.4. Isomerization

[0292] The soluble starch hydrolysate produced by treatment with AfGATR or a variant thereof can be converted to a high fructose starch-based syrup (HFSS), such as high fructose corn syrup (HFCS). This conversion can be achieved using a glucose isomerase, particularly a glucose isomerase immobilized on a solid support. The pH is increased to about 6.0 to about 8.0, such as pH 7.5, and the Ca is removed by ion exchange. 2+ Suitable isomerases include IT (Novozymes A / S); IMGI, and G993, G993, G993 liquid and IGI. After isomerization, the mixture typically contains about 40-45% fructose, such as 42% fructose.

[0293] 4.5. Fermentation

[0294] Soluble starch hydrolysates, especially glucose-rich syrups, can be fermented by contacting the starch hydrolysate with a fermenting organism, typically at a temperature of about 32° C., such as 30° C. to 35° C. EOF products include metabolites such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, itaconic acid and other carboxylic acids, glucono-δ-lactone, sodium isoascorbate, lysine and other amino acids, ω3 fatty acids, butanol, isoprene, 1,3-propanediol, and biomaterials.

[0295] Ethanol producing microorganisms include yeasts such as Saccharomyces cerevisiae and bacteria such as Zymomonas moblis that express alcohol dehydrogenase and pyruvate decarboxylase. Ethanol producing microorganisms can express xylose reductase and xylitol dehydrogenase that can convert xylose into xylulose. Improved strains of ethanol producing microorganisms (e.g., that can withstand higher temperatures) are known in the art and can be used. See Liu et al., (2011) Journal of Biotechnology (Sheng Wu Gong Cheng Xue Bao) 27 (7): 1049-56. Commercial sources of yeast include ETHANOL (LeSaffre); (Lallemand); RED (Red Star); (DSM Specialties); and (Alltech). Microorganisms that produce other metabolites such as citric acid and lactic acid by fermentation are also known in the art. See, for example, Papagianni (2007) "Advances in citric acid fermentation by Aspergillus niger: biochemical aspects, membrane transport and modeling", Biotechnol. Adv., 25(3): 244-63; John et al., (2009) "Direct lactic acid fermentation: focus on simultaneous saccharification and lactic acid production", Biotechnol. Adv., 27(2): 145-52.

[0296] The saccharification and fermentation process can be performed as an SSF process. The fermentation can include, for example, subsequent ethanol purification and recovery. During the fermentation process, the ethanol content of the fermentation broth or "beer" can reach about 8-18% v / v, such as 14-15% v / v. The fermentation broth can be distilled to produce an enriched (e.g., 96% pure) ethanol solution. In addition, the CO produced by the fermentation 2 Available CO 2 The scrubber collects, compresses and sells it for other uses, such as carbonating beverages or making dry ice. Solid waste from the fermentation process can be used as a protein-rich product, such as livestock feed.

[0297] As mentioned above, the SSF process can be performed with fungal cells, such as Trichoderma reesei, that continuously express and secrete AfGATR or a variant thereof throughout the SSF period. The fungal cell expressing AfGATR or a variant thereof can also be a fermenting microorganism, such as an ethanologenic microorganism. Thus, ethanol production can be performed with fungal cells that express enough AfGATR or a variant thereof so that less or no enzyme needs to be added from the outside. The fungal host cell can be from a properly engineered fungal strain. Fungal host cells that express and secrete other enzymes in addition to AfGATR or a variant thereof can also be used. Such cells can express alpha-amylases and / or pullulanases, phytases, alpha-glucosidases, isoamylases, beta-amylases, cellulases, xylanases, other hemicellulases, proteases, beta-glucosidases, pectinases, esterases, oxidoreductases, transferases, glucoamylases in addition to AfGATR, or other enzymes.

[0298] A variation of the process is the "fed-batch fermentation" system, in which substrate is added incrementally as fermentation proceeds. Fed-batch systems are useful when degradation product repression may inhibit the metabolism of the cells and when it is desirable to have a limited amount of substrate in the culture medium. The actual substrate concentration in a fed-batch system is determined by factors such as pH, dissolved oxygen, and waste gases (such as CO). 2 ) The change of the measurable factor of partial pressure is estimated. Batch fermentation and fed-batch fermentation are common and well known in the art.

[0299] Continuous fermentation is an open system, in which the fermentation medium defined is continuously added to the bioreactor, and an equal amount of conditioned medium is pipetted for processing simultaneously. Continuous fermentation is usually maintained at a constant high density culture, wherein the cells are mainly in the logarithmic growth phase. Continuous fermentation enables cell growth and / or product concentration to be regulated. For example, limiting nutrients such as carbon source or nitrogen source are maintained at a fixed rate, and all other parameters are allowed to be regulated. Because growth remains in a steady state, the cell loss caused by the extraction of the medium should be balanced relative to the cell growth rate in the fermentation. The method for optimizing the continuous fermentation process and maximizing the product formation rate is well known in the field of industrial microbiology.

[0300] 4.6. Compositions containing AfGATR or variants thereof

[0301] AfGATR or a variant thereof may be combined with an alpha-amylase (EC 3.2.1.1). In some embodiments, the alpha-amylase is an acid-stable alpha-amylase that has activity in a pH range of 3.0 to 7.0 and preferably 3.5 to 6.5 when added in an effective amount. The alpha-amylase may be a fungal alpha-amylase or a bacterial alpha-amylase. In addition, the alpha-amylase may be a wild-type alpha-amylase or a variant thereof.

[0302] Preferred examples of fungal alpha amylases include those obtained from filamentous fungal strains, including but not limited to strains of Aspergillus (e.g., Aspergillus niger, Aspergillus kawachii and Aspergillus oryzae); Trichoderma species, Rhizopus species, Mucor species and Penicillium species, Lactobacillus species and Streptomyces species. Acid-stable alpha-amylases can be derived from bacterial strains. Preferred bacterial strains include Bacillus species, such as Bacillus licheniformis, Bacillus stearothermophilus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus lentus and Bacillus coagulans. Particularly preferred are Bacillus licheniformis, Bacillus stearothermophilus and Bacillus amyloliquefaciens. One of the bacterial alpha amylases used in the compositions and methods of the present invention can include one of the alpha-amylases described in the following documents: U.S. Patent Nos. 5,093,257, 5,763,385, 5,824,532, 5,958,739, 6,008,026, 6,093,563, 6,187,576, 6,361,809, 6,867,031; U.S. Publication No. 2006 / 0014265; and International PCT Nos. WO 96 / 23874, WO 96 / 39528, WO 97 / 141213, WO 99 / 19467, and WO 05 / 001064.

[0303] Exemplary α-amylases include AkAA or AcAmy1 and variants thereof, which have excellent specific activity and thermostability. Suitable variants of AkAA include those having α-amylase activity and having at least 80%, 90%, 95%, 98% or at least 99% sequence identity with wild-type AkAA. Suitable variants of AcAmy1 include those having α-amylase activity and having at least 80%, at least 90% or at least 95% sequence identity with wild-type AcAmy1. AfGATR and variants thereof advantageously increase the yield of glucose produced in the saccharification process catalyzed by AnGA or Tr-GA.

[0304] Commercially available alpha amylases contemplated for use in the compositions and methods include: SPEZYME TM AA; SPEZYME TM FRED; SPEZYME TM XTRA;GZYME TM 997; and CLARASE TM L (Genencor International Inc.); TERMAMYL TM 120-L, LC and SC and SUPRA (Novozymes Biotech); LIQUOZYME TM X and SAN TM SUPER (Novozymes A / S) and Fuelzyme TM LF (Diversa). In some embodiments, the alpha amylase will include an alpha amylase derived from Bacillus stearothermophilus, such as SPEZYME TM AA, SPEZYME TM FRED OR SPEZYME TM In some embodiments, the enzyme composition will include BP-WT, SPEZYME TM XTRA and optionally SPEZYME TM In other embodiments, the composition will include BP-17, SPEZYME TM XTRA and optionally SPEZYME TM FRED.

[0305] Other suitable enzymes that can be used with AfGATR or variants thereof include non-AfGATR glucoamylases, phytases, proteases, pullulanases, beta-amylases, isoamylases, alpha-amylases, alpha-glucosidases, cellulases, xylanases, other hemicellulases, beta-glucosidases, transferases, pectinases, lipases, cutinases, esterases, oxidoreductases, or combinations thereof.

[0306] For example, debranching enzymes such as pullulanases (EC 3.2.1.41), e.g. Can be added in effective amounts well known to those skilled in the art. Pullulanase is usually added at 100U / kg dry solids. Pullulanase is usually secreted by Bacillus species. Exemplary pullulanases are described for Bacillus deramificans (U.S. Patent #5,817,498; 1998), Bacillus acidopullulyticus (European Patent #0 063 909) and Bacillus naganoensis (U.S. Patent #5,055,403). Commercial enzymes with pullulanase activity are produced, for example, from Bacillus species (trade name OPTIMAX TM L-1000, from Danisco Genenco; and Promozyme TM , from Novozymes).

[0307] Bacillus megaterium amylase / transferase (BMA): Bacillus megaterium is able to convert debranched sugars into a form that is readily hydrolyzed by glucoamylase. (Habeda RE, Styrlund CR and Teague, WM; 1988 Starch / Starke, 40, 33-36). The enzyme exhibits maximum activity at pH 5.5 and a temperature of 75°C (David, MH, Gunther H and Vilvoorde, HR; 1987, Starch / Starke, 39 436-440). The enzyme has been cloned, expressed in genetically engineered Bacillus subtilis and produced on a commercial scale (Brumm, PJ, Habeda RE and Teague WM, 1991 Starch / Starke, 43 315-329). The enzyme is available as MEGADEX TM Sold under the trade name AGENT(R) for enhancing glucose yield during saccharification of enzymatically liquefied starch using Aspergillus niger glucoamylase.

[0308] Isoamylase (EC 3.2.1.68) can also be added in an effective amount known to those skilled in the art. Pullulanase (EC 3.2.1.41), e.g. Also suitable. Pullulanase is typically added at 100 U / kg dry solids. Other suitable enzymes include proteases, such as fungal and bacterial proteases. Fungal proteases include those obtained from Aspergillus, such as Aspergillus niger, Aspergillus awamori, Aspergillus oryzae; Mucor (e.g., Mucor miehei); Rhizopus; and Trichoderma.

[0309] β-amylase (EC 3.2.1.2) is an exo-acting maltogenic amylase that catalyzes the hydrolysis of 1,4-α-glycosidic bonds into amylopectin and related glucose polymers, thereby releasing maltose. β-amylases have been isolated from a variety of plants and microorganisms. See Fogarty et al., (1979), PROGRESS IN INDUSTRIAL MICROBIOLOGY, Vol. 15, pp. 112-115. These β-amylases have an optimum temperature in the range of 40°C to 65°C and an optimum pH in the range of about 4.5 to about 7.0. Contemplated β-amylases include, but are not limited to, β-amylases from barley. BBA1500, DBA, Optimalt TM ME, Optimalt TM BBA (Danisco America); and Novozym TM WBA (Novozymes).

[0310] 5. Compositions and methods for baking and food preparation

[0311] The present invention also relates to "food compositions" comprising AfGATR or variants thereof, including but not limited to food, animal feed and / or food / feed additives, and methods of preparing such food compositions, comprising mixing AfGATR or variants thereof with one or more food ingredients, and uses of said food compositions and methods.

[0312] The AfGATR or variant thereof can be used to prepare a food composition, wherein the food composition is baked after the polypeptide is added. The term "baking composition" as used herein means any composition and / or additive prepared in a process for providing a baked food, including but not limited to baking flour, dough, baking additives and / or baked products. The food composition or additive can be liquid or solid.

[0313] The term "flour" as used herein means ground or milled grains. The term "flour" may also mean ground or mashed sago or tuber products. In certain embodiments, flour may contain a variety of components in addition to ground or mashed grains or plant matter. An example of an additional component (non-limiting) is a leavening agent. Grains include wheat, oats, rye and barley. Tuber products include tapioca flour, cassava flour and custard flour. The term "flour" also includes ground corn flour, corn meal, rice flour, whole-meal flour, self-rising flour, cassava flour, cassava flour, ground rice, enriched flowers and custard flour.

[0314] For commercial and domestic use of flour for baking and food production, it is important to maintain an appropriate level of glucoamylase activity in the flour. Too high an activity level may result in a sticky and / or clumping product that cannot be marketed. Flour with insufficient glucoamylase activity may not contain enough sugars for the yeast to function properly, resulting in a dry, brittle bread or baked product. Therefore, AfGATR or a variant thereof may be added to flour by itself or in combination with one or more alpha amylases to increase the level of endogenous glucoamylase activity in the flour.

[0315] Amylases can be added alone or in combination with other amylases to prevent or delay staling of baked products, i.e., crumb firming. The amount of anti-staling amylase will typically be in the range of 0.01-10 mg enzyme protein / kg flour, e.g., 0.5 mg / kg dry solids. Additional anti-staling amylases that can be used in combination with AfGATR or a variant thereof include endoamylases, e.g., bacterial endoamylases from Bacillus. The additional amylase can be another maltogenic alpha-amylase (EC 3.2.1.133), e.g., from Bacillus. is an exemplary maltogenic alpha-amylase from Bacillus stearothermophilus strain NCIB 11837 and is described, for example, in Christophersen et al., (1997), Starch, 50:39-45. Other examples of resistant endo-amylases include bacterial alpha-amylases derived from Bacillus, such as Bacillus licheniformis or Bacillus amyloliquefaciens. resistant staling amylases can be exo-amylases, such as beta-amylases, for example from plant sources such as soybeans, or from microbial sources such as Bacillus.

[0316] The baking composition comprising AfGATR or a variant thereof may further comprise a phospholipase or an enzyme having phospholipase activity. The activity of the enzyme having phospholipase activity may be measured in lipase units (LU). The phospholipase may have A 1 or A 2 Phospholipids can be active to remove fatty acids from phospholipids, thereby forming lysophospholipids. It may or may not have lipase activity, i.e., activity on triglyceride substrates. Phospholipases typically have an optimum temperature in the range of 30-90° C., for example 30-70° C. The added phospholipases can be of animal origin, for example from pancreas such as bovine or porcine pancreas, snake venom or bee venom. Alternatively, the phospholipases can be of microbial origin, for example from filamentous fungi, yeast or bacteria.

[0317] The phospholipase is added in an amount that improves the softness of the bread in the initial period after baking, especially in the first 24 hours. The amount of the phospholipase will generally be in the range of 0.01-10 mg enzyme protein / kg flour, for example 0.1-5 mg / kg. That is, the phospholipase activity will generally be in the range of 20-1000 LU / kg flour, wherein a lipase unit is defined as the amount of enzyme required to release 1 micromole of butyric acid per minute at 30°C and pH 7.0 with gum arabic as an emulsifier and tributyrin as a substrate.

[0318] Dough compositions typically comprise wheat meal or wheat flour and / or other types of meal, flour or starch, such as corn flour, corn starch, rye meal, rye flour, oat flour, oat meal, soy flour, sorghum meal, sorghum flour, potato meal, potato flour or potato starch. The dough may be fresh, frozen or prebaked. The dough may be a puffed dough or a dough to be puffed. The dough may be puffed in various ways, such as by adding a chemical leavening agent such as sodium bicarbonate, or by adding a leavening agent, i.e., a fermented dough. The dough may also be puffed by adding a suitable yeast culture, such as a culture of Saccharomyces cerevisiae (baker's yeast), such as a commercially available strain of Saccharomyces cerevisiae.

[0319] The dough may also include other conventional dough ingredients, such as proteins, such as milk powder, gluten and soy; eggs (e.g., whole eggs, egg yolks or egg whites); antioxidants, such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate; amino acids, such as L-cysteine; sugars; or salts, such as sodium chloride, calcium acetate, sodium sulfate or calcium sulfate. The dough may also include fats, such as triglycerides, such as granulated fat or shortening. The dough may also include emulsifiers, such as monoglycerides or diglycerides, diacyl tartaric acid esters of monoglycerides or diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetate esters of monoglycerides, polyoxyethylene stearates or lysolecithin. For example, the dough may be prepared without the addition of an emulsifier.

