Brewing with thermostable AMG variants

Abstract

The present invention relates to a method of producing beer wort comprising adding to the mash a mature thermostable variant of a parent glucoamylase having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 10, before, during or after starch liquefaction, and a starch saccharification or brewing composition comprising said mature thermostable variant of the parent glucoamylase.

Description

[0001] References to sequence listings

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Technical Field

[0003] The present invention relates to a method for producing beer wort, the method comprising adding to the mash a mature thermostable variant of a parent glucoamylase having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 10; and a saccharification or brewing composition comprising the variant. Background Art

[0004] Cereal starch consists of glucose polymers in which the glucose residues are linked by α-1,4 or α-1,6 bonds. In brewing, the extraction of cereal components is called saccharification. During this step, water is adsorbed by starch granules and the intermolecular bonds of starch are gradually broken until the starch is irreversibly dissolved in water. The temperature required to start this process (called swelling) depends on the type of starch. Some cereal starches such as barley or wheat starch begin to swell at 58°C-65°C, while other cereal starches such as corn, rice, sorghum starch begin to swell between 60°C-85°C. In the case of brewing with cereals with a high gelatinization temperature, a parallel process is typically used, the so-called liquefaction or grain cooking, in which the starch is exposed to a temperature of 85°C-100°C for a period of time to ensure the dissolution and liquefaction of the starch (typically achieved using a thermostable α-amylase). After this step, the liquefied starch is adjusted (by cooling or mixing with cold mash) to a temperature at which the saccharifying enzymes can hydrolyze the existing non-fermentable dextrins into fermentable sugars. The main fermentable sugars are glucose, maltose and maltotriose, with trace amounts of sucrose and fructose also present.

[0005] Wort produced without the addition of exogenous enzymes typically consists of 65%-80% fermentable sugars and 20%-35% dextrins. The extent of fermentable sugars depends on the amylolytic activity of the endogenous amylases of the malt and the amylolytic scheme employed. Due to the natural limitation of the endogenous amylolytic activity of the malt (i.e., β-amylase), there will always be residual unfermentable dextrins that cannot be converted into ethanol by brewing yeast.

[0006] The goal of breweries is to increase the ethanol yield per unit of raw material without significantly changing the sensory perception of the final beer. Commercial pullulanase, amyloglucosidase, α-amylase, β-amylase, maltogenic amylase and combinations thereof can be used in the saccharification step of liquefied starch to overcome this limitation. In addition, low-calorie beer and low-residual carbohydrate beer are very popular in the US beer market and have recently attracted widespread attention worldwide. The principle of obtaining such beer is the same as using commercial saccharifying enzymes, but the goal is to maximize the hydrolysis of dextrins.

[0007] Another area of ​​application of commercial saccharifying enzymes is the production of wort using 100% unmalted raw materials such as barley, wheat, sorghum, corn, rice, cassava, etc. Since such raw materials lack or contain only insufficient endogenous amylolytic activity for brewing purposes, commercial amylolytic enzymes must be used. With regard to the saccharification of barley malt, the saccharification protocol design needs to ensure that the grain starch is completely gelatinized and liquefied, and that the subsequent saccharification has been carried out to a certain extent so that the fermentable sugars in the wort can reach the target actual fermentation degree by the yeast.

[0008] For feedstocks that exhibit high gelatinization temperatures (e.g., rice, corn, sorghum), saccharification protocols typically include a liquefaction step at a temperature of 85° C.-100° C., followed by saccharification at a lower temperature of 62° C.-70° C. Such methods typically require more time and may require additional equipment.

[0009] In order to simplify and shorten the starch saccharification process without affecting the efficiency of the process, improved and more thermostable amylolytic enzymes are needed.

[0010] US 3379534 describes the preparation of low dextrin beer by using amyloglucosidase.

[0011] US 4536477 describes thermostable glucoamylases which are particularly useful for the preparation of glucose-containing syrups from starch.

[0012] WO 2009 / 075682 describes the use of certain pullulanases for the production of beer wort, wherein the starch saccharification is achieved using relatively small amounts of enzyme protein.

[0013] Matthews et al., 2001, Journal of Institute of brewing 107(3) pp185-194 discloses the preparation of low carbohydrate beer by saccharification of starch at high temperature with glucoamylase derived from Aspergillus niger.

[0014] WO 2012 / 140075 (Novozymes A / S, Denmark) discloses a method for producing beer wort by adding certain glucoamylases. Summary of the invention

[0015] The inventors have found that certain thermostable variants of glucoamylases show greatly improved performance in saccharification (i.e. brewing). Another improved performance of thermostable variants is that they increase the sweetness or sweetness of the product, thereby reducing the amount of sugar added in traditional recipes.

[0016] Therefore, in a first aspect, the present invention relates to a method for producing beer wort, the method comprising adding to the mash a mature thermostable variant of a parent glucoamylase having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 10 before, during or after liquefaction of starch.

[0017] The second aspect of the invention relates to starch saccharification or brewing compositions, which comprise a mature thermostable variant of a parent glucoamylase as defined in the first aspect; preferably further comprising one or more additional enzymes selected from the group consisting of alpha amylase, maltogenic amylase, raw starch degrading alpha amylase, beta amylase, aminopeptidase, carboxypeptidase, catalase, cellobiose oxidase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1,4-dihydropyranose, 1,2 ... - α-maltotetraose hydrolase, glucanase, β-glucanase, galactanase, α-galactosidase, β-galactosidase, cellobiose oxidase, glucose oxidase, α-glucosidase, β-glucosidase, haloperoxidase, hemicellulolytic enzyme, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinase, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, protease, pullulanase, ribonuclease, transglutaminase and xylanase. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A multiple alignment of the amino acid sequences of the following mature proteins is shown:

[0019] - Wild-type AMG from Penicillium oxalicum (PoAMG) of SEQ ID NO: 1

[0020] - PoAMG variant denoted "AMG NL" of SEQ ID NO: 2

[0021] - A PoAMG variant of SEQ ID NO: 3 denoted "AMG anPAV498"