[0320] The dough product can be any processed dough product, including fried, deep fried, baked, baked, steamed or boiled dough, such as steamed bread and rice crackers. In one embodiment, the food is a baked product. Typical baked (baked) products include breads, such as buns, rolls, buns, bagels, pizza bases, etc., yeasted pastries, pretzels, unleavened tortillas, cakes, cookies, biscuits, crackers, etc.

[0321] Optionally, additional enzymes can be used together with the anti-staling amylase and the phospholipase. The additional enzyme can be a second amylase such as an amyloglucosidase, a β-amylase, a cyclodextrin glucanotransferase, or the additional enzyme can be a peptidase, especially an exopeptidase, a transglutaminase, a lipase, a cellulase, a xylanase, a protease, a protein disulfide isomerase such as a protein disulfide isomerase disclosed in WO95 / 00636, for example a glycosyltransferase, a branching enzyme (1,4-α-glucan branching enzyme), a 4-α-glucanotransferase (dextrin glycosyltransferase) or an oxidoreductase, for example a peroxidase, a laccase, a glucose oxidase, a pyranose oxidase, a lipoxygenase, an L-amino acid oxidase or a carbohydrate oxidase. The additional enzyme can be from any source, including mammals and plants, in particular microbial (bacterial, yeast or fungal) sources, and can be obtained by techniques conventionally used in the art.

[0322] Xylanases are typically derived from a microbial source, such as from bacteria or fungi, such as a strain of Aspergillus. Xylanases include, for example, Novozym They are commercially available xylanase preparations produced from Trichoderma reesei. The amyloglucosidase may be an Aspergillus niger amyloglucosidase (eg Other available amylase products include A1000 or A 5000 (Grindsted Products, Denmark) and H or P(DSM). The glucose oxidase may be a fungal glucose oxidase, in particular Aspergillus niger glucose oxidase (eg ). Exemplary proteases are

[0323] The process can be used for any kind of baked product prepared from dough, whether soft or crisp, whether white, light or dark, for example bread, in particular white, whole meal or rye bread, usually in the form of pillows or rolls, such as but not limited to baguette type bread, pita bread, unleavened tortillas, cakes, pancakes, crackers, cookies, pie crusts, crisp bread, steamed bread, pizza, etc.

[0324] AfGATR or a variant thereof can be used in a premix comprising flour and an anti-staling amylase, a phospholipase and / or a phospholipid. The premix may contain other dough-improving and / or bread-improving additives, such as any additive, including the enzymes described above. AfGATR or a variant thereof can be a component of an enzyme preparation comprising an anti-staling amylase and a phospholipase for use as a baking additive.

[0325] The enzyme preparation is optionally in the form of granules or agglomerated powders. The preparation may have a narrow particle size distribution, with more than 95% (by weight) of the particles being in the range of 25-500 μm. Granules and agglomerated powders may be prepared by conventional methods, such as by spraying AfGATR or a variant thereof onto a carrier in a fluidized bed granulator. The carrier may consist of a granular core having a suitable particle size. The carrier may be soluble or insoluble, for example a salt (such as NaCl or sodium sulfate), a sugar (such as sucrose or lactose), a sugar alcohol (such as sorbitol), starch, rice, corn residue or soy.

[0326] Encapsulated particles, i.e. glucoamylase particles, may comprise AfGATR or a variant thereof. To prepare encapsulated glucoamylase particles, the enzyme may be contacted with a food-grade lipid in an amount sufficient to suspend all glucoamylase particles. The food-grade lipid used herein may be any natural organic compound that is insoluble in water but soluble in a non-polar organic solvent (such as a hydrocarbon or diethyl ether). Suitable food-grade lipids include, but are not limited to, saturated or unsaturated triglycerides in the form of fats or oils. Examples of various fatty acids and combinations thereof constituting saturated triglycerides include, but are not limited to, butyric acid (derived from milk fat), palmitic acid (derived from animal and plant fats) and / or stearic acid (derived from animal and plant fats). Examples of fatty acids and combinations thereof constituting unsaturated triglycerides include, but are not limited to, palmitoleic acid (derived from animal and plant fats), oleic acid (derived from animal and plant fats), linoleic acid (derived from plant fats) and / or linolenic acid (derived from linseed oil). Other suitable food-grade lipids include, but are not limited to, monoglycerides and diglycerides derived from the triglycerides discussed above, as well as phospholipids and glycolipids.

[0327] Food-grade lipids, especially food-grade lipids in liquid form, are contacted with the glucoamylase particles in powder form so that the lipid material covers at least the majority, for example at least a portion of the surface of 100% of the glucoamylase particles. Therefore, each glucoamylase particle is encapsulated in lipid individually. For example, all or substantially all of the glucoamylase particles are provided in the form of thin, continuous lipid encapsulation films. This can be achieved by: first a certain amount of lipid is poured into a container, and then the glucoamylase particles are slurried so that the lipid thoroughly wets the surface of the glucoamylase particles. After short-term stirring, the encapsulated glucoamylase particles carrying a large amount of lipids are recovered on their surface. The thickness of the coating applied to the glucoamylase particles in this way can be controlled by selecting the lipid type used and by repeating the operation to form a thicker film when necessary.

[0328] Storage, handling and blending of the loaded delivery medium can be accomplished with the aid of a packaging mix. The packaging mix can include the encapsulated glucoamylase. However, the packaging mix can also contain additional ingredients as desired by the manufacturer or baker. After the encapsulated glucoamylase is blended into the dough, the baker continues with the normal production process for the product.

[0329] The advantage of the encapsulated glucoamylase granules is twofold. First, for those heat-labile enzymes, the food-grade lipids can protect the enzyme from thermal denaturation during the baking process. Therefore, although the glucoamylase is stabilized and protected in the proofing and baking stages, it is released from the protective coating in the final baked product to hydrolyze the glycosidic bonds in the polyglucan in the product. The loaded delivery medium also provides a sustained release of the active enzyme into the baked product. That is, after the baking process, the active glucoamylase continues to be released from the protective coating at a rate that can hinder the staling mechanism thereby reducing the staling mechanism.

[0330] In general, the amount of lipid applied to the glucoamylase particles can vary from a few percent to many times the total weight of the glucoamylase, depending on the nature of the lipid, the manner in which the lipid is applied to the glucoamylase particles, the composition of the dough mixture to be treated, and the severity of the dough mixing operations involved.

[0331] The delivery medium loaded, i.e., lipid-encapsulated enzyme, is added to the ingredients used to prepare the baked product in an amount that effectively prolongs the shelf life of the baked product. The baker will calculate the amount of the encapsulated alpha-amylase prepared as described above that is needed to achieve the desired anti-staling effect. The amount of the required encapsulated glucoamylase is calculated based on the concentration of the encapsulated enzyme and based on the ratio of the specified glucoamylase to flour. It has been found that a wide range of concentrations is effective, but as discussed, there is no linear correspondence between the observable improvement in anti-staling effect and the glucoamylase concentration, and above some minimum levels, a large increase in the glucoamylase concentration only brings very little additional improvement. The glucoamylase concentration actually used in a specific baking production may be much higher than the necessary minimum amount to provide a certain insurance to the baker to prevent the baker's unintentional low measurement error. The lower limit of the enzyme concentration is determined by the minimum anti-staling effect that the baker wishes to achieve.

[0332] A method of preparing a baked product may comprise: a) preparing lipid-encapsulated glucoamylase particles, wherein substantially all of the glucoamylase particles are encapsulated; b) mixing a dough containing flour; c) adding the lipid-encapsulated glucoamylase to the dough before mixing is complete and terminating the mixing before the lipid envelope is removed from the alpha-amylase; d) proofing the dough; and e) baking the dough to provide a baked product, wherein the glucoamylase is inactive during the mixing, proofing and baking stages and is active in the baked product.

[0333] The encapsulated glucoamylase can be added to the dough during the mixing cycle, for example, near the end of the mixing cycle. The encapsulated glucoamylase is added at a specific moment in the mixing stage to allow the encapsulated glucoamylase to be fully distributed throughout the dough; however, the mixing stage is terminated before the protective coating is stripped from the glucoamylase particles. Depending on the type and volume of the dough and the mixer action and speed, it may take anywhere from one minute to six minutes or more to mix the encapsulated glucoamylase into the dough, but on average two to four minutes. Thus, several variables may determine the exact procedure. First, the amount of encapsulated glucoamylase should have a total volume that is sufficient to allow the encapsulated glucoamylase to be distributed throughout the dough mixture. If the preparation of the encapsulated glucoamylase is highly concentrated, it may be necessary to add additional oil to the premix before adding the encapsulated glucoamylase to the dough. The formulation and production method may require specific modifications; however, good results are generally obtained when 25% of the oil specified in the bread dough recipe is left out of the dough to serve as a carrier for the concentrated encapsulated glucoamylase added near the end of the mixing cycle. In bread or other baked products, especially those with a low fat content such as French bread, a mixture of about 1% of the encapsulated glucoamylase by weight of dry flour is sufficient to allow the encapsulated glucoamylase to mix properly with the dough. The range of suitable percentages is wide and depends on the formulation, the final product, and the production method requirements of each baker. Secondly, the encapsulated glucoamylase suspension should be added to the mix for a sufficient time to be thoroughly mixed into the dough, but not for a time that causes excessive mechanical action to strip the protective lipid coating from the encapsulated glucoamylase particles.

[0334] A food composition is contemplated, wherein the food is an oil, meat, lard composition comprising AfGATR or a variant thereof. In this context, the term "[oil / meat / lard] composition" means any composition based on, prepared from and / or containing oil, meat or lard, respectively. A method of preparing an oil or meat or lard composition and / or an additive comprising AfGATR or a variant thereof is contemplated, the method comprising mixing a polypeptide of the invention with an oil / meat / lard composition and / or an additive component.

[0335] The food composition may be an animal feed composition, an animal feed additive and / or a pet food comprising AfGATR and variants thereof. A method of preparing such an animal feed composition, an animal feed additive composition and / or a pet food is contemplated, the method comprising mixing AfGATR and variants thereof with one or more animal feed ingredients and / or animal feed additive ingredients and / or pet food ingredients. AfGATR and variants thereof may be used to prepare an animal feed composition and / or an animal feed additive composition and / or a pet food.

[0336] The term "animal" includes all non-ruminants and ruminants. In a specific embodiment, the animal is a non-ruminant, such as a horse and a monogastric animal. Examples of monogastric animals include, but are not limited to, pigs (pig / swine), such as piglets, growing pigs, sows; poultry such as turkeys, ducks, chickens, broilers, laying hens; fish such as salmon, trout, tilapia, catfish and carp; and crustaceans such as river shrimp and prawns. In another embodiment, the animal is a ruminant, including but not limited to cattle, calves, goats, sheep, giraffes, bison, moose, elk, yaks, buffalo, deer, camels, alpacas, llamas, antelopes, pronghorns and blue cattle.

[0337] In the context of the present invention, the term "pet food" is intended to be understood as meaning food for domestic animals such as, but not limited to, dogs, cats, gerbils, hamsters, chinchillas, brown rats, guinea pigs; avian pets such as canaries, parakeets and parrots; reptile pets such as turtles, lizards and snakes; and aquatic pets such as tropical fish and frogs.

[0338] The terms "animal feed composition", "feed", and "fodder" are used interchangeably and may comprise one or more feed materials selected from the group consisting of: a) cereals, such as small grains (e.g., wheat, barley, rye, oats, and combinations thereof) and / or large grains (e.g., maize or sorghum); b) cereal by-products, such as corn gluten meal, distillers dried grains with solubles (DDGS), especially corn distillers dried grains with solubles (cDDGS), wheat bran, wheat semolina, wheat short meal, rice bran, rice hulls, oat hulls, palm kernels, and citrus pulp; c) proteins from sources such as soy, sunflower, peanut, lupine, pea, bean, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, coconut kernel, sesame; d) oils and fats from plant and animal sources; e) minerals and vitamins.

[0339] 6. Textile desizing composition and use

[0340] Compositions and methods for treating fabrics (e.g., desizing textiles) using AfGATR are also contemplated. Fabric treatment methods are well known in the art (see, e.g., U.S. Patent No. 6,077,316). For example, the feel and appearance of a fabric can be improved by a method comprising contacting the fabric with AfGATR in a solution. The fabric can be treated with the solution under pressure.

[0341] AfGATR can be applied during or after weaving of textiles, or during the desizing stage or one or more additional fabric processing steps. During weaving of textiles, the yarns are exposed to considerable mechanical strain. Before weaving on mechanical looms, warp yarns are usually coated with sizing starch or starch derivatives to increase their tensile strength and prevent breakage. AfGATR can be applied during or after weaving to remove these sizing starches or starch derivatives. After weaving, before further processing of the fabric, AfGATR can be used to remove the sizing coating to ensure a uniform and wash-resistant result.

[0342] AfGATR can be used as a washing additive (e.g., in an aqueous composition) alone or with other desizing chemicals and / or desizing enzymes to desize fabrics, including cotton-containing fabrics. AfGATR can also be used in compositions and methods for producing a stonewashed look on indigo-dyed denim fabrics and garments. For the production of clothing, the fabric can be cut and sewn into garments or garments, which are then finished. In particular, for the production of denim jeans, different enzymatic finishing methods have been developed. The finishing of denim garments typically begins with an enzymatic desizing step, during which amylolytic enzymes act on the garments to soften the fabric and make the cotton more receptive to subsequent enzymatic finishing steps. AfGATR can be used in methods for finishing denim garments (e.g., "bio-polishing"), enzymatic desizing, and imparting fabric softness and / or finishing processes.

[0343] 7. Cleaning composition

[0344] One aspect of the compositions and methods of the present invention is a cleaning composition comprising AfGATR or a variant thereof as a component.Amylase polypeptides can be used as a component in detergent compositions for hand washing, laundry washing, dishwashing and other hard surface cleaning.

[0345] 7.1. Overview

[0346] Preferably, AfGATR or a variant thereof is incorporated into a detergent at a concentration equal to or close to that of amylases conventionally used in detergents. For example, a glucoamylase polypeptide may be added in an amount corresponding to 0.00001-1 mg (calculated as pure enzyme protein) of amylase per liter of washing liquid / dishwashing liquid. Exemplary formulations are provided herein, as shown below:

[0347] Glucoamylase polypeptides can be components of detergent compositions as the sole enzyme or together with other enzymes (including other amylolytic enzymes). As such, they can be included in detergent compositions in the form of dust-free granules, stabilized liquids or protected enzymes. Dust-free granules can be produced, for example, as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452, and can optionally be coated by methods known in the art. Examples of waxy coating materials are polyethylene oxide products (polyethylene glycol, PEG) having an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols, wherein the alcohol contains 12 to 20 carbon atoms and wherein there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides and triglycerides of fatty acids. For example, GB1483591 gives examples of film-forming coating materials suitable for application by fluidized bed technology. Liquid enzyme preparations can be stabilized, for example, by adding polyols (such as propylene glycol), sugars or sugar alcohols, lactic acid or boric acid, according to established methods. Other enzyme stabilizers are known in the art. Protected enzymes can be prepared according to the methods disclosed in, for example, EP 238 216. Polyols have long been recognized as stabilizers for proteins and for improving the solubility of proteins.

[0348] The detergent composition may be in any useful form, for example as a powder, granules, paste or liquid. A liquid detergent may be aqueous, typically containing up to about 70% water, and 0% to about 30% organic solvent. It may also be in the form of a compact gel type containing about 30% water.

[0349] The detergent composition comprises one or more surfactants, each of which may be anionic, nonionic, cationic or zwitterionic. The detergent will typically contain 0% to about 50% of anionic surfactants, such as linear alkylbenzene sulfonates (LAS); α-olefin sulfonates (AOS); alkyl sulfates (fatty alcohol sulfates) (AS); alcohol ethoxysulfates (AEOS or AES); secondary alkyl sulfonates (SAS); α-sulfo fatty acid methyl esters; alkyl or alkenyl succinic acid; or soap. The composition may also contain 0% to about 40% of nonionic surfactants, such as alcohol ethoxylates (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylates, alkyl polyglycosides, alkyl dimethyl amine oxides, ethoxylated fatty acid monoethanolamides, fatty acid monoethanolamides or polyhydroxyalkyl fatty acid amides (as described, for example, in WO 92 / 06154).

[0350] The detergent composition may additionally comprise one or more other enzymes, such as a protease, another amylolytic enzyme, a cutinase, a lipase, a cellulase, a pectate lyase, a perhydrolase, a xylanase, a peroxidase and / or a laccase, in any combination.

[0351] The detergent may contain from about 1% to about 65% of a detergent builder or complexing agent, such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl or alkenyl succinic acid, soluble silicate or layered silicate (e.g., SKS-6 from Hoechst). The detergent may also be builder-free, i.e., substantially free of detergent builder. The enzyme may be used in any composition compatible with the stability of the enzyme. Enzymes may generally be protected from harmful components by known encapsulation forms (e.g., by granulation or separation in a hydrogel). Enzymes, particularly amylases (with or without a starch binding domain), may be used in a variety of compositions including laundry and dishwashing applications, surface cleaners, and in compositions for generating ethanol from starch or biomass.

[0352] The detergent may contain one or more polymers. Examples include carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polycarboxylates such as polyacrylates, maleic acid / acrylic acid copolymers and lauryl methacrylate / acrylic acid copolymers.

[0353] The detergent may contain a bleach system which may comprise H 2 O which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS). 2 O 2 Alternatively, the bleaching system may comprise a peroxyacid (eg an amide, imide or sulfone peroxyacid). The bleaching system may also be an enzyme bleaching system, such as a perhydrolase, as described in PCT International Patent Application WO 2005 / 056783.

[0354] Conventional stabilizers may be used to stabilize the enzymes of the detergent composition, such as polyols, such as propylene glycol or glycerol; sugars or sugar alcohols; lactic acid; boric acid or boric acid derivatives, such as aromatic boric acid esters; and the composition may be formulated as described, for example, in WO 92 / 19709 and WO 92 / 19708.

[0355] The detergent may also contain other conventional detergent ingredients such as fabric conditioners including clays, suds boosters, foam suppressors, corrosion inhibitors, soil suspending agents, anti-soil redeposition agents, dyes, bactericides, tarnish inhibitors, optical brighteners or perfume.

[0356] The pH (measured in aqueous solution at use concentration) is typically neutral or alkaline, for example, pH about 7.0 to about 11.0.

[0357] Specific forms for detergent compositions comprising the glucoamylase of the present invention are described below.

[0358] 7.2. Heavy Duty Liquid (HDL) Laundry Detergent Compositions

[0359] Exemplary HDL laundry detergent compositions include detersive surfactants (10%-40% w / w) including anionic detersive surfactants (selected from linear or branched or random chain, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates and / or mixtures thereof), and optionally nonionic surfactants (selected from linear or branched or random chain, substituted or unsubstituted alkyl alkoxylated alcohols, such as C 8 -C 18 Alkyl ethoxylated alcohol and / or C 6 -C 12 Alkylphenol alkoxylate), wherein the weight ratio of anionic detersive surfactant (hydrophilic index (HIc) of 6.0-9) to nonionic detersive surfactant is greater than 1: 1. Suitable detersive surfactants also include cationic detersive surfactants (selected from the group of alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds and / or mixtures thereof); zwitterionic and / or amphoteric detersive surfactants (selected from the group of alkanolamine sulfonobetaine); amphoteric surfactants; semi-polar nonionic surfactants and mixtures thereof.

[0360] The composition may optionally include a surfactant enhancing polymer, the polymer being composed of an amphiphilic alkoxylated lipid cleaning polymer (selected from alkoxylated polymers having branched hydrophilic and hydrophobic properties, such as alkoxylated polyalkyleneimines, in the range of 0.05 wt% to 10 wt%) and / or a random grafted polymer (generally composed of a hydrophilic backbone and hydrophobic side chains, the hydrophilic backbone comprising monomers selected from the group consisting of: unsaturated C 1 -C 6 Carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, saturated polyols such as glycerol, and mixtures thereof; the hydrophobic side chains are selected from: C 4 -C 25 Alkyl, polypropylene, polybutene, saturated C 1 -C 6 Vinyl esters of monocarboxylic acids, acrylic acid or methacrylic acid 1 -C 6 alkyl esters and mixtures thereof).

[0361] The composition may include additional polymers such as soil release polymers (including anionic end-capped polyesters such as SRP1, polymers comprising at least one monomer unit selected from saccharides, dicarboxylic acids, polyols and combinations thereof, in random or block configurations, ethylene terephthalate-based polymers and copolymers thereof, in random or block configurations such as Repel-o-tex SF, SF-2 and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325, Marloquest SL), anti-redeposition polymers (0.1 wt%-10 wt%, including carboxylic acid polymers, such as polymers containing at least one monomer selected from acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylenemalonic acid and any mixture thereof, vinyl pyrrolidone homopolymer and / or polyethylene glycol, with a molecular weight ranging from 500 to 100,000 Daltons); cellulosic polymers (including those selected from the following: alkyl cellulose, alkyl alkoxyalkyl cellulose, carboxyalkyl cellulose, alkyl carboxyalkyl cellulose, examples of which include carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose and mixtures thereof) and polymeric carboxylates (e.g., maleate / acrylate random copolymers or polyacrylate homopolymers).

[0362] The composition may also include saturated or unsaturated fatty acids, preferably saturated or unsaturated C 12 -C 24 Fatty acids (0 to 10 wt %); deposition aids, examples of which include polysaccharides, preferably cellulosic polymers, polydiallyldimethylammonium halides (DADMAC) and copolymers of DAD MAC with vinyl pyrrolidone, acrylamide, imidazole, imidazolinium halides and mixtures thereof, in random or block configurations, cationic guar gum, cationic cellulose such as cationic hydroxyethyl cellulose, cationic starch, cationic polyacrylamide and mixtures thereof.

[0363] The composition may also include dye transfer inhibitors, examples of which include manganese phthalocyanine, peroxidase, polyvinyl pyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinyl pyrrolidone and N-vinylimidazole, polyvinyl oxazole dione and polyvinylimidazole and / or mixtures thereof; chelating agents, examples of which include ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentamethylenephosphonic acid (DTPMP), hydroxyethanediphosphonic acid (HEDP), ethylenediamine N,N'-disuccinic acid (EDDS), methylglycine diacetic acid (MGDA), diethylenetriaminepentaacetic acid (DTPA), propylenediaminetetraacetic acid (PDT A), 2-hydroxypyridine-N-oxide (HPNO), or methylglycine diacetic acid (MGDA), glutamic acid N,N-diacetic acid (N,N-dicarboxymethylglutamate tetrasodium salt (GLDA), nitrilotriacetic acid (NTA), 4,5-dihydroxy-m-benzenedisulfonic acid, citric acid and any salt thereof, N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP) and their derivatives.

[0364] The composition preferably includes an enzyme (typically about 0.01 wt % active enzyme to 0.03 wt % active enzyme) selected from the group consisting of proteases, amylases, lipases, cellulases, choline oxidases, peroxidases / oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferases, perhydrolases, arylesterases, and any mixtures thereof. The composition may include an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or a boric acid derivative, such as an aromatic borate ester, or a phenylboronic acid derivative such as 4-formylphenylboronic acid).

[0365] The composition optionally includes silicone or fatty acid based foam suppressors; heuing dyes, calcium and magnesium cations, visual signal ingredients, foam suppressors (0.001 wt % to about 4.0 wt %) and / or structurants / thickeners (0.01 wt % to 5 wt %, selected from di- and tri-glycerides, ethylene glycol distearate, microcrystalline cellulose, cellulose based materials, microfiber cellulose, biopolymers, xanthan gum, gellan gum and mixtures thereof).

[0366] The composition may be in any liquid form, such as a liquid or gel form, or any combination thereof. The composition may be in any unit dosage form, such as a pouch.

[0367] 7.3. Heavy Duty Dry / Solid (HDD) Laundry Detergent Compositions

[0368] Exemplary HDD laundry detergent compositions include detersive surfactants, including anionic detersive surfactants (e.g., linear or branched or random chain, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates and / or mixtures thereof), nonionic detersive surfactants (e.g., linear or branched or random chain, substituted or unsubstituted C 8 -C 18 Alkyl ethoxylate and / or C 6 -C 12 alkylphenol alkoxylates), cationic detersive surfactants (e.g., alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds, and mixtures thereof), zwitterionic and / or amphoteric detersive surfactants (e.g., alkanolamine sulfonobetaines), amphoteric surfactants, semi-polar nonionic surfactants, and mixtures thereof; builders, including phosphate-free builders (e.g., zeolite builders, examples of which include zeolite A, zeolite X, zeolite P, and zeolite MAP, ranging from 0 wt % to less than 10 wt %), phosphoric acid Salt builders (e.g., sodium tripolyphosphate, ranging from 0 wt % to less than 10 wt %), citric acid, citrates and nitrilotriacetic acid, silicates (e.g., sodium silicate or potassium silicate or sodium metasilicate, ranging from 0 wt % to less than 10 wt %, or layered silicate (SKS-6)); carbonates (e.g., sodium carbonate and / or sodium bicarbonate, ranging from 0 wt % to less than 80 wt %); and bleaches, including photobleaches (e.g., sulfonated zinc phthalocyanine, sulfonated aluminum phthalocyanine, xanthene dyes, and mixtures thereof), hydrophobic or hydrophilic bleach activators (e.g., dodecanoyl hydroxybenzene sulfonate, decanoylhydroxybenzene sulfonate, decanoylhydroxybenzoic acid or its salt, 3,5,5-trimethylhexanoylhydroxybenzene sulfonate, tetraacetylethylenediamine-TAED, nonanoyloxybenzene sulfonate-NOBS, nitrile quaternary ammonium compounds, and mixtures thereof), a hydrogen peroxide source (e.g., an inorganic perhydrate salt, examples of which include monohydrate or tetrahydrate sodium salts of perborate, percarbonate, persulfate, perphosphate or persilicate), a preformed hydrophilic and / or hydrophobic peracid (e.g., percarboxylic acids and their salts, percarbonic acids and their salts, perimidic acids and their salts, permonosulfuric acids and their salts, Acids and their salts, and mixtures thereof), and / or bleach catalysts (e.g., imine bleach boosters (examples of which include imine cations and polyions), imine zwitterions, modified amines, modified amine oxides, N-sulfonyl imines, N-phosphonyl imines, N-acyl imines, thiadiazole dioxides, perfluoroimines, cyclic sugar ketones, and mixtures thereof, and metal-containing bleach catalysts (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations and auxiliary metal cations such as zinc or aluminum and chelating agents such as ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and water-soluble salts thereof).

[0369] The composition preferably includes enzymes such as proteases, amylases, lipases, cellulases, choline oxidases, peroxidases / oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferases, perhydrolases, arylesterases and any mixtures thereof.

[0370] The composition may optionally include additional detergent ingredients including perfume microcapsules, starch encapsulated perfume accords, hueing agents, additional polymers including fabric integrity and cationic polymers, dye locking ingredients, fabric softeners, brighteners (e.g. CI fluorescent brighteners), flocculants, chelants, alkoxylated polyamines, fabric deposition aids and / or cyclodextrins.

[0371] 7.4. Automatic Dishwashing (ADW) Detergent Compositions

[0372] Exemplary ADW detergent compositions include nonionic surfactants, including ethoxylated nonionic surfactants, alcohol alkoxylated surfactants, epoxy-terminated poly(oxyalkylated) alcohols or amine oxide surfactants, present in an amount of 0 to 10% by weight; builders, ranging from 5-60%, including phosphate builders (e.g., monophosphates, diphosphates, tripolyphosphates, other oligomeric polyphosphates, sodium tripolyphosphate-STPP) and phosphate-free builders (e.g., amino acid-based compounds, including methyl-glycine-diacetic acid (MGDA) and its salts and derivatives, glutamic acid-N,N-diacetic acid (GLDA) and its salts and derivatives, iminodisuccinic acid (IDS) and its salts and derivatives, carboxymethyl Inulin and its salts and derivatives, nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), B-alaninediacetic acid (B-ADA) and its salts, polycarboxylic acids and their partially or completely neutralized salts, homopolymers and copolymers of monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts, ranging from 0.5 wt % to 50 wt %); sulfonated / carboxylated polymers, ranging from about 0.1 wt % to about 50 wt % to provide dimensional stability; drying aids, ranging from about 0.1 wt % to about 10 wt % (e.g., polyesters, especially anionic polyesters, optionally and other monomers having 3 to 6 functional groups (usually acid, alcohol or ester functional groups that favor polycondensation), polycarbonate-polyorganosiloxanes, polyurethane-polyorganosiloxanes and / or polyurea-polyorganosiloxane compounds or precursor compounds thereof, particularly reactive cyclic carbonate and urea types); silicates ranging from about 1 wt % to about 20 wt % (including sodium or potassium silicates, such as sodium disilicate, sodium metasilicate and crystalline layered silicates); inorganic bleaches (e.g., perhydrate salts such as perborates, percarbonates, perphosphates, persulfates and persilicates) and organic bleaches (e.g., organic peroxyacids, including diacyl and tetraacyl peroxides, such as diperoxydodecanedioic acid, diperoxytetradecandioic acid and diperoxyhexadecanedioic acid); bleach activators (i.e., organic peracid precursors, ranging from about 0.1 wt % to about 10 wt %); bleach catalysts (e.g., triazacyclononane manganese and phase % to 5 wt % (e.g., benzotriazoles, metal salts and complexes, and / or silicates); enzymes ranging from about 0.01 to 5.0 mg active enzymes per gram of the automatic dishwashing detergent composition (e.g., proteases, amylases, lipases, cellulases, choline oxidases, peroxidases / oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferases, perhydrolases, arylesterases, and mixtures thereof); and enzyme stabilizer components (e.g., oligosaccharides, polysaccharides, and inorganic divalent metal salts).

[0373] 7.5. Additional detergent compositions

[0374] Additional exemplary detergent formulations to which the amylases of the present invention can be added are described in the numbered paragraphs below.

[0375] 1) a detergent composition in granular form formulated to have a bulk density of at least 600 g / L, comprising from about 7% to about 12% of a linear alkylbenzene sulfonate (calculated as acid); from about 1% to about 4% of an alcohol ethoxysulfate (e.g., C 12-18 alcohol 1-2 ethylene oxide (EO)) or alkyl sulfate (e.g., C 16-18 ); from about 5% to about 9% of an alcohol ethoxylate (e.g., C 14-15 alcohol, 7EO); about 14% to about 20% sodium carbonate (e.g., Na 2 CO 3 ); about 2% to about 6% soluble silicate (e.g., Na 2 O,2SiO 2 ); about 15% to about 22% zeolite (e.g., NaA1SiO 4 ); 0% to about 6% sodium sulfate (e.g., Na 2 SO 4 ); about 0% to about 15% sodium citrate / citric acid (e.g., C 6 H 5 Na 3 O 7 / C 6 H 8 O 7 ); about 11% to about 18% sodium perborate (e.g., NaBO 3 H 2 O); about 2% to about 6% TAED; 0% to about 2% carboxymethyl cellulose (CMC); 0-3% polymers (e.g., maleic acid / acrylic acid copolymers, PVP, PEG); 0.0001-0.1% enzymes (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., foam inhibitors, perfumes, optical brighteners, photobleaches).

[0376] 2) a detergent composition formulated in granular form having a bulk density of at least 600 g / L, comprising from about 6% to about 11% of a linear alkylbenzene sulfonate (calculated as acid); from about 1% to about 3% of an alcohol ethoxysulfate (e.g., C 12-18 Alcohol 1-2EO) or alkyl sulfate (e.g., C 16-18 ); from about 5% to about 9% of an alcohol ethoxylate (e.g., C 14-15 alcohol, 7EO); about 15% to about 21% sodium carbonate (e.g., Na 2 CO 3); about 1% to about 4% soluble silicate (e.g., Na 2 O,2SiO 2 ); about 24% to about 34% zeolite (e.g., NaA1SiO 4 ); about 4% to about 10% sodium sulfate (e.g., Na 2 SO 4 ); 0% to about 15% sodium citrate / citric acid (e.g., C 6 H 5 Na 3 O 7 / C 6 H 8 O 7 ); 0% to about 2% carboxymethyl cellulose (CMC); 1-6% polymers (e.g., maleic acid / acrylic acid copolymers, PVP, PEG); 0.0001-0.1% enzymes (calculated as pure enzyme protein); 0-5% minor ingredients (e.g., foam inhibitors, fragrances).