[0022] - PoAMG variant denoted "AMG JPO001" of SEQ ID NO:4

[0023] - A PoAMG variant of SEQ ID NO:5 designated as "AMG JPO124"

[0024] - A PoAMG variant of SEQ ID NO: 6 denoted "AMG JPO-172"

[0025] - Wild-type AMG from Penicillium miczynskii (PoAMG) of SEQ ID NO: 7

[0026] - Wild-type AMG from Penicillium russellii (PoAMG) of SEQ ID NO: 8

[0027] - Wild-type AMG from Penicillium glabrum (PoAMG) of SEQ ID NO: 9 DETAILED DESCRIPTION

[0028] definition

[0029] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0030] For the purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453), as implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Genetics Trend] 16: 276-277) (preferably version 5.0.0 or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the "longest identity" marked with Needle (obtained using the non-simplified (-no brief) option) is used as the identity percentage and is calculated as follows:

[0031] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0032] Variant: The term "variant" means a polypeptide comprising an alteration (i.e., a substitution, insertion, and / or deletion) at one or more (e.g., several) positions. Substitution means replacing the amino acid occupying a position with a different amino acid; deletion means removing the amino acid occupying a position; and insertion means adding one or more amino acids adjacent to and immediately following the amino acid occupying a position. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function (such as a polyhistidine stretch, antigenic epitope, or binding domain). Examples of conservative substitutions are within the group consisting of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0033] Improved thermostability: Thermostability improvement (Td) in °C is a measure of how much a variant is improved in thermostability relative to its parent glucoamylase under the same conditions, determined as exemplified herein.

[0034] As used herein, the term "cracked malt" is understood to mean the starch- or sugar-containing material, such as barley malt and adjuncts, which is the basis for beer production. Typically, cracked malt does not contain any added water.

[0035] The term "malt" is understood to mean any malted cereal, in particular barley.

[0036] The term "adjunct" is understood to mean the non-barley malt portion of the ground malt. Adjuncts may be any starch-rich plant material, e.g., unmalted grains such as, but not limited to, barley, corn, rice, sorghum, and wheat, and also include readily fermentable sugars and / or syrups. The starch of some adjuncts has a relatively low gelatinization temperature, which allows them to be saccharified with malt, while other adjuncts such as rice, corn, and sorghum have higher gelatinization temperatures, and such adjuncts are typically cooked separately and solubilized with alpha-amylase before being added to the mash.

[0037] The term "mash" is understood to be a starch-containing slurry comprising ground barley malt, ground unmalted grains, other starch-containing materials, or combinations thereof, which is steeped in water to make wort.

[0038] The term "wort" is understood to mean the unfermented liquid which flows out after extraction of the grist during mashing.

[0039] The term "stillers grains" is understood to mean the drained solids remaining after extraction of the grist and separation of the wort.

[0040] The term "beer" is understood here as fermented wort, i.e. an alcoholic beverage brewed from barley malt, optionally adjuncts and hops. As used herein, the term "beer" is intended to encompass at least beers prepared from mashes prepared from unmalted grains, all mashes prepared from malted grains, and all mashes prepared from mixtures of malted and unmalted grains. The term "beer" also encompasses beers prepared with adjuncts, as well as beers of all possible alcohol contents.

[0041] The term "starch gelatinization" is understood as the irreversible order-disorder transition that starch undergoes when heated in the presence of water. Differential scanning calorimetry (DSC) is a method that can be used to study the gradual process of starch gelatinization (describing the onset temperature and peak temperature (T o and T p The term "initial gelatinization temperature (T o The term "peak gelatinization temperature (T p The term "end gelatinization temperature (T c )” is understood as the temperature at which gelatinization is terminated.

[0042] Wort production

[0043] A first aspect of the invention relates to a method for producing beer wort, the method comprising adding to the mash a mature thermostable variant of a parent glucoamylase having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 10 before, during or after starch liquefaction.

[0044] Preferably, the method of the first aspect comprises adding to the mash, before, during or after liquefaction of the starch, a mature thermostable variant of a parent glucoamylase having at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 10.

[0045] The starch saccharification process generally uses a controlled step-wise increase in temperature, wherein each step favors an enzymatic action, ultimately degrading proteins, cell walls and starch. Starch saccharification temperature profiles are generally known in the art. In the present invention, the saccharification (starch degradation) step in the starch saccharification process is preferably carried out between 60°C and 66°C, more preferably between 61°C and 65°C, even more preferably between 62°C and 64°C, and most preferably between 63°C and 64°C. In a specific embodiment of the invention, the saccharification temperature is 64°C.

[0046] In one aspect, the starch saccharification method of the present invention includes but is not limited to a cooking step. In one aspect, the cooking step includes but is not limited to incubating the mash at a temperature of at least 65°C for at least 20 minutes. In one aspect, the cooking step comprises incubating the mash at a temperature of at least 65°C, such as at least 66°C, at least 67°C, at least 68°C, at least 69°C, at least 70°C, at least 71°C, at least 72°C, at least 73°C, at least 74°C, or at least 75°C, at least 76°C, at least 77°C, at least 78°C, at least 79°C, at least 80°C, at least 81°C, at least 82°C, at least 83°C, at least 84°C, or at least 85°C for at least 20 minutes, such as at least 25 minutes, at least 30 minutes, at least 35 minutes. In a specific embodiment, the slurry is cooked at 75° C. for 120 minutes.

[0047] In one embodiment, the present invention relates to a method for producing beer wort, the method comprising adding to the mash a mature thermostable variant of a parent glucoamylase, which mature thermostable variant is active during mashing conditions at a temperature of at least 65° C. On the other hand, the mature thermostable variant of the parent glucoamylase is active during mashing conditions at least 68° C., such as 70° C., such as 72° C., such as 75° C., such as 76° C., such as 77° C., such as 78° C., such as 79° C., such as 80° C.