[0377] 3) a detergent composition formulated in granular form having a bulk density of at least 600 g / L, comprising from about 5% to about 9% of a linear alkylbenzene sulfonate (calculated as acid); from about 7% to about 14% of an alcohol ethoxylate (e.g., C 12-15 alcohol, 7EO); about 1% to about 3% soap such as fatty acids (e.g., C 16-22 fatty acids); about 10% to about 17% sodium carbonate (such as Na 2 CO 3 ); about 3% to about 9% soluble silicates (e.g., Na 2 O,2SiO 2 ); about 23% to about 33% zeolite (such as NaA1SiO 4 ); 0% to about 4% sodium sulfate (e.g., Na 2 SO 4 ); about 8% to about 16% sodium perborate (e.g., NaBO 3 H 2 O); about 2% to about 8% TAED; 0% to about 1% phosphonate (e.g., EDTMPA); 0% to about 2% carboxymethyl cellulose (CMC); 0-3% polymer (e.g., maleic acid / acrylic acid copolymer, PVP, PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); 0-5% minor ingredients (e.g., foam inhibitors, fragrances, optical brighteners).

[0378] 4) a detergent composition formulated in granular form having a bulk density of at least 600 g / L, comprising from about 8% to about 12% of a linear alkylbenzene sulfonate (calculated as acid); from about 10% to about 25% of an alcohol ethoxylate (e.g., C12-15 alcohol, 7EO); about 14% to about 22% sodium carbonate (such as Na 2 CO 3 ); about 1% to about 5% soluble silicate (e.g., Na 2 O,2SiO 2 ); about 25% to about 35% zeolite (e.g., NaAlSiO 4 ); 0% to about 10% sodium sulfate (e.g., Na 2 SO 4 ); 0% to about 2% carboxymethyl cellulose (CMC); 1-3% polymers (e.g., maleic acid / acrylic acid copolymers, PVP, PEG); 0.0001-0.1% enzymes (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., foam inhibitors, fragrances).

[0379] 5) An aqueous liquid detergent composition comprising from about 15% to about 21% of a linear alkylbenzene sulfonate (calculated as acid); from about 12% to about 18% of an alcohol ethoxylate (e.g., C 12-15 Alcohol, 7EO or C 12-15 alcohol, 5EO); about 3% to about 13% soap such as fatty acids (e.g., oleic acid); 0% to about 13% alkenyl succinic acid (C 12-14 ); about 8% to about 18% aminoethanol; about 2% to about 8% citric acid; 0% to about 3% phosphonate; 0% to about 3% polymer (e.g., PVP, PEG); 0% to about 2% borate (e.g., B 4 O 7 ); 0% to about 3% ethanol; about 8% to about 14% propylene glycol; 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., dispersants, foam inhibitors, fragrances, optical brighteners).

[0380] 6) an aqueous structured liquid detergent composition comprising from about 15% to about 21% of a linear alkylbenzene sulfonate (calculated as acid); 3-9% of an alcohol ethoxylate (e.g., C 12-15 Alcohol, 7EO or C 12-15 alcohol, 5EO); about 3% to about 10% soap such as fatty acid (e.g., oleic acid); about 14% to about 22% zeolite (e.g., NaAlSiO 4 ); about 9% to about 18% potassium citrate; 0% to about 2% borate (e.g., B 4 O 7); 0% to about 2% carboxymethyl cellulose (CMC); 0% to about 3% polymers (e.g., PEG, PVP); 0% to about 3% anchoring polymers (e.g., lauryl methacrylate / acrylic acid copolymer; molar ratio of 25:1, MW 3800); 0% to about 5% glycerol; 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., dispersants, foam inhibitors, fragrances, optical brighteners).

[0381] 7) A detergent composition formulated in granular form having a bulk density of at least 600 g / L, comprising from about 5% to about 10% fatty alcohol sulfate; from about 3% to about 9% ethoxylated fatty acid monoethanolamide; 0-3% soap such as fatty acid; from about 5% to about 10% sodium carbonate (e.g., Na 2 CO 3 ); about 1% to about 4% soluble silicate (e.g., Na 2 O,2SiO 2 ); about 20% to about 40% zeolite (e.g., NaAlSiO 4 ); about 2% to about 8% sodium sulfate (e.g., Na 2 SO 4 ); about 12% to about 18% sodium perborate (e.g., NaBO 3 H 2 O); about 2% to about 7% TAED; about 1% to about 5% polymer (e.g., maleic acid / acrylic acid copolymer, PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., optical brighteners, foam inhibitors, fragrances).

[0382] 8) A detergent composition formulated in granular form comprising from about 8% to about 14% of a linear alkylbenzene sulfonate (calculated as acid); from about 5% to about 11% of an ethoxylated fatty acid monoethanolamide; from 0% to about 3% of a soap such as a fatty acid; and from about 4% to about 10% of sodium carbonate (e.g., Na 2 CO 3 ); about 1% to about 4% soluble silicate (Na 2 O,2SiO 2 ); about 30% to about 50% zeolite (e.g., NaAlSiO 4 ); about 3% to about 11% sodium sulfate (e.g., Na 2 SO 4 ); about 5% to about 12% sodium citrate (e.g., C 6 H 5 Na 3 O 7); about 1% to about 5% polymer (e.g., PVP, maleic acid / acrylic acid copolymer, PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., foam inhibitor, fragrance).

[0383] 9) A detergent composition formulated in granular form comprising from about 6% to about 12% of a linear alkylbenzene sulfonate (calculated as acid); from about 1% to about 4% of a nonionic surfactant; from about 2% to about 6% of a soap such as a fatty acid; and from about 14% to about 22% of sodium carbonate (e.g., Na 2 CO 3 ); about 18% to about 32% zeolite (e.g., NaAlSiO 4 ); about 5% to about 20% sodium sulfate (e.g., Na 2 SO 4 ); about 3% to about 8% sodium citrate (e.g., C 6 H 5 Na 3 O 7 ); about 4% to about 9% sodium perborate (e.g., NaBO 3 H 2 O); about 1% to about 5% bleach activator (e.g., NOBS or TAED); 0% to about 2% carboxymethyl cellulose (CMC); about 1% to about 5% polymer (e.g., polycarboxylate or PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., optical brighteners, perfume).

[0384] 10) An aqueous liquid detergent composition comprising from about 15% to about 23% of a linear alkylbenzene sulfonate (calculated as acid); from about 8% to about 15% of an alcohol ethoxysulfate (e.g., C 12-15 alcohol, 2-3EO); about 3% to about 9% of alcohol ethoxylate (e.g., C 12-15 Alcohol, 7EO or C 12-15 alcohol, 5EO); 0% to about 3% soap such as fatty acids (e.g., lauric acid); about 1% to about 5% aminoethanol; about 5% to about 10% sodium citrate; about 2% to about 6% hydrotropes (e.g., sodium toluene sulfonate); 0% to about 2% borates (e.g., B 4 O 7 ); 0% to about 1% carboxymethyl cellulose; about 1% to about 3% ethanol; about 2% to about 5% propylene glycol; 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., polymers, dispersants, fragrances, optical brighteners).

[0385] 11) An aqueous liquid detergent composition comprising from about 20% to about 32% of a linear alkylbenzene sulfonate (calculated as acid); 6-12% of an alcohol ethoxylate (e.g., C 12-15 Alcohol, 7EO or C 12-15 about 2% to about 6% aminoethanol; about 8% to about 14% citric acid; about 1% to about 3% borate (e.g., B 4 O 7 ); 0% to about 3% polymer (e.g., maleic acid / acrylic acid copolymer, anchoring polymer such as lauryl methacrylate / acrylic acid copolymer); about 3% to about 8% glycerol; 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., hydrotropes, dispersants, fragrances, optical brighteners).

[0386] 12) A detergent composition formulated in granular form with a bulk density of at least 600 g / L, comprising from about 25% to about 40% anionic surfactant (linear alkylbenzene sulfonate, alkyl sulfate, α-olefin sulfonate, α-sulfo fatty acid methyl ester, alkyl sulfonate, soap); from about 1% to about 10% nonionic surfactant (e.g., alcohol ethoxylate); from about 8% to about 25% sodium carbonate (e.g., Na 2 CO 3 ); about 5% to about 15% soluble silicate (e.g., Na 2 O,2SiO 2 ); 0% to about 5% sodium sulfate (e.g., Na 2 SO 4 ); about 15% to about 28% zeolite (NaA1SiO 4 ); 0% to about 20% sodium perborate (e.g., NaBO 3 ·4H 2 O); about 0% to about 5% bleach activator (TAED or NOBS); 0.0001-0.1% enzyme (calculated as pure enzyme protein); 0-3% minor ingredients (e.g., perfume, optical brightener).

[0387] 13) A detergent composition as described in the above compositions 1) to 12), wherein all or part of the linear alkylbenzene sulfonate is replaced by (C 12 -C 18 ) alkyl sulfate instead.

[0388] 14) A detergent composition in granular form formulated to have a bulk density of at least 600 g / L, comprising from about 9% to about 15% of (C 12 -C 18) alkyl sulfate; about 3% to about 6% alcohol ethoxylate; about 1% to about 5% polyhydroxy alkyl fatty acid amide; about 10% to about 20% zeolite (e.g., NaAlSiO 4 ); about 10% to about 20% of a layered disilicate (e.g., SK56 from Hoechst); about 3% to about 12% of sodium carbonate (e.g., Na 2 CO 3 ); 0% to about 6% soluble silicates (e.g., Na 2 O,2SiO 2 ); about 4% to about 8% sodium citrate; about 13% to about 22% sodium percarbonate; about 3% to about 8% TAED; 0% to about 5% polymers (e.g., polycarboxylates and PVP); 0.0001-0.1% enzymes (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., optical brighteners, photobleaches, perfumes, foam inhibitors).

[0389] 15) A detergent composition in granular form formulated to have a bulk density of at least 600 g / L, comprising from about 4% to about 8% of (C 12 -C 18 ) alkyl sulfate; about 11% to about 15% alcohol ethoxylate; about 1% to about 4% soap; about 35% to about 45% zeolite MAP or zeolite A; about 2% to about 8% sodium carbonate (such as Na 2 CO 3 ); 0% to about 4% soluble silicates (e.g., Na 2 O,2SiO 2 ); about 13% to about 22% sodium percarbonate; 1-8% TAED; 0% to about 3% carboxymethyl cellulose (CMC); 0% to about 3% polymers (e.g., polycarboxylates and PVP); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-3% minor ingredients (e.g., optical brighteners, phosphonates, fragrances).

[0390] 16) Detergent formulations as described above in 1) to 15), which contain stabilized or encapsulated peracids as an additional component or as a replacement for the bleaching systems already mentioned.

[0391] 17) A detergent composition as described in 1), 3), 7), 9) and 12) above, wherein perborate is replaced by percarbonate.

[0392] 18) A detergent composition as described in 1), 3), 7), 9), 12), 14) and 15) above, further comprising a manganese catalyst. For example, the manganese catalyst is one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching", Nature, 369: 637-639 (1994).

[0393] 19) Detergent compositions formulated as non-aqueous detergent liquids comprising a liquid nonionic surfactant, such as a linear alkoxylated primary alcohol, a builder system (e.g. phosphates), an enzyme and an alkali. The detergent may also contain an anionic surfactant and / or a bleaching system.

[0394] As mentioned above, the amylase polypeptides of the invention may be incorporated at concentrations conventionally employed in detergents. It is presently envisaged that in detergent compositions, the enzyme may be added in an amount corresponding to 0.00001-1.0 mg (calculated as pure enzyme protein) of amylase polypeptide per liter of wash liquor.

[0395] The detergent composition may also contain other conventional detergent ingredients such as deflocculant materials, filler materials, defoamers, corrosion inhibitors, soil suspending agents, chelating agents, anti-soil redeposition agents, dehydrating agents, dyes, bactericides, fluorescent agents, thickeners and perfumes.

[0396] The detergent compositions may be formulated as hand (manual) or machine (automatic) laundry detergent compositions, containing a laundry additive composition suitable for pre-treatment of soiled fabrics and a rinse added fabric softener composition, or may be formulated as detergent compositions for general household hard surface cleaning operations, or may be formulated for use in manual or automatic dishwashing operations.

[0397] Any cleaning composition described herein can include any number of additional enzymes. Generally, the enzyme should be compatible with the selected detergent (e.g., in terms of optimal pH, compatibility with other enzyme components or non-enzyme components, etc.), and the enzyme should be present in an effective amount. The following enzymes are provided as examples.

[0398] Proteases: Suitable proteases include those of animal, plant or microbial origin. Chemically modified or protein engineered mutants are included, as well as naturally made proteins. Proteases may be serine proteases or metalloproteases, alkaline microbial proteases, trypsin-like proteases or chymotrypsin-like proteases. Examples of alkaline proteases are subtilisins, such as those derived from Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (see, e.g., WO 89 / 06279). Examples of trypsin-like proteases are trypsin (e.g., porcine or bovine origin) and Fusarium proteases (see, e.g., WO 89 / 06270 and WO 94 / 25583). Examples of available proteases also include, but are not limited to, variants described in WO 92 / 19729, WO 98 / 20115, WO 98 / 20116 and WO 98 / 34946. Commercially available proteases include, but are not limited to: Primase TM 、Duralase TM , Kannase TM and BLAZE TM (Novo Nordisk and Novozymes); Maxacal TM 、Maxapem TM , Purafect OxP TM 、FN2 TM and FN3 TM (Danisco, Inc., USA). Other exemplary proteases include NprE from Bacillus amyloliquifaciens and ASP from Cellulomonas sp. strain 69B4.

[0399] Lipases: Suitable lipases include those of bacterial or fungal origin, including chemically modified, proteolytically modified or protein engineered mutants. Examples of useful lipases include, but are not limited to, lipases from the genus Humicola (synonym thermophilic fungi), e.g., from H. lanuginosa (T. lanuginosus) (see, e.g., EP 258068 and EP 305216), from H. insolens (see, e.g., WO 96 / 13580); Pseudomonas lipases (e.g., from Pseudomonas alcaligenes or P. pseudoalcaligenes (see, e.g., EP 218272), Pseudomonas cepacia (see, e.g., EP 331 376), Pseudomonas stutzeri (see, e.g., GB 1,372,034), Pseudomonas fluorescens, Pseudomonas strain SD 705 (see, e.g., WO 96 / 13580); 95 / 06720 and WO 96 / 27002), P. wisconsinensis (see, e.g., WO 96 / 12012)); Bacillus lipase (e.g., from Bacillus subtilis (see, e.g., Dartois et al., Biochemica et Biophysica Acta, 1131:253-360 (1993)), Bacillus stearothermophilus (see, e.g., JP 64 / 744992), or Bacillus pumilus (see, e.g., WO 91 / 16422)). Other lipase variants contemplated for use in formulations include, for example, those described in WO 92 / 05249, WO 94 / 01541, WO 95 / 35381, WO 96 / 00292, WO 95 / 30744, WO 94 / 25578, WO 95 / 14783, WO 95 / 22615, WO 97 / 04079, WO 97 / 07202, EP 407225, and EP 260105. Some commercially available lipases include and Lipolase Ultra TM (Novo Nordisk and Novozymes).

[0400] Polyesterase: Suitable polyesterases may be included in the composition, such as those described in WO 01 / 34899, WO 01 / 14629 and US6933140.

[0401] Amylase: The composition can be combined with an amylase (e.g., a non-production-enhanced amylase). These can include commercially available amylases such as, but not limited to and BAN TM (Novo Nordisk and Novozymes); and (From Danisco USA).

[0402] Cellulase: Cellulase can be added to the composition. Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, such as, for example, fungal cellulases produced by Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757, and WO 89 / 09259. Exemplary cellulases contemplated for use are those having color care benefits for textiles. Examples of such cellulases are cellulases described in, for example, EP 0495257, EP 0531372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are cellulase variants, such as those described in WO 94 / 07998, WO 98 / 12307, WO 95 / 24471, PCT / DK98 / 00299, EP 531315, U.S. Pat. Nos. 5,457,046, 5,686,593, and 5,763,254. Commercially available cellulases include and (Novo Nordisk and Novozymes); and Puradax (Danisco Corporation); and KAC-500(B) TM (Kao Corporation).