[0048] Preferably, the mature thermostable variant of the parent glucoamylase is active during clarification, saccharification and / or mash filtration.

[0049] According to one aspect, the mash can be obtained by grinding ground malt comprising malt and / or adjuncts. Preferably, water can be added to the ground malt, preheated, so that the mash reaches the desired mash temperature at the moment the mash is formed. If the temperature of the formed mash is lower than the desired saccharification temperature, additional heat is preferably supplied in order to reach the desired process temperature. Preferably, the desired saccharification temperature is reached within 15 minutes after the mash is formed, or more preferably within 10 minutes, for example within 9, 8, 7, 6, 5, 4, 3, 2 minutes, or even more preferably within 1 minute, or most preferably the desired saccharification temperature is reached when the mash is formed. The temperature profile of the saccharification process may be a profile from a conventional saccharification process, in which the temperature is set to achieve optimal degradation of the ground malt dry matter by the maltase.

[0050] The starch saccharification process generally uses a controlled step-wise increase in temperature, wherein each step favors an enzymatic action, ultimately degrading proteins, cell walls and starch. Starch saccharification temperature profiles are generally known in the art. In the present invention, the saccharification (starch degradation) step in the starch saccharification process is preferably carried out between 60°C and 66°C, more preferably between 61°C and 65°C, even more preferably between 62°C and 64°C, and most preferably between 63°C and 64°C. In a specific embodiment of the invention, the saccharification temperature is 64°C.

[0051] In a preferred embodiment of the first aspect, the saccharification comprises an incubation step at 65°C or higher for at least 20 minutes; preferably at 67°C or higher for at least 20 minutes; even more preferably at 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C or most preferably at 87°C or higher for at least 20 minutes.

[0052] In one aspect, the pH of the mash is in the range of about 4.6 to about 6.4. In another aspect, the pH is in the range of about 4.6 to 6.2, such as a pH in the range of about 4.8 to about 6.0, preferably a pH in the range of about 5.0 to about 6.0, more preferably a pH in the range of about 5.0 to about 5.6, and even more preferably a pH in the range of about 5.0 to about 5.4.

[0053] In another preferred embodiment of the first aspect, the pH of the mash is about 4.6 to about 6.4.

[0054] The malt is preferably derived from one or more of the cereals selected from the list consisting of: corn, barley, wheat, rye, sorghum, millet and rice. Preferably, the malt is barley malt. The ground malt preferably comprises from 0.5% to 99%, preferably from 1% to 95%, more preferably from 5% to 90%, even more preferably from 10% to 80% malt.

[0055] Preferably, the wort of the first aspect has higher than 80% glucose compared to the total carbohydrate content of the wort.

[0056] In addition to malted grains, the crushed malt may preferably include adjuncts such as unmalted corn, or other unmalted grains such as barley, wheat, rye, oats, corn, rice, milo, millet and / or sorghum, or unprocessed and / or refined starch and / or sugar (containing materials derived from plants such as wheat, rye, oats, corn, rice, milo, millet, sorghum, potato, sweet potato, cassava, tapioca, sago, banana, beet and / or sugar cane). For the present invention, adjuncts may be obtained from tubers, roots, stems, leaves, beans, cereals and / or whole grains. Preferred are adjuncts obtained from corn and / or rice, more preferably the adjuncts are rice starch, corn starch and / or corn grits. The mash preferably comprises from 1% to 60%, preferably from 5% to 45%, more preferably from 10% to 40% adjunct starch. Adjuncts may also include readily fermentable carbohydrates (e.g., sugars or syrups) and may be added to the malt mash before, during, or after the saccharification process of the present invention but are preferably added after the saccharification process. Before forming the mash, the malt and / or adjuncts are preferably ground and most preferably dry or wet ground. In one aspect, the adjuncts have a high gelatinization temperature, more particularly, a high initial gelatinization temperature for, for example, corn, rice, and sorghum. In one aspect, the adjuncts are gelatinized before saccharification. In another aspect, the adjuncts are not gelatinized before saccharification.

[0057] In one embodiment, the mash consists of at least 20% adjuncts having a starch gelatinization temperature, preferably an initial gelatinization temperature, of at least 65° C. In another aspect, the mash consists of at least 25%, such as at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, such as at least 65% adjuncts having a starch gelatinization temperature, preferably an initial gelatinization temperature, of at least 65° C.

[0058] In one embodiment, the mash comprises at least 10% unmalted grains compared to whole ground malt. In another aspect, the mash comprises at least 15%, such as at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, such as at least 60% unmalted grains.

[0059] Preferably, the adjunct comprises corn. In another aspect of the invention, the mash comprises at least 20% corn adjunct. In one aspect, the mash comprises at least 25%, such as at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, such as at least 60% corn adjunct; preferably, the corn adjunct is ungelatinized when added to the mash.

[0060] In another preferred embodiment, the adjunct comprises rice. In another aspect of the invention, the mash comprises at least 20% rice adjunct. In one aspect, the mash comprises at least 25%, such as at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, such as at least 60% rice adjunct; preferably, the rice adjunct is not gelatinized when added to the mash.

[0061] In a preferred embodiment, practicing the method of the invention results in the formation of additional glucose at temperatures between 65° C. and 90° C. In one aspect, the additional glucose formed at temperatures between 65° C. and 90° C. is at least 1%, such as at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, compared to wort produced in the absence of such glucoamylase.

[0062] Preferably, practicing the method of the invention results in a reduction in the concentration of maltose in the wort. In one aspect, the concentration of maltose is reduced by at least 0.5%, such as at least 1%, such as at least 2%, at least 3%, at least 4%, at least 5%, compared to wort produced in the absence of such glucoamylase.

[0063] In preferred embodiments, the thermostable variant glucoamylase is provided exogenously and / or is present in the mash. In one aspect, the glucoamylase is introduced at the beginning of saccharification. In another aspect, the glucoamylase is introduced during saccharification. In another aspect, the glucoamylase is introduced during clarification.

[0064] As will be apparent from the following examples, when using the thermostable glucoamylase variants of the first aspect of the invention, the saccharification or amyloamylation step in brewing can be shortened, which is obviously of commercial interest because it will reduce capacity costs and energy costs. In other words, the present invention allows brewers to reach a desired predetermined target glucose concentration more quickly.

[0065] Therefore, a preferred embodiment of the present invention relates to the method of the first aspect, wherein the target glucose concentration is achieved in a shorter saccharification time compared to adding an AMG enzyme having a lower thermal stability than claim 1.

[0066] Preferably, the methods of the invention result in a shortened saccharification time; more preferably, these methods result in a saccharification time that is reduced by at least 5 minutes, e.g., at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, more preferably by at least 30 minutes, compared to a method carried out without the addition of a thermostable glucoamylase variant of the invention.

[0067] Glucoamylase

[0068] Glucoamylases are also called amyloglucosidases and glucan 1,4-alpha-glucosidases (EC 3.2.1.3), more commonly they are known as AMG.

[0069] According to the present invention, different types of amyloglucosidases can be used as parents for producing thermostable amyloglucosidase variants, for example, the amyloglucosidase can be a polypeptide encoded by a DNA sequence found in a fungal strain of Aspergillus, Rhizopus, or Talaromyces (Rasamsonia) or Penicillium; preferably a DNA sequence found in a fungal strain of Penicillium, even more preferably a DNA sequence found in a fungal strain of Penicillium oxysporum, Penicillium oxalicum, Penicillium mikrokeri, Penicillium roqueforti or Penicillium luminosporum. Preferably, the parent glucoamylase is from a species of Penicillium, preferably from Penicillium oxalicum, Penicillium mikrokeri, Penicillium roqueforti or Penicillium luminosporum.

[0070] Examples of other suitable fungi include Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Rhizopus delemar, Rhizopus niveus, Rhizopus oryzae, and Talaromyces emersonii (Rasamsonia emersonii).

[0071] The following shows Figure 1 The % identity between the AMG amino acid sequences aligned in , and are also provided in the sequence listing:

[0072]

[0073] Thermostable variants of PoAMG have been generated (see Table 2 below).In preferred embodiments, the mature thermostable glucoamylase variants of the invention comprise one or more or all combinations of the amino acid substitutions listed in Table 2 below.

[0074] In a preferred embodiment, the mature variant of the present invention comprises at least one amino acid modification at one or more or all positions corresponding to positions 1, 2, 4, 6, 7, 11, 31, 34, 50, 65, 79, 103, 132, 327, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595 in SEQ ID NO: 1; preferably, the at least one amino acid modification comprises a substitution at one or more or all positions corresponding to positions 1, 2, 4, 11, 65, 79 and 327 in SEQ ID NO: 1, preferably, the at least one amino acid modification comprises a substitution at one or more or all positions corresponding to R1A, P2N, P4S, P11F, T65A, K79V and Q327F in SEQ ID NO: 1; or preferably, the at least one amino acid modification comprises a substitution at one or more or all positions corresponding to SEQ ID NO: NO:1 comprises a substitution at one or more or all of positions 1, 6, 7, 31, 34, 79, 103, 132, 445, 447, 481, 566, 568, 594 and 595, preferably, the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to R1A, G6S, G7T, R31F, K34Y, K79V, S103N, A132P, D445N, V447S, S481P, D566T, T568V, Q594R and F595S in SEQ ID NO:1; or preferably, the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to SEQ ID NO:1. NO:1 comprises a substitution at one or more or all of positions 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595, preferably, the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO:1;or preferably, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595 in SEQ ID NO: 1, preferably, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO: 1; or preferably, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to SEQ ID NO: IDNO: 1 comprises a substitution at one or more or all of positions 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595, preferably, the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO: 1. ;

[0075] The improved thermostability (Td) of the variants in Table 2 are listed in Table 3, wherein the Td of the PoAMG variant denoted "anPAV498" (parent) is set to zero. In preferred embodiments, the mature thermostable variants of the invention have an improved thermostability (Td) of at least 5°C, preferably at least 6°C, 7°C or 8°C relative to their parent, preferably as determined as exemplified herein.

[0076] In another preferred embodiment, the mature thermostable variant of the present invention has a relative activity of at least 150, preferably at least 200, more preferably at least 250, most preferably at least 300 at 91°C compared to its parent.

[0077] Other enzymes

[0078] In a preferred embodiment of the first aspect, one or more additional enzymes are added to the mash, and the additional enzymes may be selected from the group consisting of: alpha amylase, maltogenic amylase, raw starch degrading alpha amylase, beta amylase, aminopeptidase, carboxypeptidase, catalase, cellobiose oxidase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1,4-alpha-maltotetraose hydrolase, glucanase, beta glucanase, galactoside. Polysaccharidase, α-galactosidase, β-galactosidase, cellobiose oxidase, glucose oxidase, α-glucosidase, β-glucosidase, haloperoxidase, hemicellulolytic enzyme, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinase, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, protease, pullulanase, raw starch degrading α-amylase, ribonuclease, transglutaminase and xylanase.

[0079] Raw starch degrading α-amylase

[0080] As used herein, "raw starch degrading α-amylase" refers to an enzyme that can directly degrade raw starch granules below the gelatinization temperature of starch.

[0081] Examples of raw starch degrading α-amylases include those disclosed in WO 2005 / 003311, U.S. Patent Publication No. 2005 / 0054071, and U.S. Patent No. 7,326,548. Examples also include those disclosed in Tables 1-5 of the Examples in U.S. Patent No. 7,326,548 and in U.S. Patent Publication No. 2005 / 0054071 (Table 3 on page 15), and enzymes disclosed in WO 2004 / 020499, WO 2006 / 06929, and WO 2006 / 066579.

[0082] In one embodiment, the raw starch degrading alpha-amylase is a GH13-1 amylase.