[0403] Peroxidase / oxidase: Suitable peroxidase / oxidase enzymes contemplated for use in the composition include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of peroxidases that can be used include peroxidases from Coprinus, e.g., C. cinereus, and variants thereof as described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257. Commercially available peroxidases include, for example, GuardzymeTM (Novo Nordisk and Novozymes).

[0404] The detergent composition may also comprise 2,6-β-D-fructan hydrolase, which may be effective for removing / cleaning biofilm present on household and / or industrial textiles / laundry.

[0405] Detergent enzymes can be included in detergent compositions by adding a single additive containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. The detergent additive (i.e., a single additive or a combined additive) can be formulated, for example, as a granule, a liquid, a slurry, etc. Exemplary detergent additive formulations include, but are not limited to, granules, especially non-dusting granules, liquids, especially stable liquids, or slurries.

[0406] Dust-free granules can be produced, for example, as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452, and can optionally be coated by methods known in the art. Examples of waxy coating materials are polyethylene oxide products (e.g., polyethylene glycol, PEG) having an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols, wherein the alcohol contains 12 to 20 carbon atoms and wherein there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. For example, GB 1483591 gives examples of film-forming coating materials suitable for application by fluidized bed technology. Liquid enzyme preparations can be stabilized, for example, by adding polyols (e.g., propylene glycol), sugars or sugar alcohols, lactic acid or boric acid, according to established methods. Protected enzymes can be prepared according to the method disclosed in EP 238,216.

[0407] The detergent composition can be in any convenient form, such as a bar, tablet, powder, granule, paste or liquid. Liquid detergents can be aqueous, typically containing up to about 70% water, and 0% to about 30% organic solvent. Compact detergent gels containing about 30% or less water are also contemplated. The detergent composition can optionally contain one or more surfactants, which can be nonionic, including semi-polar and / or anionic and / or cationic and / or zwitterionic. The surfactant can be present in a wide range (about 0.1% by weight to about 60% by weight).

[0408] When included in a detergent, the detergent will typically contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzene sulfonate, α-olefin sulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkyl sulfonate, α-sulfo fatty acid methyl ester, alkyl or alkenyl succinic acid or soap.

[0409] When included in a detergent, the detergent will typically contain from about 0.2% to about 40% of a nonionic surfactant such as alcohol ethoxylates, nonylphenol ethoxylates, alkyl polyglycosides, alkyl dimethylamine oxides, ethoxylated fatty acid monoethanolamides, fatty acid monoethanolamides, polyhydroxyalkyl fatty acid amides, or N-acyl-N-alkyl derivatives of glucosamine ("glucamides").

[0410] The detergent may contain 0% to about 65% of a detergent builder or complexing agent such as zeolites, diphosphates, triphosphates, phosphonates, carbonates, citrates, nitrilotriacetic acid, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid, alkyl or alkenyl succinic acid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst Corporation).

[0411] The detergent may comprise one or more polymers. Exemplary polymers include carboxymethylcellulose (CMC), poly(vinyl pyrrolidone) (PVP), poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine-N-oxide), poly(vinylimidazole), polycarboxylates (e.g., polyacrylates), maleic acid / acrylic acid copolymers, and lauryl methacrylate / acrylic acid copolymers.

[0412] Conventional stabilizers may be used to stabilize the enzymes of the detergent composition, such as polyols (e.g., propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives (e.g., aromatic borate esters) or phenylboronic acid derivatives (e.g., 4-formylphenylboronic acid). The composition may be formulated as described in WO 92 / 19709 and WO 92 / 19708.

[0413] It is envisaged that in detergent compositions, inter alia, the enzyme variant may be added in an amount corresponding to about 0.01 to about 100 mg enzyme protein per liter of wash liquor, for example about 0.05 to about 5.0 mg enzyme protein per liter of wash liquor or 0.1 to about 1.0 mg enzyme protein per liter of wash liquor.

[0414] While the compositions and methods of the present invention have been described with reference to the following details, it will be understood that various modifications may be made.

[0415] 7.6. Methods for Assessing Amylase Activity in Detergent Compositions

[0416] Many glucoamylase cleaning assays are known in the art, including sample assays and microsample assays. The attached examples describe only a few of these assays.

[0417] 8. Brewing composition

[0418] AfGATR or a variant thereof can be a component of a brewing composition used in a process for providing a fermented beverage such as brewing. It is believed that non-fermentable carbohydrates constitute the majority of the dissolved solids in the final beer. This residue remains because malt amylase is unable to hydrolyze the α-1,6-bonds in the starch. Non-fermentable carbohydrates contribute about 50 calories per 12 ounces (about 340 grams) of beer. AfGATR or a variant thereof is typically combined with a glucoamylase and optionally with a pullulanase and / or an isoamylase, which can help convert starch into dextrins and fermentable sugars, reducing residual non-fermentable carbohydrates in the final beer.

[0419] The main raw materials used to prepare these beverages are water, hops and malt. In addition and exclusively, adjuncts such as common coarse ground corn meal, refined coarse ground corn meal, brewery milled yeast, rice, sorghum, refined corn starch, barley, barley starch, hulled barley, wheat, wheat starch, baked cereals, cereal flakes, rye, oats, potatoes, cassava and slurries such as corn syrup, cane syrup, invert syrup, barley and / or wheat syrup, etc. can be used as starch sources.

[0420] For many reasons, malt, mainly produced from selected barley varieties, has an important impact on the overall characteristics and quality of beer. First, malt is the main flavoring agent in beer. Secondly, malt provides the vast majority of fermentable sugars. Thirdly, malt provides protein, which will contribute to the body and foam characteristics of beer. Fourthly, malt provides the necessary enzyme activity in the pulping process. Hops also make a significant contribution to the quality of beer, including flavor. Specifically, hops (or hop ingredients) add desirable bitter substances to beer. In addition, hops can act as a protein precipitant, establish a preservative and help foam formation and stability.

[0421] Cereals (grains) such as barley, oats, wheat, as well as corn and rice are commonly used for industrial brewing and home brewing, but other plant components such as hops are also commonly added. The components used for brewing can be unmalted or can be malted, i.e. partially germinated, resulting in increased levels of enzymes including alpha amylase. For successful brewing, sufficient levels of alpha amylase activity are necessary to ensure that there are appropriate levels of sugars during the fermentation process. AfGATR or a variant thereof can also be added to the components used for brewing.

[0422] The term "stock" as used herein means crushed or broken grains and plant components. For example, barley used for beer production is a grain that has been coarsely ground or crushed to produce a consistency suitable for producing a fermentation mash. The term "stock" as used herein includes any of the aforementioned types of plants and grains in crushed or coarsely ground form. The methods described herein can be used to determine the level of alpha-amylase activity in flour and raw materials.

[0423] The process of preparing beer is well known in the art. See, for example, Wolfgang Kunze (2004), "Technology Brewing and Malting", Research and Teaching Institute of Brewing, Berlin (VLB), 3rd edition. Briefly, the process involves: (a) preparing a mash, (b) filtering the mash to prepare wort, and (c) fermenting the wort to obtain a fermented beverage such as beer. Typically, ground or crushed malt, malt and adjuncts, or adjuncts are mixed with water and kept at a controlled temperature for a period of time to allow the enzymes present in the malt and / or adjuncts to convert the starch present in the malt into fermentable sugars. The mash is then transferred to a mash filter, where the liquid is separated from the grain residue. This sweet liquid is called "wort" and the remaining grain residue is called "waste". The mash is usually extracted, which involves adding water to the mash to recover the remaining soluble extract from the waste. The wort is then boiled vigorously to sterilize the wort and help develop color, flavor, and aroma. Hops are added at some point during the boil. The wort is cooled and transferred to a fermenter.

[0424] The wort is then contacted with yeast in a fermentation tank. The fermentation tank may be cooled to stop fermentation. Any yeast that may flocculate is removed. Finally, the beer is cooled and stored for a period of time, during which the beer clarifies and develops flavor, and any substances that may impair the appearance, flavor, and shelf life of the beer are precipitated. Beer typically contains about 2% to about 10% v / v of alcohol, but beer with a higher alcohol content (e.g., 18% v / v) may also be obtained. Before packaging, the beer is charged with carbon dioxide and optionally filtered and pasteurized.

[0425] A brewing composition comprising an alpha-amylase, typically but not necessarily in combination with one or more exogenous enzymes such as one or more glucoamylases (e.g. AfGATR or variants thereof), one or more pullulanases and / or one or more isoamylases and any combination thereof may be added to the mash of step (a) above (i.e. during mash preparation). Alternatively, or in addition, the brewing composition may be added to the mash of step (b) above (e.g. during mash filtration). Alternatively, or in addition, the brewing composition may be added to the wort of step (c) above (e.g. during wort fermentation).

[0426] Particular embodiments relate to any of the above-mentioned uses, methods or fermented beverages, wherein the fermented beverage is beer, such as all-malt beer, beer brewed under the "purity method", ale, India pale ale, lager, bitter beer, low-malt beer (second beer), third beer, dry beer, thin beer, light beer, low-alcohol beer, low-calorie beer, porter, bock, strong beer, malt liquor, non-alcoholic beer, non-alcoholic malt liquor, etc., but there are also alternative grain and malt beverages, such as fruit-flavored malt beverages, for example citrus-flavored malt beverages such as lemon, orange, lime or berry flavored malt beverages; alcohol-flavored malt beverages, such as vodka, rum or tequila flavored malt liquor; or coffee-flavored malt beverages, such as caffeine-flavored malt liquor, etc.

[0427] 9. Reduction of iodine-positive starch

[0428] When used in a liquefaction and / or saccharification process, AfGATR and its variants can reduce iodine-positive starch (IPS). One source of IPS is from amylose that escapes hydrolysis and / or from retrograded starch polymers. Starch retrograde occurs spontaneously in starch pastes or gels during aging because starch molecules tend to associate with each other and then crystallinity increases. As starch molecules gradually associate into larger particles, low concentration solutions become increasingly turbid. Spontaneous precipitation occurs, and the precipitated starch appears to return to its initial cold water insoluble state. Higher concentration pastes solidify into gels when cooled, which steadily solidify as they age due to the increasing association of starch molecules. This is due to the strong tendency to form hydrogen bonds between hydroxyl groups on adjacent starch molecules. See JARadley, ed., STARCH ANDITS DERIVATIVES, pp. 194-201, Chapman and Hall, London, (1968)).

[0429] The presence of IPS in the sugar solution can adversely affect the final product quality and is a major problem in downstream processing. IPS can block or slow down the filtration system and clog the carbon column used for purification. When IPS reaches a sufficiently high level, it may leak out of the carbon column and reduce production efficiency. In addition, it may cause a turbid final product during storage, which is unacceptable for the final product quality. The amount of IPS can be reduced by isolating the saccharification tank and backmixing the contents. Nevertheless, IPS will accumulate in particular in the carbon column and filtration system. Therefore, it is expected that the use of AfGATR or its variants will improve the overall process performance by reducing the amount of IPS.

[0430] To further illustrate the compositions and methods and their advantages, the following specific examples are given, which should be understood to be illustrative rather than limiting.

[0431] Examples

[0432] Example 1: Cloning of AfGA1 .

[0433] The genomic DNA of Aspergillus fumigatus Af293 was purchased from Fungal Genetics Stock Center, Kansas City, MO (FGSC A1100). The genome of Aspergillus fumigatus was sequenced. The nucleic acid sequence of the AfGA1 gene (in the disclosed genome in the NCBI reference sequence NC_007195) and the amino acid sequence of the predicted glucan 1,4-α-glucosidase (NCBI deposit number XP_749206) encoded by the AfGA1 gene were obtained in the NCBI database. As determined by BLAST search, AfGA1 is homologous to other fungal glucoamylases. See Figure 1. The nucleotide sequence of the AfGA1 gene contains three introns, and the nucleotide sequence is shown in SEQ ID NO:8.

[0434] The AfGA1 gene was amplified from genomic DNA of Aspergillus fumigatus using the following primers: Primer 1: AfGA1-Fw 5'-GCGGCGGCCGC ACC atgcctcgcctttcctacgc-3' (SEQ ID NO:9), and Primer 2: AfGA1-Rv 5'-ccggcgcgccc TTA tcactgccaagtatcattctcg-3' (SEQ ID NO:10). The forward primer contained a NotI restriction site, and the reverse primer contained an AscI restriction site. After digestion with NotI and AscI, the PCR product was cloned into the pTrex3gM expression vector (described in published U.S. patent application 2011 / 0136197A1) digested with the same restriction enzymes, and the resulting plasmid was labeled pJG222. The plasmid map of pJG222 is provided at Figure 2 The sequence of the AfGA1 gene was confirmed by DNA sequencing.

[0435] Example 2: Expression and purification of AfGA1TR .

[0436] Plasmid pJG222 (Trex3gM-AfGA1) was transformed into a quadruple deletion Trichoderma reesei strain (described in WO 05 / 001036) using a gene gun method (Te'o et al., J. Microbiol. Methods, 51:393-99, 2002). Transformed colonies (about 50) appeared within about 1 week. After 5 days of growth on acetamide plates, colonies were inoculated in 250 ml shake flasks containing 30 ml of glucose / agarose defined medium for protein expression. The protein AfGA1TR was secreted into the extracellular medium, and the filtered medium was used for SDS-PAGE and glucoamylase activity assay for DP7 to confirm enzyme expression.

[0437] The stable strain was then grown in a 7L fermentor in a defined medium. The fermentation broth was collected by centrifugation. After centrifugation, filtration and concentration, a 450ml concentrated sample was obtained. By using the BCA method (protein quantification kit, Shanghai Generay Biotech CO., Ltd), the total protein concentration in the sample was measured to be 83.70g / L. SDS-PAGE analysis showed that 80% of the total protein was the target protein. Therefore, the target protein concentration in the concentrated sample was estimated to be 66.96g / L.

[0438] AfGA1TR was purified by affinity chromatography using an AKTA Explorer 100FPLC system (GE Healthcare). β-cyclodextrin (Sigma-Aldrich, 856088) was coupled to epoxy-activated agarose beads (GE Healthcare, 17-0480-01) and used for purification. The pH of 40 ml concentrated fermentation broth from a 7L fermentor was adjusted to 4.3 and the solution was loaded onto a 30 ml β-CD-agarose column pre-equilibrated with 25 mM pH 4.3 sodium acetate (buffer A). The column was washed with 2 column volumes of buffer A. The target protein was eluted with three column volumes of buffer B, which contained buffer A and 10 mM α-cyclodextrin (Sigma-Aldrich, C4642). The fractions were analyzed by SDS-PAGE gel, and glucoamylase activity was determined. The fractions containing the target protein were pooled and passed through a Hiprep 26 × 10 desalting column to remove β-cyclodextrin. The purity of the resulting samples was higher than 95%, and the solution was concentrated using a 10K Amicon Ultra-15 device and stored in 40% glycerol at -80 °C.

[0439] Example 3: Determination of AfGA1TR substrate specificity .

[0440] Glucoamylase activity was determined based on the release of glucose from different substrates using glucoamylase, AfGA1TR, AnGA or wild-type AfGA, including maltose, isomaltose, maltoheptaose (DP7), maltodextrin (DE4-DE10), potato amylopectin and soluble starch. The glucose release rate was measured using the coupled glucose oxidase / peroxidase (GOX / HRP) method (Anal. Biochem., 105 (1980), 389-397). Glucose was quantified by the oxidation rate of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) by peroxide, which was generated from the coupled GOX / HRP enzyme reacting with glucose.

[0441] Substrate solutions were prepared by mixing 9 mL of each substrate (1% in water, w / w) and 1 mL of 0.5 M pH 5.0 sodium acetate buffer in a 15 mL conical flask. ABTS-containing coupled enzyme (GOX / HRP) solution was prepared by dissolving GOX / HRP in 50 mM sodium acetate buffer (pH 5.0) with final concentrations of 2.74 mg / mL ABTS, 0.1 U / mL HRP, and 1 U / mL GOX.

[0442] Glucoamylase samples, benchmark AnGA (Genenco product, Optidex L-400), wild-type AfGA and serial dilutions of glucose standards were also prepared in 50mM sodium acetate buffer (pH 5.0). Each glucoamylase sample (10 μl) was transferred to a new microtiter plate (Corning 3641) containing 90 μl substrate solutions pre-incubated for 5 minutes at 600rpm at 50°C. Reacted 10 minutes at 50°C in a thermomixer (Eppendorf) under vibration (600rpm), 10 μl reaction mixtures and serial dilutions of 10 μl glucose standards were quickly transferred to a new microtiter plate (Corning 9017), followed by the addition of 100 μl ABTS / GOX / HRP solutions. The microtiter plate containing the reaction mixture was measured immediately at 405nm for 5 minutes with 11 seconds time intervals on a SoftMax Pro plate reader (Molecular Device). Output is the reaction rate Vo for each enzyme concentration. Linear regression is used to determine the slope of the curve of Vo relative to enzyme dosage. The specific activity of glucoamylase activity is calculated based on the glucose standard curve using the following equation 1:

[0443] Specific activity (unit / mg) = slope (enzyme) / slope (standard) × 100 (1),

[0444] Where 1 unit = 1 micromole glucose / minute.

[0445] Representative specific activities of AfGA1TR and the benchmark glucoamylases AnGA and wild-type AfGA are shown in Table 1 .

[0446] Table 1. Specific activity of purified glucoamylase on various substrates .

[0447]

[0448] Example 4: Effect of pH on AfGA1TR Glucoamylase Activity .

[0449] The effect of pH (3.0 to 10.0) on the activity of AfGA1TR was monitored using the ABTS assay protocol as described in Example 3. The buffer working solution consisted of a combination of glycine / sodium acetate / HEPES (250 mM) with the pH varied from 3.0 to 10.0. The substrate solution was prepared by mixing soluble starch (1% in water, w / w) with 250 mM buffer solution in a ratio of 9:1. Enzyme working solutions were prepared in water at specific doses (curves showing signals in the linear range according to dose response). All incubations were performed at 50°C for 10 minutes following the same protocol as described in Example 3 for the glucoamylase activity assay. The enzyme activity at each pH is reported as the relative activity compared to the enzyme activity at the optimal pH. The pH curves of AfGA1TR are presented in Tables 2 and Figure 3 It was found that AfGA1TR has an optimal pH of about 5.0 and maintains more than 70% of the maximum activity at a pH of 3.5 to 7.5.

[0450] Table 2. pH profile of purified glucoamylase

[0451]

[0452] Example 5: Effect of temperature on AfGA1TR glucoamylase activity .

[0453] The effect of temperature (40°C to 84°C) on the activity of AfGA1TR was monitored using the ABTS assay protocol as described in Example 3. The substrate solution was prepared by mixing 3.6 mL of soluble starch (1% in water, w / w) and 0.4 mL of 0.5 M buffer (pH 5.0 sodium acetate) in a 15 mL conical flask. Enzyme working solutions were prepared in water at specific doses (curves showing signals in the linear range according to dose response). The incubations were carried out at temperatures of 40°C to 84°C for 10 minutes respectively following the same protocol as described in Example 3 for the glucoamylase activity assay. The enzyme activity at each temperature is reported as the relative activity compared to the enzyme activity at the optimal temperature. The temperature profiles of AfGA1TR are shown in Tables 3 and Figure 4It was found that AfGA1TR had an optimum temperature of 68°C and maintained more than 70% of the maximum activity at 56°C to 74°C.

[0454] Table 3. Temperature profile of glucoamylase .

[0455]

[0456] Example 6: AfGA1TR product profile analysis .

[0457] To determine the fungal glucoamylase catalytic products of polysaccharides, glucoamylase, AnGA (0.118 mg / g dry solids starch) and AfGA1TR (0.118 mg / g dry solids or 0.059 mg / g dry solids) were mixed with 34% DS Super liquefied starch (CPI, Stockton, CA) was incubated at 60°C, pH 4.2 to 4.5 for 2 days. Pullulanase (PU) and acid-stable α-amylase from Aspergillus kawachii were added. (AkAA) was provided at a dosage of 0.256 ASPU / g dry solids and 0.35 SSU / g dry solids, respectively, together with purified AfGA1TR. Samples were taken at different time intervals and analyzed for sugar composition by HPLC.

[0458] Table 4 shows the distribution of oligosaccharides saccharified by AnGA / PU / AkAA and AfGA1TR / PU / AkAA at concentrations of 100% and 50% AnGA. Figure 6 The distribution of oligosaccharides saccharified by AnGA and AfGA1TR in the presence and absence of PU and AkAA is shown at concentrations of 100%, 50% and 40% AnGA. ​​Only oligosaccharides at DP1, DP2, DP3 and HS are shown. The numbers in Table 4 reflect the weight percentage of each DPn as a portion of the total DP1, DP2, DP3 and HS.

[0459] Table 4. Product distribution of fungal glucoamylase on liquefied starch .

[0460]

[0461]

[0462] Table 4 shows that AfGA1TR produced >95.5% DP1 in 24 hours, compared to 48.5 hours for AnGA at an equal dose of protein.The data in Table 4 show that at 50% dose equivalent on a protein basis (under the same conditions of supplemental enzyme dose), AfGA1TR exhibits improved performance compared to AnGA.

[0463] Example 7: Comparison of DP2 levels .

[0464] The DP2 levels of liquefied starch from AfGA1TR treatment were compared to those from AnGA treatment based on the same DP1 level (96%). The comparison showed that at equal DP1 levels, the DP2 level was statistically significantly lower by about 0.2%, which may be due to the lower glucoamylase dosage. The reversal reaction by AnGA and AfGA1TR (in triple blend form) was measured by calculating the isomaltose / maltose ratio via ion chromatography.

[0465] Table 5. Product distribution of fungal glucoamylase on liquefied starch .

[0466]

[0467] ΔRatio = [AnGA ratio - AfGA1TR ratio] at 48 hours and 70 hours

[0468] Table 5 shows that for both glucoamylases, isomaltose accumulated over time as the ratio of isomaltose:maltose increased. However, with AfGA1TR, isomaltose formation appeared to be slightly lower as the difference in ratio between AnGA and AfGA1TR increased from 48 to 70 hours, which may support a lower reversal reaction of AfGA1TR.

[0469] Example 8: Titration of AkAA .

[0470] To determine the fungal glucoamylase catalytic products of polysaccharides using different doses of auxiliary α-amylase, AfGA1TR (0.059 mg / g dry solids) and pullulanase (PU, L-1000) (0.256 ASPU / g dry solids) and different concentrations of acid-stable α-amylase from Aspergillus kawachii AkAA was added in increments of 0.1 SSU at 0 to 0.3 SSU / dry solids, and 34% DS Super liquefied starch (CPI, Stockton, CA) at 60°C, pH 4.5 for 2 days. Pullulanase (PU) and (AkAA) was provided at a dosage of 0.256 ASPU / g dry solids and 0.35 SSU / g dry solids, respectively, together with purified AfGA1TR. Samples were taken at different time intervals and analyzed for sugar composition by HPLC.

[0471] Table 6 and Figure 7The distribution of oligosaccharides saccharified by AfGA1TR, PU and different doses of AkAA is disclosed. Only oligosaccharides at DP1, DP2, DP3 and HS are shown. The numbers in Table 6 reflect the weight percentage of each DPn as a part of the total DP1, DP2, DP3 and HS.

[0472] Table 6. Effect of AkAA during enzymatic liquefaction saccharification using AfGA1TR .

[0473]

[0474] Table 6 shows that in the presence of at least 0.1 SSU / g dry solids, AfGA1TR was able to achieve >96% DP1 in 45 hours, while the absence of AkAA resulted in a statistically significant decrease in saccharification rate. The results indicate that a significant increase in final glucose yield was achieved by adding AkAA during enzymatic liquefaction saccharification using AfGA1TR.

[0475] Example 9: Solubilization and desaturation of granular amylase by an enzyme blend containing α-amylase, AfGA1TR and pullulanase hydrolysis

[0476] A granular corn starch slurry with 35% dry solids starch in distilled water was prepared and the pH was adjusted to pH 5.0 using NaOH. 10 AAU / g dry solids of α-amylase ( XTRA) and 0.047 mg / g dry solids of AfGA1TR purified protein and 0.15 ASPU / g dry solids of pullulanase ( L-1000) was added to the starch slurry. The starch slurry was then kept in a water bath maintained at 60°C under constant stirring. Aliquots were extracted and centrifuged at different time intervals. The clarified supernatant was used for refractive index (RI) to calculate the solubilization percentage and analyzed for sugar composition by HPLC.

[0477] Table 7: Product distribution of fungal glucoamylase on starch during liquefaction .

[0478]

[0479] Table 7 shows that, using granular starch in the presence of α-amylase and PU, AfGA1TR was able to achieve >95.5% DP1 within 68 hours, with 86% of the granular starch being solubilized.

[0480] Example 10: Effect of residual α-amylase activity on DP3 levels .

[0481] Analysis by adding 0.066KG / MT dry solids Supra (NZ) back to α-killed starch liquefact, using AfGA1TR, the effect of single pH (5.5) on DP1 and DP3 and the effect of residual α-amylase activity at pH 4.5 on DP1 and DP3. 0.066 mg / g dry solids of AfGA1TR and 0.25 ASPU / g dry solids of L-1000 (pullulanase) and 0.1 SS U / g dry solids of AkAA were blended. Table 8 and Figure 5 The AfGA1TR and Distribution of oligosaccharides saccharified by 4060 VHP (AnGA / Pullulanase blend).

[0482] Table 8: Product distribution of fungal glucoamylase on liquefied starch .

[0483]

[0484] The AfGA1TR triple blend showed a significant loss in saccharification rate at pH 5.5 compared to pH 4.5, likely due to L-1000, but was able to achieve >95.5% DP1 in 48 hours. Due to the expected higher DP3, AfGA1TR and 4060VHP were all negatively affected by the small amount of residual α-amylase activity to maximize glucose yield. In the case of α-amylase killing liquefact, AfGA1TR was compared to 4060 VHP resulted in a significant decrease in DP3 by 0.1%. The level of AfGA1TR in liquefacts with α-amylase activity was comparable to that in liquefacts with α-“killed” 4060VHP is also low.

[0485] Example 11: Comparison of AfGA1TR and wild-type Aspergillus fumigatus glucoamylase

[0486] Starch liquefact was prepared by dilution with water to have 34% dry solids and saccharified using 2 different glucoamylases at pH 4.4 and 60°C; 1) AfGA1TR at 0.067 mg / g dry solids starch and 2) purified protein of wild-type AfGA (expressed in Aspergillus fumigatus) from GLUCOTEAM DB (Nagase Co. & Ltd., Japan) at 0.065 mg / g dry solids. In addition, pullulanase (PU) and acid-stable α-amylase (A-AM) were added at 0.14 ASPU / g dry solids and 0.9 SSU / g dry solids, respectively. (AkAA) was administered with each glucoamylase. Samples were taken at different time intervals and analyzed for sugar composition by HPLC.

[0487] Table 9 shows that AfGA1TR produced >95.5% DP1 in 48 hours, while the commercial A. fumigatus took longer saccharification time. Both glucoamylases were able to achieve >96% DP1, while DP2 was less than 3%.

[0488] Table 9: Product distribution of fungal glucoamylase blends on liquefied starch .

[0489]

[0490] Example 12: Solubilization and characterization of granular starch by an enzyme blend containing α-amylase, pullulanase and Aspergillus fumigatus GA hydrolysis

[0491] In a typical example, a granular corn starch slurry with 35% dry solids starch in distilled water was prepared and the pH was adjusted to pH 5.0 using sodium hydroxide. Purified proteins of XTRA and AfGA1TR or wild-type Aspergillus fumigatus from GLUCOTEAM DB (Nagase Sangyo Co., Ltd., Japan) were added to 0.15 ASPU / g dry solids. L-1000 was added to the starch slurry. Then, the starch slurry was kept in a water bath maintained at 60°C under constant stirring. Aliquots were extracted and centrifuged at different time intervals. The clarified supernatant was used for refractive index (RI) to calculate the solubilization percentage and analyzed by HPLC for sugar composition. Table 10 shows that in the presence of α-amylase and PU, using granular starch, two Aspergillus fumigatus glucoamylases were able to reach>95.5% DP1 in 68 hours, with>86% of granular starch dissolved.

[0492] Table 10. Effect of α-amylase and pullulanase on solubilization of granular starch

[0493]

[0494] Example 13: Expression and purification of AfGA2TR .

[0495] The nucleic acid sequence of the AfGA2 gene (NCBI reference sequence DS499595, 145382 to 147441) was taken from Aspergillus fumigatus A1163, and the amino acid sequence of the hypothetical protein encoded by the AfGA2 gene was found in the NCBI database (NCBI accession number EDP53734). The nucleotide sequence of the AfGA2 gene from Aspergillus fumigatus A1163 was optimized and synthesized by Generay (Shanghai Jierui Biotechnology Co., Ltd., China).

[0496] The DNA sequence of AfGA2 was optimized for its expression in Trichoderma reesei and then synthesized by Generay (Shanghai Jierui Biotechnology Co., Ltd., China) and inserted into the pTrex3gM expression vector (described in U.S. Published Application No. 2011 / 0136197A1), generating pJG313 ( Fig. 9 ).

[0497] Plasmid pJG313 was transformed into a quadruple deletion Trichoderma reesei strain (described in WO 05 / 001036) using a gene gun method (Te'o et al., J. Microbiol. Methods, 51:393-99, 2002). Transformants were selected on a medium containing acetamide as the sole nitrogen source (acetamide 0.6 g / L; cesium chloride 1.68 g / L; glucose 20 g / L; potassium dihydrogen phosphate 15 g / L; magnesium sulfate heptahydrate 0.6 g / L; calcium chloride dihydrate 0.6 g / L; iron (II) sulfate 5 mg / L; zinc sulfate 1.4 mg / L; cobalt (II) chloride 1 mg / L; manganese (II) sulfate 1.6 mg / L; agar 20 g / L; pH 4.25). Transformed colonies (approximately 50-100) appeared within about 1 week. After growth on acetamide plates, transformants were picked up and transferred individually to acetamide agar plates. After 5 days of growth on acetamide plates, transformants showing stable morphology were inoculated in 200 μl of glucose / agarose defined medium in a 96-well microtiter plate. The microtiter plate was incubated at 28°C for 5 days in an oxygen growth chamber. The supernatants from these cultures were used to confirm protein expression and determine enzyme activity by SDS-PAGE analysis. Stable strains were then grown in a 7L fermentor in a defined medium containing 60% glucose-agarose feed. Glucose / agarose defined medium (per liter) consisted of 5g (NH 4 ) 2 SO 4 , 33g PIPPS buffer, 9g casamino acids, 4.5g KH 2 PO 4 1gCaCl 2 (anhydrous), 1g MgSO 4 7H 2 O, adjusted the pH to 5.5 with 50% NaOH, and washed with Milli-Q H 2 After sterilization, add the following: 26 mL of 60% glucose / agarose, and 2.5 mL of 400× T. reesei trace metals.

[0498] The protein AfGA2TR was purified via two steps of chromatography. Ammonium sulfate was added to 600 mL of fermentation broth until the final concentration of ammonium sulfate reached 1 M. The sample was loaded onto a 50 mL hydrophobic interaction chromatography Phenyl HP column pre-equilibrated with 20 mM sodium phosphate pH 7.0 (buffer A) containing 1 M ammonium sulfate. The column was washed with a linear salt gradient from 1 to 0 M ammonium sulfate. The active fractions were pooled and applied to affinity chromatography. The sample after hydrophobic interaction chromatography was exchanged in 25 mM pH 4.3 sodium acetate buffer (buffer B) and loaded onto a β-cyclodextrin-coupled Sepharose 6B column pre-equilibrated with buffer B. The target protein was eluted using 25 mM pH 4.3 sodium acetate (buffer C) containing 10 mM α-cyclodextrin. The eluate was concentrated by using a 10K Amicon Ultra-15 device. The final product purity was higher than 98% and was stored in 40% glycerol at -80°C for further study.

[0499] Example 14: Glucoamylase activity of AfGA2TR

[0500] AfGA2TR belongs to the glycosyl hydrolase 15 family (GH15, CAZy numbering). 1% w / w soluble starch (SigmaS9765) was used as a substrate to measure the glucoamylase activity of AfGA2TR. The assay was performed in 50 mM sodium acetate buffer pH 5.0 at 50°C for 10 minutes. The glucose release rate was measured using the glucose oxidase / peroxidase (GOX / HRP) technique disclosed in Example 3. Glucose was quantified by the oxidation rate of 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) by an excess of coupled GOX / HRP enzyme. Enzyme activity was calculated based on a glucose standard curve. In this assay, one glucoamylase unit is defined as the amount of enzyme required to generate 1 micromole of glucose per minute under the assay conditions. Using the above method, the specific activity of soluble starch of purified AfGA2TR was determined to be 214 units / mg.