[0083] In an embodiment the raw starch degrading alpha-amylase has at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to the raw starch degrading alpha-amylase in European Patent No. 2981170 (Novozymes) or shown in SEQ ID NO: 11 herein.

[0084] Amylase

[0085] α-Amylases (α-1,4-glucan-4-glucanohydrolases, EC.3.2.1.1) constitute a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycosidic oligo- and polysaccharides.

[0086] Termamylase is a type of α-amylase. TM , SC and "Termamyl TM -like α-amylases" and are known from, for example, WO 90 / 11352, WO 95 / 10603, WO 95 / 26397, WO 96 / 23873 and WO 96 / 23874.

[0087] Another group of α-amylases is called Fungamyl TM and "Fungamyl TM -like α-amylases", which are α-amylases related to the α-amylase derived from Aspergillus oryzae disclosed in WO 01 / 34784.

[0088] Protease

[0089] Suitable proteases include microbial proteases, such as fungal proteases and bacterial proteases. Preferred proteases are acidic proteases, i.e., proteases characterized by being able to hydrolyze proteins under acidic conditions below pH 7. The role of proteases is to shorten the total length of high molecular weight proteins in the mash to low molecular weight proteins. Low molecular weight proteins are essential for yeast nutrition, while high molecular weight proteins ensure foam stability. Therefore, it is well known to those skilled in the art that proteases should be added in a balanced amount so as to provide sufficient free amino acids for yeast at the same time and leave enough high molecular weight proteins to stabilize the foam. On the one hand, the protease activity is provided by a proteolytic enzyme system having a suitable FAN producing activity, including endoproteases, exopeptidases or any combination thereof, preferably metalloproteases. Preferably, the protease has at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% or even 100% identity to the amino acid sequence shown in SEQ ID NO: 6 as described in WO 9967370. In another aspect, the protease is available from Novozymes AG The amount of protease added can be 0.0001-1000AU / kg DS, preferably 1-100AU / kg DS and most preferably 5-25AU / kg dry weight grain. Proteolytic activity can be determined by using denatured hemoglobin as a substrate. In the Anson-Hemoglobin method for determining proteolytic activity, denatured hemoglobin is digested and undigested hemoglobin is precipitated with trichloroacetic acid (TCA). The amount of TCA-soluble products is determined by using a phenol reagent, which can produce a blue color with tyrosine and tryptophan. One Anson unit (AU) is defined as the amount of enzyme that digests hemoglobin at an initial rate under standard conditions (i.e., 25°C, pH 7.5 and 10min reaction time), so that a certain amount of TCA-soluble products is released per minute, and the color produced by the product and the phenol reagent is the same as one milliequivalent of tyrosine.

[0090] Enzyme composition

[0091] The mature thermostable variant glucoamylase of the invention and any one or more additional enzymes can be added in any suitable form, for example in the form of a liquid (particularly a stabilized liquid), or can be added as a substantially dry powder or granules.

[0092] For example, granules may be produced as disclosed in U.S. Pat. No. 4,106,991 and U.S. Pat. No. 4,661,452. Liquid enzyme preparations may be stabilized, for example, by adding sugars or sugar alcohols or lactic acid according to established procedures. Other enzyme stabilizers are well known in the art.

[0093] The one or more enzymes can be added in any suitable manner, such as as separate components (adding the enzymes separately or sequentially), or adding the enzymes together in one step or one composition.

[0094] Granules and agglomerated powders can be prepared by conventional methods, for example, by spraying the enzyme onto a carrier in a fluidized bed granulator. The carrier can be composed of microparticle cores with a suitable particle size. The carrier can be soluble or insoluble, for example salt (such as NaCl or sodium sulfate), sugar (such as sucrose or lactose), sugar alcohol (such as sorbitol), starch, rice, corn residue, or soybean.

[0095] One aspect of the present invention relates to starch saccharification or brewing compositions comprising mature thermostable variants of parent glucoamylases as defined in the first aspect of the present invention and preferred embodiments thereof. Preferably, the starch saccharification or brewing composition further comprises one or more additional enzymes selected from the group consisting of alpha amylase, maltogenic amylase, raw starch degrading alpha amylase, beta amylase, aminopeptidase, carboxypeptidase, catalase, cellobiose oxidase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1,4-alpha-maltotetraose hydrolase, glucanase, beta glucanase, esterase, alpha amylase, maltogenic amylase, raw starch degrading alpha amylase, beta glucan ... Enzymes, galactanases, α-galactosidases, β-galactosidases, glucose oxidases, α-glucosidases, β-glucosidases, haloperoxidases, hemicellulolytic enzymes, invertases, laccases, lipases, mannanases, mannosidases, oxidases, pectinolytic enzymes, peptidylglutaminases, peroxidases, phospholipases, phytases, polyphenol oxidases, proteases, pullulanases, ribonucleases, transglutaminases and xylanases.

[0096] Examples

[0097] Example 1: Construction of PoAMG library

[0098] The PoAMG library was constructed as follows:

[0099] Design the forward or reverse primer with NNK or one or more desired mutations at one or more target sites, and these target sites have an overlap of 15bp each other. Use appropriate template plasmid DNA (for example, plasmid DNA containing JPO-0001 gene) to carry out inverse PCR under the following conditions, i.e., amplify the whole plasmid DNA sequence by reverse directional primer. Purify the obtained PCR fragment by QIAquick gel extraction kit [Qiagen (QIAGEN)], then introduce it into Escherichia coli ECOS competent Escherichia coli DH5α [Japan Gene Co., Ltd. (NIPPON GENE CO., LTD.)]. Extract plasmid DNA from Escherichia coli transformant by MagExtractor plasmid extraction kit [Toyo Company (TOYOBO)], then introduce it into Aspergillus niger competent cell.