[0501] Example 15: pH distribution of AfGA2TR

[0502] The effect of pH (3.0 to 10.0) on the activity of AfGA2TR was monitored by following the ABTS assay protocol as described in Example 3. The buffer working solution consisted of a combination of glycine / sodium acetate / HEPES (250 mM) with the pH varied from 3.0 to 10.0. The substrate solution was prepared by mixing soluble starch (1% in water, w / w) with 250 mM buffer solution in a ratio of 9:1. Enzyme working solutions were prepared in water at specific doses (curves showing signals in the linear range according to the dose response). All incubations were performed at 50°C for 10 minutes using the same protocol as described in Example 3 for the glucoamylase activity assay. The enzyme activity at each pH is reported as the relative activity relative to the enzyme activity at the optimal pH. The pH curve of AfGA2TR was Fig.10 It was found that AfGA2TR has an optimal pH of about 5.3 and maintains more than 70% of the maximum activity at a pH of 3.3 to 7.3.

[0503] Example 16: Temperature curve of AfGA2TR

[0504] The effect of temperature (40°C to 84°C) on the activity of AfGA2TR was monitored by following the ABTS assay protocol as described in Example 3. The substrate solution was prepared by mixing 9 mL of soluble starch (1% in water, w / w) and 1 mL of 0.5 M buffer (pH 5.0 sodium acetate) in a 15 mL conical flask. Enzyme working solutions were prepared in water at specific doses (curves showing signals in the linear range according to dose response). Incubations were performed at temperatures of 40°C to 84°C for 10 minutes respectively. After incubation, the activity was determined following the same protocol as described in Example 3 for the glucoamylase activity assay. The activity is reported as the relative activity relative to the enzyme activity at the optimal temperature. The temperature profile of AfGA2TR was Fig.11 It was found that AfGA2TR had an optimum temperature of 69°C and maintained more than 70% of the maximum activity at 61°C to 74°C.

[0505] Example 17: Thermal stability of AfGA2TR

[0506] The thermal stability of AfGA2TR was determined in 50 mM sodium acetate buffer (pH 5.0). The enzyme was incubated in a PCR machine at the desired temperature for 2 hours and then added to the substrate. The residual activity of the samples was measured as described in Example 3. The activity of the samples kept on ice was defined as 100% activity. Fig.12 As shown in , at temperatures below 63°C, AfGA2TR maintained more than 50% activity during a 2-hour incubation period.

[0507] Example 18: Comparison of AfGA1TR and AfGA2TR

[0508] Starch liquefact was prepared by diluting with water to have 34% dry solids and saccharified at pH 4.4 and 60°C using 2 different glucoamylases; 1) AfGA1TR at 0.06 mg / g dry solids starch and 2) purified protein of AfGA2TR at 0.06 mg / g dry solids. In addition, pullulanase ( L-1000) and acid-stable α-amylase (AsAA) was administered with each glucoamylase to enhance glucose production. Samples were taken at different time intervals and analyzed for sugar composition by HPLC.

[0509] Table 11: Product distribution of AfGA1TR and AfGA2TR blends for liquefied starch .

[0510]

[0511] Table 11 shows that both amylases produced >95.5% DP1 within 48 hours, with saccharification being slightly faster using AfGA2TR.

[0512] Some embodiments of the present invention:

[0513] 1. A recombinant Trichoderma reesei host cell expressing AfGATR or a variant thereof, wherein the AfGATR or a variant thereof has at least 80% sequence identity with SEQ ID NO: 12 or 13, wherein under the same conditions, the Trichoderma reesei host cell expresses the AfGATR or variant at a level comparable to that of an Aspergillus fumigatus host cell, wherein the Aspergillus fumigatus host cell expresses AfGA or a variant thereof having the same amino acid sequence as the AfGATR or a variant thereof.

[0514] 2. A recombinant Trichoderma reesei host cell expressing AfGATR or a variant thereof, wherein the AfGATR or a variant thereof has at least 80% sequence identity with SEQ ID NO: 12 or 13, wherein the AfGATR or a variant thereof is more thermostable than AfGA or a variant thereof having the same amino acid sequence as AfGATR or a variant thereof, and wherein the AfGA or a variant thereof is expressed in an Aspergillus fumigatus host cell.

[0515] 3. A method for producing recombinant AfGATR or a variant thereof, comprising:

[0516] (a) providing a Trichoderma reesei host cell expressing a recombinant AfGATR or a variant thereof, wherein the AfGATR or a variant thereof has at least 80% sequence identity to SEQ ID NO: 12 or 13;

[0517] (b) culturing the host cell under conditions that allow production of the recombinant AfGATR or variant thereof; and

[0518] Wherein the AfGATR or variant thereof is more thermostable than AfGA or variant thereof having the same amino acid sequence as AfGATR or variant thereof, and wherein the AfGA or variant thereof is expressed in an Aspergillus fumigatus host cell.

[0519] 4. A recombinant AfGATR or a variant thereof, produced by the host cell of embodiment 1 or embodiment 2, or the method according to embodiment 3.

[0520] 5. The recombinant AfGATR or variant thereof according to embodiment 4, wherein the AfGATR or variant thereof has at least 70% activity at 74°C at pH 5.0 within 10 minutes.

[0521] 6. The recombinant AfGATR or variant thereof according to embodiment 5, wherein the AfGATR or variant thereof is AfGA1TR or variant thereof.

[0522] 7. The recombinant AfGA1TR or variant thereof according to embodiment 6, wherein the AfGA1TR or variant thereof has at least 70% activity within 10 minutes at pH 5.0 within a temperature range of 55°C to 74°C.

[0523] 8. The recombinant AfGA1TR or variant thereof according to embodiment 7, wherein the AfGATR or variant thereof has an optimal temperature of about 68°C.

[0524] 9. The recombinant AfGATR or variant thereof according to embodiment 5, wherein the AfGATR or variant thereof is AfGA2TR or variant thereof.

[0525] 10. The recombinant AfGA2TR or variant thereof according to embodiment 9, wherein the AfGA2TR or variant thereof has at least 70% activity within 10 minutes at pH 5.0 within a temperature range of 61°C to 74°C.

[0526] 11. The recombinant AfGA2TR or variant thereof according to embodiment 10, wherein the AfGA2TR or variant thereof has an optimal temperature of about 69°C.

[0527] 12. The recombinant AfGATR or variant thereof according to any one of embodiments 4-8, wherein the AfGATR or variant thereof comprises an amino acid sequence having at least 90%, 95% or 99% amino acid sequence identity to SEQ ID NO: 12.

[0528] 13. The recombinant AfGATR or variant thereof according to embodiment 12, wherein the AfGATR or variant thereof comprises SEQ ID NO:12.

[0529] 14. The recombinant AfGATR or variant thereof according to any one of embodiments 4-8, wherein the AfGATR or variant thereof consists of an amino acid sequence having at least 80%, 90%, 95% or 99% amino acid sequence identity to SEQ ID NO:12.

[0530] 15. The recombinant AfGATR or variant thereof according to embodiment 14, wherein the AfGA1TR or variant thereof consists of SEQ ID NO:12.

[0531] 16. The recombinant AfGATR or variant thereof according to any one of embodiments 4-5 and 9-11, wherein the AfGATR or variant thereof comprises an amino acid sequence having at least 90%, 95% or 99% amino acid sequence identity to SEQ ID NO: 13.

[0532] 17. The recombinant AfGATR or variant thereof according to embodiment 16, wherein the AfGATR or variant thereof comprises SEQ ID NO:13.

[0533] 18. The recombinant AfGATR or variant thereof according to any one of embodiments 4-5 and 9-11, wherein the AfGATR or variant thereof consists of an amino acid sequence having at least 80%, 90%, 95% or 99% amino acid sequence identity to SEQ ID NO: 13.

[0534] 19. The recombinant AfGATR or variant thereof according to embodiment 18, wherein the AfGA1TR or variant thereof consists of SEQ ID NO:13.

[0535] 20. A method of saccharifying a composition comprising starch to produce a composition comprising glucose, wherein the method comprises:

[0536] (i) contacting a starch composition with an isolated AfGATR or variant thereof according to any one of embodiments 4-19; and

[0537] (ii) saccharifying the starch composition to produce the glucose composition; wherein the AfGA1TR or variant thereof catalyzes the saccharification of the composition comprising starch into a composition comprising glucose.

[0538] 21. The method according to embodiment 20, wherein the composition comprising glucose is enriched in DP1 compared to a second composition comprising DP1 produced by saccharification of AnGA for 24 hours under the same conditions.

[0539] 22. The method of embodiment 20, wherein the composition comprising glucose is enriched in DP1 compared to a second composition comprising DP1 produced by wild-type AfGA under the same conditions.

[0540] 23. The method of embodiment 20, wherein the AfGATR or variant thereof is AfGATR2 and wherein the composition comprising glucose is enriched in DP1 compared to a second composition comprising DP1 produced by AfGA1TR under the same conditions.

[0541] 24. The method according to embodiment 20, wherein the dosage of AfGA1TR or its variant is about 40%-50% of the dosage of AnGA to produce the same DP1 yield after saccharification for 24 hours under the same conditions.

[0542] 25. The method according to any one of embodiments 20-24, wherein the composition comprising starch comprises liquefied starch, gelatinized starch, or granular starch.

[0543] 26. The method of any one of embodiments 20-25, wherein saccharification is performed at a temperature in the range of about 30°C to about 65°C.

[0544] 27. The method of embodiment 26, wherein the temperature ranges from 47°C to 60°C.

[0545] 28. The method according to any one of embodiments 20-27, wherein saccharification is performed in a pH range of pH 2.0 to pH 6.0.

[0546] 29. The method of embodiment 28, wherein the pH range is pH 3.5 to pH 5.5.

[0547] 30. The method of embodiment 29, wherein the pH range is pH 4.0 to pH 5.0.

[0548] 31. The method of any one of embodiments 20-30, wherein the method further comprises contacting the starch composition with an alpha-amylase.

[0549] 32. The method of embodiment 31, wherein the α-amylase is AkAA.

[0550] 33. The method of any one of embodiments 20-32, wherein the method further comprises contacting the starch composition with a pullulanase.

[0551] 34. The method of any one of embodiments 20-33, further comprising fermenting the glucose composition to produce an end-of-fermentation (EOF) product.

[0552] 35. The method of embodiment 34, wherein the fermentation is a simultaneous saccharification and fermentation (SSF) reaction.

[0553] 36. The method according to embodiment 34 or 35, wherein the fermentation is carried out at a pH of 2 to 8 and at a temperature ranging from 25°C to 70°C for 24 to 70 hours.

[0554] 37. The method of any one of embodiments 34-36, wherein the EOF product comprises ethanol.

[0555] 38. The method of any one of embodiments 34-37, wherein the EOF product comprises 8% to 18% (v / v) ethanol.

[0556] 39. The method of any one of embodiments 34-38, wherein the method further comprises contacting the mash and / or wort with a pullulanase, an alpha-amylase and the AfGATR or variant thereof.

[0557] 40. The method according to embodiment 39, wherein the method further comprises:

[0558] (a) preparing mash;

[0559] (b) filtering the mash to obtain wort; and

[0560] (c) fermenting the wort to obtain a fermented beverage,

[0561] wherein the pullulanase, the α-amylase and the AfGATR or variant thereof are added to:

[0562] (i) the mash of step (a) and / or

[0563] (ii) the wort of step (b) and / or

[0564] (iii) the wort of step (c).

[0565] 41. The method of any one of embodiments 34-40, wherein the EOF product comprises metabolites.

[0566] 42. The method of embodiment 41, wherein the metabolite is citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, sodium isoascorbate, omega-3 fatty acids, butanol, amino acids, lysine, itaconic acid, 1,3-propanediol, or isoprene.

[0567] 43. The method of any one of embodiments 20-42, further comprising adding to the starch composition an additional glucoamylase, a hexokinase, a xylanase, a glucose isomerase, a xylose isomerase, a phosphatase, a phytase, a protease, a pullulanase, a β-amylase, an additional α-amylase, a protease, a cellulase, a hemicellulase, a lipase, a cutinase, a trehalase, an isoamylase, an oxidoreductase, an esterase, a transferase, a pectinase, an α-glucosidase, a β-glucosidase, a lyase, a hydrolase, or a combination thereof.

[0568] 44. The method of any one of embodiments 20-43, comprising adding the AfGATR or variant thereof at a dosage of 0.1 to 2 glucoamylase units (GAU) / g dry solids.

[0569] 45. The method of embodiment 44, wherein the AfGATR or variant thereof is added at a dose of about 49.5 μg protein / g solid.

[0570] 46. ​​The method of any one of embodiments 20-45, wherein the isolated AfGATR or variant thereof is secreted by the Trichoderma reesei host cell.

[0571] 47. The method of embodiment 46, wherein the host cell also expresses and secretes α-amylase.

[0572] 48. The method of embodiment 47, wherein the host cell also expresses and secretes pullulanase.

[0573] 49. The method of any one of embodiments 46-48, wherein the composition comprising starch is contacted with the host cell.

[0574] 50. A composition comprising glucose produced according to the method of any one of embodiments 20-49.

[0575] 51. A liquefied starch produced according to the method of any one of embodiments 34-49.

[0576] 52. A fermented beverage produced according to the method of any one of embodiments 20-49.

[0577] 53. A composition for saccharifying a composition comprising starch, comprising the isolated AfGATR or variant thereof according to any one of embodiments 4-19.

[0578] 54. A composition according to embodiment 53, wherein the composition is a cultured cell material.

[0579] 55. The composition of embodiment 53 or 54, wherein the composition comprises an alpha-amylase.

[0580] 56. The composition of any one of embodiments 53-55, wherein the AfGATR or variant thereof is purified.

[0581] 57. The composition of any one of embodiments 53-56, wherein the AfGATR or variant thereof is secreted by a host cell.

[0582] 58. A use of AfGATR or a variant thereof according to any one of embodiments 4-19 for producing a composition comprising glucose.

[0583] 59. A use of AfGATR or a variant thereof according to any one of embodiments 4-19 for producing liquefied starch.

[0584] 60. Use of AfGATR or a variant thereof according to any one of embodiments 4-19 for producing a fermented beverage.

[0585] 61. The method according to any one of embodiments 34-42, the fermented beverage according to embodiment 52, or the use according to embodiment 60, wherein the fermented beverage or fermentation end product is selected from:

[0586] i) beer selected from the group consisting of all-malt beer, beer brewed according to the "Purity Law", ale, India pale ale, lager, bitter beer, low-malt beer (second beer), third beer, dry beer, thin beer, pale beer, low-alcohol beer, low-calorie beer, porter, bock, stout, malt liquor, non-alcoholic beer and non-alcoholic malt liquor; and

[0587] ii) a cereal or malt beverage selected from the group consisting of a fruit-flavored malt beverage, an alcohol-flavored malt beverage, and a coffee-flavored malt beverage.

[0588] 62. A method for producing a food composition comprising combining the following substances

[0589] (i) one or more food ingredients, and

[0590] (ii) the isolated AfGATR or variant thereof according to embodiments 4-19,

[0591] Wherein the pullulanase and the isolated AfGATR or variant thereof catalyze the hydrolysis of starch components present in the food ingredient to produce glucose.

[0592] 63. The method of embodiment 62, wherein the food composition is selected from the group consisting of food, baking compositions, food additives, animal food, feed products, feed additives, oils, meats and lard.

[0593] 64. The method according to any one of embodiments 62 or 63, and wherein the one or more food ingredients comprise a baking ingredient or additive.

[0594] 65. The method of any one of embodiments 62-64, wherein the one or more food ingredients are selected from: flour; anti-staling amylase; phospholipase; phospholipids; malting alpha-amylase or a variant, homolog or mutant thereof having malting alpha-amylase activity; xylanase for bakery products (EC 3.2.1.8); and lipase.

[0595] 66. The method according to embodiment 65, wherein the one or more food ingredients are selected from:

[0596] (i) a maltogenic α-amylase from Bacillus stearothermophilus,

[0597] (ii) a xylanase for bakery products from Bacillus, Aspergillus, a thermophilic fungus or Trichoderma,

[0598] (iii) Glycolipase from Fusarium heterosporum.

[0599] 67. The method according to any one of embodiments 62-66, wherein the food composition comprises a dough or a dough product, preferably a processed dough product.

[0600] 68. The method according to any one of embodiments 62-67, comprising baking the food composition to produce a baked product.

[0601] 69. The method according to any one of embodiments 62-66, wherein the method further comprises:

[0602] (i) providing a starch medium;

[0603] (ii) adding the pullulanase and the AfGATR or variant thereof to the starch medium; and

[0604] (iii) applying heat to the starch medium during or after step (b) to produce a baked product.

[0605] 70. A composition for use in the production of a food composition comprising the AfGATR or a variant thereof according to any one of embodiments 4-19.

[0606] 71. Use of AfGATR or a variant thereof according to any one of embodiments 4-19 for preparing a food composition.

[0607] 72. The use according to embodiment 71, wherein the food composition comprises dough or a dough product, preferably a processed dough product.

[0608] 73. The use according to embodiment 71 or 72, wherein the food composition is a baking composition.

[0609] 74. Use of the AfGATR or variant thereof according to any one of embodiments 4-19 in a dough product for delaying or reducing staling of the dough product, preferably delaying or reducing adverse retrogradation thereof.

[0610] Although the compositions and methods of making and using the same have been described in detail with reference to the above examples, it is understood that various modifications may be made without departing from the spirit of these compositions and methods and that such modifications will be apparent to those skilled in the art.

[0611] All cited patents and publications mentioned in this application are incorporated herein by reference in their entirety and for all purposes.

[0612] Sequence Listing

[0613] SEQ ID NO:1-AfGA1 precursor

[0614] MPRLSYALCALSLGHAAIAAPQLSARATGSLDSWLGTETTVALNGILANIGADGAYAKSAKPGIIIASPSTSEPDYYYTWTRDAALVTKVLVDLFRNGNLGLQKVITEYVNSQAYLQTVSNPSGGLASGGLAEPKYNVDMTAFTGAWGRPQRDGPALRATALIDFGNWLIDNGYSSYAVNNIWPIVRNDLSYVSQYWSQSGFDLWEEVNSMSFFTVAVQHRALVEGSTFAKRVGASCSWCDSQAPQILCYMQSFWTGSYINANTGGGRSGKDANTVLASIHTFDPEAGCDDTTFQPCSPRALANHKVYTDSFRSVYAINSGIPQGAAVSAGRYPEDVYYNGNPWFLTTLAAAEQLYDAIYQWKKIGSISITSTSLAFFKDIYSSAAVGTYASSTSTFTDIINAVKTYADGYVSIVQAHAMNNGSLSEQFDKSSGLSLSARDLTWSYAAFLTANMRRNGVVPAPWGAASANSVPSSCSMGSATGTYSTATATSWPSTLTSGSPGSTTTVGTTTSTTSGTAAETACATPTAVAVTFNEIATTTYGENVYIVGSISELGNWDTSKAVALSASKYTSSNNLWYVSVTLPAGTTFEYKYIRKESDGSIVWESDPNRSYTVPAACGVSTATENDTWQ

[0615] SEQ ID NO:2 - AfGA2 precursor

[0616] MPRLSYALCALSLGHAAIAAPQLSARATGSLDSWLGTETTVALNGILANIGADGAYAKSAKPGIIIASPSTSEPDYYYTWTRDAALVTKVLVDLFRNGNLGLQKVITEYVNSQAYLQTVSNPSGGLASGGLAEPKYNVDMTAFTGAWGRPQRDGPAL RATALIDFGNWLIDNGYSSYAVNNIWPIVRNDLSYVSQYWSQSGFDLWEEVNSMSFFTVAVQHRALVEGSTFAKRVGASCSWCDSQAPQILCYMQSFWTGSYNANTGGGRSGKDANTVLASIHTFDPEAGCDDTTFQPCSPALANHKVYTDSFRSV YAINSGIPQGAAVSAGRYPEDVYYNGNPWFLTTLAAAEQLYDAIYQWKKIGSISITSTSLAFFKDIYSSAAVGTYASSTSFTDIINAVKTYADGYVSIVQAHAMNNGSLSEQFDKSSGLSLSARDLTWSYAAFLTANMRRNGVVPAPWGAASANSVPSSCSMGSATGTYSTATATSWPSTLTSGSPGSTTTVGTTTSTTSGTATETACATPTAVAVTFNEIATTTYGENVYIVGSISELGNWDTSKAVALSASKYTSSNNLWYVSVTLPAGTTFEYKYIRKESDGSIVWESDPNRSYTVPAACGVSTATENDTWR

[0617] SEQ ID NO:3-Nf_NRRL_181_GA

[0618] MPRLSYALCALSLGHAAIAAPQLSPRATGSLDSWLATESTVSLNGILANIGADGAYAKSAKPGIIIASPSTSDPDYYYTWTRDAALVTKVLVDLFRNGNLGLQKVITEYVNSQAYLQTVSTPSGGLSSGGLAEPKYNVDMTAFTGAWGRPQRDGPALRATALIDFGNWLIDNGYSSYAVNNIWPIVRNDLSYVSQYWSQSGFDLWEEVNSMSFFTVAVQHRALVEGSTFAKRVGASCSWCDSQAPQILCYMQSFWTGSYINANTGGGRSGKDANTVLASIHTFDPEAGCDDTTFQPCSPRALANHKVYTDSFRSVYAINSGIPQGVAVSAGRYPEDVYYNGNPWFLTTLAAAEQLYDAIYQWKKIGSISITSTSLAFFKDIYSSVAVGTYASSSSTFTAIIDAVKTYADGYVSIVEAHAMTNGSLSEQFDKSSGMSLSARDLTWSYAALLTANMRRNGVVPAPWGAASANSVPSSCSMGSATGTYSTATATSWPSTLTSGSPSDTTSGTTPGTTTTTSACTTPTSVAVTFDEIATTTYGENVYIIGSISQLGSWDTSKAVPLSSSKYTSSNNLWYVTINLPAGTTFEYKYIRKESDGSIEWESDPNRSYTVPSACGVSTATEKDTWR

[0619] SEQ ID NO:4-Ts_ATCC0_10500_GA

[0620] MTRLSSVLCALAALGQTALAAPGLSPRASTSLDAWLATETTVSLSGILANIGADGAYSKSAKPGVVIASPSTDNPNYYYTWTRDSALTLKVLIDLFRNGNLGLQTVIEEYVNAQAYLQTVSNPSGDLSSGAGLAEPKFNVDMSAFTGSWGRPQRDGPALRAIALIDFGNWLIENGYTSLAANNIWPIVRNDLSYVAQYWSQSGFDLWEEVNSMSFFTVANQHRSLVEGSTFAAKVGASCSWCDSQAPQILCYMQTFWTGSYMNANTGGGRSGKDANTVLTSIATFDPEATCDDVTFQPCSPRALANHKVYTDSFRSVYGLNSGIAEGVAVAVGRYPEDSYYNGNPWFLSNLAAAEQLYDAIYQWNKIGSITITSTSLAFFKDVYSSAAVGTYASGSSAFTSIINAVKTYADGYISVVQSHAMNNGSLSEQFDKNTGAELSARDLTWSYAALLTANMRRNGVVPPSWGAASATSIPSSCTTGSAIGTYSTPTATSWPSTLTSGTGSPGSTTSATGSVSTSVSATTTSAGSCTTPTSVAVTFDEIATTSYGENVYIVGSISQLGSWNTANAIALSASKYTTSNNLWYVTINLPAGTTFQYKYIRKESDGTVKWESDPNRSYTVPSACGVSTATENDTWR

[0621] SEQ ID NO:5-Pm_ATCC_18224_GA

[0622] MTFSRLSSSVLCALAALGHNALAAPQFSPRATVGLDAWLASETTFSLNGILANIGSSGAYSASAKPGVVIASPSTNNPNYYYTWTRDSALTLKVLIDLFGNGNLSLQTVIEEYINAQAYLQTVSNPSGDLSSGAGLAEPKYNVDMSPFTGGWGRPQRDGPALRAIALIEFGNWLIDNGYSSYAVNNIWPIVRNDLSYVSQYWSQSGFDLWEEVNSMSFFTVANQHRALVQGSTFAARVGASCSWCDSQAPQILCYMQTFWTGSYINANTGGGRSGKDSNTVLTTIHTFDPEATCDDVTFQPCSPRALANHKVYTDSFRSIYGVNSGIAQGVAVSVGRYPEDSYYGGNPWFLSNLAAAEQLYDAIYQWNKIGSITITSTSLAFFKDVYSSAAVGTYASGSTAFTSIISAVKTYADGYVSIVQGHAAANGSLSEQFDRNSGVEISARDLTWSYAALLTANLRRNGVMPPSWGAASANSVPSSCSMGSATGTYSTPTATAWPSTLTSATGIPVTTSATASVTKATSATSTTTSATTCTTPTSVAVTFDEIATTTYGENVFIVGSISQLGSWDTSKAIALSASQYTSSNHLWFATLSLPAGTTFQYKYIRKESNGSIVWESDPNRSYTVPSGCGVSTATENDTWR

[0623] SEQ ID NO:6-An_FGSC_A4_GA

[0624] MPTTILKITLFPLIDSIFSVQLSPVRIAMLTLSKVLPVLALSHAVAAAPQLSARATASLNTWLSTEASFALDGILTNIGANGAYAKTAKAGADYYTWTRDAALTVKVLVDLFHNGDLSLQTILEEYTNSQAYLQTVSNPSGGLASGGLAEPKFYVDMTAFTGSWGRPQRDGPALRATTLIGFGNWLIDNGYSSYASNNIWPIVRNDLTYVAQYWSKSGYDLWEEVNSMSFFTVAVQHRALVEGSTFAHRVGASCPWCDSQAPQILCYMQNFWTGSYINANTGGGRSGKDANTVLASIHTFDPDAACDDITFQPCSSRALANHKVYTDSFRSVYSLNTGIAQGVAVAAGRYPEDSYYNGNPWFLTTLAAAEQLYDAIYQWQKARSISITSTSLAFFKDIYSSAAVGTYASGSSAFTAIIDAVKTYADGYVSIVKAHAMANGSLSEQFDKTYGTCVSARDLTWSYAALLTASMRRNGVVPPSWDAASANTLPSSCSTGSATGTYSTATVTTWPSTLTSGSASATTTIMATSTATSSSTTTSTTTACTTPSTVAVTFNVIATTTYGENVYIVGSISQLGNWDTGSAVALSASKNTSSNNLWYVDINLPGGTAFEYKYIRKETDGSIVWESDPNRSYTVPSSCGVSTATESDTWRCTLETQSVRN

[0625] SEQ ID NO:7 - AfGA1 and AfGA2 CBM

[0626] FNEIATTTYGENVYIVGSISELGNWDTSKAVALSASKYTSSNNLWYVSVTLPAGTTFEYKYIRKESDGSIVWESDPNRSYTVPAACGVSTATENDTW

[0627] SEQ ID NO:8 - pTrex3gM - AfGA1's AfGA1 gene

[0628]

[0629] SEQ ID NO:9 - AfGA1

[0630] GCGGCGGCCGCACCATGCCTCGCCTTTCCTACGC

[0631] SEQ ID NO:10 - AfGA1

[0632] CCGGCGCGCCCTTATCACTGCCAAGTATCATTCTCG

[0633] SEQ ID NO:11 - AfGA1 and AfGA2 signal peptides

[0634] MPRLSYALCALSLGHAAIA

[0635] SEQ ID NO:12 - AfGA1 mature form

[0636] APQLSARATGSLDSWLGTETTVALNGILANIGADGAYAKSAKPGIIIASPSTSEPDYYYTWTRDAALVTKVLVDLFRNGNLGLQKVITEYVNSQAYLQTVSNPSGGLASGGLAEPKYNVDMTAFTGAWGRPQRDGPALRATALIDFGNWLIDNGYSSYAVNNIWPIVRNDLSYVSQYWSQSGFDLWEEVNSMSFFTVAVQHRALVEGSTFAKRVGASCSWCDSQAPQILCYMQSFWTGSYINANTGGGRSGKDANTVLASIHTFDPEAGCDDTTFQPCSPRALANHKVYTDSFRSVYAINSGIPQGAAVSAGRYPEDVYYNGNPWFLTTLAAAEQLYDAIYQWKKIGSISITSTSLAFFKDIYSSAAVGTYASSTSTFTDIINAVKTYADGYVSIVQAHAMNNGSLSEQFDKSSGLSLSARDLTWSYAAFLTANMRRNGVVPAPWGAASANSVPSSCSMGSATGTYSTATATSWPSTLTSGSPGSTTTVGTTTSTTSGTAAETACATPTAVAVTFNEIATTTYGENVYIVGSISELGNWDTSKAVALSASKYTSSNNLWYVSVTLPAGTTFEYKYIRKESDGSIVWESDPNRSYTVPAACGVSTATENDTWQ

[0637] SEQ ID NO:13 - Mature form of AfGA2

[0638] APQLSARATGSLDSWLGTETTVALNGILANIGADGAYAKSAKPGIIIASPSTSEPDYYYTWTRDAALVTKVLVDLFRNGNLGLQKVITEYVNSQAYLQTVSNPSGGLASGGLAEPKYNVDMTAFTGAWGRPQRDGPALRATALIDFGNWLIDNGYSSYAVNNIWPIVRNDLSYVSQYWSQSGFDLWEEVNSMSFFTVAVQHRALVEGSTFAKRVGASCSWCDSQAPQILCYMQSFWTGSYINANTGGGRSGKDANTVLASIHTFDPEAGCDDTTFQPCSPRALANHKVYTDSFRSVYAINSGIPQGAAVSAGRYPEDVYYNGNPWFLTTLAAAEQLYDAIYQWKKIGSISITSTSLAFFKDIYSSAAVGTYASSTSTFTDIINAVKTYADGYVSIVQAHAMNNGSLSEQFDKSSGLSLSARDLTWSYAAFLTANMRRNGVVPAPWGAASANSVPSSCSMGSATGTYSTATATSWPSTLTSGSPGSTTTVGTTTSTTSGTATETACATPTAVAVTFNEIATTTYGENVYIVGSISELGNWDTSKAVALSASKYTSSNNLWYVSVTLPAGTTFEYKYIRKESDGSIVWESDPNRSYTVPAACGVSTATENDTWR

[0639] SEQ ID NO:14 - AfGA2 gene of pTrex3gM - AfGA2

[0640]

Claims

1. A yeast cell expressing AfGA or a variant thereof, wherein the AfGA or a variant thereof comprises an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 12 or 13.

2. The yeast cell according to claim 1, wherein said yeast cell secretes said AfGA.

3. The yeast cell according to claim 1 or 2, wherein the AfGA or variant thereof comprises an amino acid sequence having at least 95% or 99% sequence identity with the sequence of SEQ ID NO: 12 or 13.

4. The yeast cell according to claim 3, wherein the AfGA or its variant comprises the sequence of SEQ ID NO: 12 or 13.

5. The yeast cell according to any one of the preceding claims, wherein the yeast cell further expresses an additional glucoamylase.

6. A simultaneous saccharification and fermentation (SSF) process, the process being performed using a yeast cell expressing AfGA or a variant thereof, wherein the AfGA or a variant thereof comprises an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 12 or 13, and wherein the yeast cell and the AfGA or a variant thereof are present in the same process step.

7. Use of a yeast cell expressing AfGA or a variant thereof for a simultaneous saccharification and fermentation (SSF) process, wherein the AfGA or a variant thereof comprises an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 12 or 13, and wherein the yeast cell and the AfGA or a variant thereof are present in the same process step.

8. The method of claim 6 or the use of claim 7, wherein the AfGA or variant thereof comprises an amino acid sequence having at least 95% or 99% sequence identity with the sequence of SEQ ID NO: 12 or 13.

9. The method or use according to any one of claims 6 to 8, wherein the yeast cell further expresses an additional glucoamylase.

10. The method or use according to any one of claims 6 to 9, wherein the method or use comprises producing ethanol.

11. The method or use according to claim 10, further comprising ethanol purification and recovery.

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