[0100] PCR reaction mixture:

[0101] PrimeSTAR Max DNA Polymerase [TaKaRa]

[0102] Total 25 μl

[0103]

[0104] PCR procedure:

[0105] 98℃ / 2min

[0106] 25x(98℃ / 10sec, 60℃ / 15sec, 72℃ / 2min)

[0107] 10℃ / maintain

[0108] Example 2: Screening for better thermal stability

[0109] The Bacillus subtilis library constructed as in Example 1 was fermented in a 96-well or 24-well MTP containing COVE liquid medium (2.0 g / L sucrose, 2.0 g / L isomaltose, 2.0 g / L maltose, 4.9 mg / L, 0.2 ml / L 5N NaOH, 10 ml / L COVE salts, 10 ml / L 1 M acetamide) at 32° C. for 3 days. AMG activity in the culture supernatant was then measured at several temperatures by the pNPG assay as described below.

[0110] pNPG thermal stability assay:

[0111] The culture supernatant containing the desired enzyme was mixed with the same volume of pH 5.0 200mM NaOAc buffer. Twenty microliters of the mixture was distributed in a 96-well plate or 8-link PCR tubes and then heated for 30 minutes at various temperatures by a thermal cycler. Those samples were mixed with 10 μl of substrate solution containing 0.1% (w / v) pNPG [Wako Co., Ltd. (wako)] in pH 5.0 200mM NaOAc buffer and incubated at 70°C for 20 minutes for enzyme reaction. After the reaction, 60 μl of 0.1M Borax buffer was added to stop the reaction. 80 μl of the reaction supernatant was taken out and its OD was read by a photometer. 405 to evaluate enzyme activity.

[0112] Table 1a. List of relative activities of PoAMG variants compared to their parents anPAV498 or JPO-0001 (anPAV498 with leader / propeptide)

[0113] name Relative activity at 80℃ / 75℃ (%) anPAV498 17％ JPO-004 32％ JPO-005 15％ JPO-006 16％ JPO-007 3％

[0114] name Relative activity at 80℃ / 75℃ (%) AnPav498 13％ JPO-009 16％ JPO-011 15％ JPO-012 15％ JPO-013 17％ JPO-020 20％

[0115] name Relative activity at 80℃ / 70℃ (%) JPO-001 10％ JPO-004 29％ JPO-009 13％ JPO-014 21％ JPO-020 16％ JPO-021 30％ JPO-052 33％

[0116] name Relative activity at 79℃ / 70℃ (%) JPO-001 23％ JPO-021 46％ JPO-022 39％ JPO-023 44％ JPO-025 51％ JPO-027 49％ JPO-029 37％

[0117] name Relative activity at 77℃ / 70℃ (%) JPO-001 72％ JPO-029 82％ JPO-047 80％ JPO-048 90％ JPO-049 84％ JPO-050 86％ JPO-064 87％

[0118]

[0119]

[0120] name Relative activity at 79℃ / 77℃ (%) JPO-001 41％ JPO-021 60％ JPO-022 48％ JPO-023 57％ JPO-025 56％ JPO-027 64％ JPO-029 66％ JPO-047 50％ JPO-048 72％ JPO-051 82％ JPO-058 73％ JPO-062 72％ JPO-063 85％ JPO-064 83％

[0121] Table 1b. List of relative activities of PoAMG variants compared to their parent JPO-022

[0122] name Relative activity at 77℃ / 70℃ (%) JPO-022 60％ JPO-027 67％ JPO-042 8％ JPO-044 86％ JPO-045 67％ JPO-046 48％

[0123] name Relative activity at 77℃ / 70℃ (%) JPO-022 76％ JPO-023 75％ JPO-025 80％ JPO-027 84％ JPO-058 92％ JPO-059 88％ JPO-060 86％ JPO-061 83％ JPO-062 87％

[0124] name Relative activity at 79℃ / 77℃ (%) JPO-022 49％ JPO-023 51％ JPO-025 52％ JPO-027 58％ JPO-058 69％ JPO-059 36％ JPO-060 41％ JPO-061 44％ JPO-062 57％

[0125] Table 1c. List of relative activities of PoAMG variants compared to their parent JPO-063 at different temperatures

[0126]

[0127]

[0128] name Relative activity at 84℃ / 80℃ (%) JPO-063 16％ JPO-065 26％ JPO-067 21％ JPO-070 12％ JPO-071 13％ JPO-074 32％ JPO-081 17％ JPO-082 24％ JPO-083 46％ JPO-084 26％ JPO-044 37％

[0129] name Relative activity at 82℃ / 70℃ (%) JPO-063 21％ JPO-093 43％ JPO-081 25％ JPO-088 39％ JPO-094 38％ JPO-096 38％ JPO-106 53％

[0130] name Relative activity at 83℃ / 80℃ (%) JPO-063 46％ JPO-051 44％ JPO-096 64％ JPO-106 88％ JPO-110 81％ JPO-111 100％ JPO-112 86％ JPO-113 83％ JPO-114 47％ JPO-115 90％

[0131] Table 1d. List of relative activities of PoAMG variants compared to their parent JPO-096

[0132] name Relative activity at 83℃ / 70℃ (%) JPO-082 53％ JPO-088 70％ JPO-091 69％ JPO-092 65％ JPO-093 62％ JPO-094 74％ JPO-095 69％ JPO-096 67％ JPO-097 65％ JPO-098 65％

[0133]

[0134]

[0135] Table 1e. List of relative activities of PoAMG variants compared to their parent JPO-129

[0136] name Relative activity at 84℃ / 80℃ (%) JPO-129 62％ JPO-156 51％ JPO-160 34％ JPO-161 41％ JPO-162 49％ JPO-163 21％ JPO-164 57％ JPO-165 77％

[0137] Table 1f. List of relative activities of PoAMG variants compared to their parent JPO-166

[0138] name Relative activity at 84℃ / 75℃ (%) JPO-166 19％ JPO-167 66％ JPO-168 58％ JPO-169 53％ JPO-171 47％ JPO-172 98％

[0139] Table 2. Amino acid substitutions in variants of the PoAMG mature sequence

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] Example 3: Fermentation of Aspergillus niger

[0146] Aspergillus niger strains were fermented in 500 ml baffled flasks containing 100 ml MU1 and 4 ml 50% urea on an orbital shaker at 220 rpm and 30° C. The culture broth was centrifuged (10,000×g, 20 min) and the supernatant was carefully decanted from the precipitate.

[0147] Example 4: Purification of PoAMG (JPO-001) variants

[0148] PoAMG variants were purified by cation exchange chromatography. The respective peak fractions were combined individually and dialyzed against 20 mM sodium acetate buffer (pH 5.0), and the samples were then concentrated using a centrifugal filter device (Vivaspin Turbo 15, Sartorius). The enzyme concentration was determined by the A280 value.

[0149] Example 5: Thermal Stability Assay (TSA)

[0150] The purified enzyme was diluted to 0.5 mg / ml with 50 mM sodium acetate buffer (pH 5.0) and mixed with an equal volume of SYPRO Orange (Invitrogen) diluted with Milli-Q water. 18 μl of the mixture solution was transferred to a LightCycler 480 multiwell plate 384 (Roche Diagnostics) and the plate was sealed.

[0151] TSA equipment parameters:

[0152] Instrument: LightCycler 480 Real-Time PCR System (Roche Applied Science)

[0153] Scan rate: 0.02℃ / sec

[0154] Scanning range: 37℃-96℃

[0155] Integration time: 1.0 sec

[0156] Excitation wavelength 465nm

[0157] Emission wavelength 580nm

[0158] The obtained fluorescence signals were normalized to the range of 0 and 1. Td was defined as the temperature at which the signal intensity was 0.5. The thermal stability improvements are listed in Table 3, where the Td of the PoAMG variants is indicated as 0 for anPAV498.

[0159] Example 6: PoAMG activity assay

[0160] Determination of maltodextrin (DE11) by GOD-POD method

[0161] Substrate solution

[0162] 30g maltodextrin (pindex #2 from MATSUTANI chemical industry Co., Ltd.)

[0163] 100 ml 120 mM sodium acetate buffer, pH 5.0

[0164] Glucose CII test kit (Wako Pure Chemical Industries, Ltd., Japan)

[0165] 20 μ l of enzyme sample was mixed with 100 μ l of substrate solution and incubated at set temperature for 2 hours. Sample was cooled on aluminum block for 3 min, then 10 ul of reaction solution was mixed with 590 ul of 1 M Tris-HCl (pH 8.0) to stop the reaction. 10 μ l of solution was mixed with 200 μ l of working solution of test kit and then left at room temperature for 15 min. Read absorbance at A505. Activity is listed in Table 3 as the relative activity of PoAMG variants represented as anPAV498.

[0166] Table 3

[0167]

[0168]

[0169]

[0170] Example 7: Starch saccharification experiment using 100% unmalted sorghum

[0171] Table 4: Raw material characteristics of sorghum used

[0172]

[0173] Sorghum was ground using a laboratory disc mill DLFU (Bühler AG, Switzerland) with a gap distance of 0.6 mm and the obtained ground malt was stored dry in a closed container until further use.

[0174] 100 ppm Ca was achieved by adding CaCl2 to deionized water. 2+ The saccharification water was prepared by dispensing 125 g of saccharification water into saccharification beakers, placing them in an LP electronic saccharification device (Lochner GmbH, Germany), and then preheating to a mash-in temperature of 55°C.

[0175] Sorghum malt was added to the saccharification water in the amount of 50 g (low tannin sorghum) or 12.5 g (low tannin) plus 37.5 g (high tannin) per beaker. The water and malt were homogenized by stirring at 150 rpm for 5 minutes. The stirring speed was then reduced to 100 ppm, and after this step, enzymes were added according to Table 5 below, and the saccharification process was started.

[0176] Table 5: Enzyme dosage used at the start of saccharification (each test was performed in duplicate)

[0177]

[0178]

[0179] The following starch saccharification scheme was used:

[0180] Starch saccharification scheme A : Stand at 55°C for 20 minutes; heat from 55°C to 78°C at a heating rate of 1°C per minute; stand at 78°C for 60 minutes; heat from 78°C to 85°C at a heating rate of 1°C per minute; stand at 85°C for 30 minutes; cool to 80°C; stand at 80°C for 15 minutes; cool to room temperature in 10 minutes. Total time without cooling = 170 minutes.

[0181] Starch saccharification scheme B : Stand at 55°C for 20 minutes; heat from 55°C to 85°C at a heating rate of 1°C per minute; stand at 85°C for 90 minutes; cool to 80°C; stand at 80°C for 15 minutes; cool to room temperature in 10 minutes. Total time without cooling = 170 minutes.

[0182] After mashing, all mashes were adjusted to the original weight of 175 g at the start of mashing for evaporation losses. The mash was filtered through a filter funnel with 240 mm (Macherey-Nagel, Germany) filter paper. The wort obtained was immediately frozen and stored until subsequent analysis.

[0183] analyze:

[0184] Finely ground sorghum malt was obtained by grinding with a laboratory hammer mill Lab mill 3100 (Perkin Elmer, Sweden) and a 0.8 mm (mm nominal diameter) retention screen, and its moisture content was determined using a HR73 halogen moisture analyzer (Mettler Toledo, Switzerland), and the drying temperature was set at 105°C.

[0185] The concentration of condensed tannins in sorghum was analyzed by the vanillin HCL colorimetric assay described in Dykes L. Tannin Analysis in Sorghum Grains. Methods Mol Biol. 2019;1931:109-120. doi:10.1007 / 978-1-4939-9039-9_8. PMID:30652286.

[0186] Glucose in wort was analyzed with a Dionex HPLC system ICS-5000 using a RI-101 differential refractive index detector (Thermo Scientific, USA) based on the Analytica-EBC method 8.7 - Fermentable carbohydrates in wort by HPLC.

[0187] Via DMA TM The density was analyzed with a 4500M density analyzer (Anton Paar, Austria) to determine the concentration of the extract.

[0188] The extract yield (expressed as % of dry matter ground malt added to the mash) was calculated based on the total water in the mash (including the moisture in the sorghum ground malt), the extract available in the wort (expressed in °Plato) and the dry matter input to the mash.

[0189] result:

[0190] Saccharification with glucoamylase JPO-172 (SEQ ID NO: 6) resulted in significantly higher final glucose concentrations than those obtained with glucoamylase AMG NL (SEQ ID NO: 2); see Tables 5 and 6. This was particularly evident when comparing the glucose concentrations obtained in saccharification protocols A and B with glucoamylase alone.

[0191] In saccharification protocol B, glucose concentration increased by 6.1%-7.4%, but when AMGJPO-172 (SEQ ID NO: 6) was used in saccharification, a 24%-27.5% decrease in glucose concentration of AMG NL (SEQ ID NO: 2) was observed; see Tables 8 and 9.

[0192] When using starch saccharification scheme B, the extract yield of two AMGs (SEQ ID NO: 2 and SEQ ID NO: 6) increased overall, which may be due to the effect of higher temperature that is conducive to solubilizing starch, and the liquefaction of the added raw starch degrading amylase JA126PE096 (SEQ ID NO: 11) and thermostable α-amylase (SEQ ID NO: 12).

[0193] Therefore, it is obvious that increasing the thermostability of saccharifying glucoamylase to a temperature that is conducive to the solubilization and liquefaction of high gelatinization temperature starch can enable starch liquefaction (by thermostable α-amylase) and saccharification (by thermostable glucoamylase) to proceed simultaneously, making the entire process simpler and more efficient.

[0194] The combination of a thermostable alpha-amylase and a thermostable glucoamylase variant JPO-172 significantly improved the simultaneous liquefaction and saccharification of starch derived from unmalted sorghum in high temperature infusion saccharification compared to the combination of a thermostable alpha-amylase and a thermostable glucoamylase variant AMG NL.

[0195] In mashing with unmalted sorghum, glucose formation increased by 10.8% to 43.6% with AMG JPO-172 compared to AMG NL, and the delta improvement was more pronounced with higher mashing temperatures. When AMG NL was used, increased mashing temperatures resulted in a 14.0% to 17.5% decrease in glucose formation, while when AMG JPO-172 was used, an increase in glucose formation of 6.1% to 7.4% was observed with increased mashing temperatures.

[0196] Table 6: Comparison of glucose concentration in wort produced by AMG NL or AMG JPO-172 using two sorghum malt compositions and protocol A for mashing

[0197]

[0198] Table 7: Comparison of glucose concentration in wort produced by AMG NL or AMG JPO-172 using two sorghum malt compositions and protocol B for mashing

[0199]

[0200] Table 8: Effect of mashing protocols A and B on glucose concentration in wort produced from low-tannin sorghum using AMG NL or AMG JPO-172

[0201]

[0202] Table 9: Effect of mashing protocols A and B on glucose concentration in wort produced from 75% high tannin and 25% low tannin sorghum using AMG NL or AMG JPO-172

[0203]

[0204] Example 8. Starch saccharification experiment using 45% malt and 55% crushed rice

[0205] Table 10: Moisture content of malt and rice used in the experiment

[0206] Moisture [%] Barley malt 7.6 Crushed Rice 12.2

[0207] Barley malt and ground rice were ground using a Lab mill 3100 (Perkin Elmer, Sweden) and a 0.8 mm (mm nominal diameter) retention screen.

[0208] 100 ppm Ca was achieved by adding CaCl2 to deionized water. 2+ -ions were used to prepare the saccharification water. The enzyme dilution was prepared in the saccharification water with the goal of adding 0.2 g of the enzyme dilution to 17.8 g of saccharification water. 3.3 g of rice and 2.7 g of ground malt were added to an RVA aluminum can and manually mixed with 17.8 g of preheated (55°C) saccharification water (containing calcium ions and enzymes according to Table 11 below) and immediately placed in a rapid viscosity analyzer RVA 4500 (Perkin Elmer, Sweden).

[0209] Table 11: Enzyme dosage used at the beginning of saccharification

[0210]

[0211] The starch saccharification was carried out according to the following starch saccharification scheme:

[0212] Starch saccharification scheme A : Stand at 55°C for 20 minutes; heat from 55°C to 78°C at a heating rate of 1°C per minute; stand at 78°C for 127 minutes; cool to room temperature in 6 minutes. Total time without cooling = 170 minutes.

[0213] Starch saccharification scheme B : Stand at 55°C for 20 minutes; heat from 55°C to 80°C at a heating rate of 1°C per minute; stand at 80°C for 125 minutes; cool to room temperature in 6 minutes. Total time without cooling = 170 minutes.

[0214] Starch saccharification scheme C: Stand at 55°C for 20 minutes; heat from 55°C to 85°C at a heating rate of 1°C per minute; stand at 85°C for 120 minutes; cool to room temperature in 6 minutes. Total time without cooling = 170 minutes.

[0215] Starch saccharification scheme D : Stand at 55°C for 20 minutes; heat from 55°C to 87°C at a heating rate of 1°C per minute; stand at 87°C for 118 minutes; cool to room temperature in 6 minutes. Total time without cooling = 170 minutes.

[0216] After saccharification, all mashes were adjusted to an original weight of 24.0 g at the start of saccharification for evaporation losses. The mashes were then centrifuged at 4000 rpm for 10 min at 8°C using a Heraeus 3S-R benchtop centrifuge (Heraeus, Germany), and the supernatant was immediately frozen and stored until subsequent analysis.

[0217] analyze:

[0218] Finely ground malt and rice malt were obtained by grinding using a laboratory hammer mill Lab mill 3100 (Perkin Elmer, Sweden) and a 0.8 mm (mm nominal diameter) retention screen, and their moisture content was determined using a HR73 halogen moisture analyzer (Mettler Toledo, Switzerland), and the drying temperature was set at 105°C.

[0219] Glucose in wort was analyzed with a Dionex HPLC system ICS-5000 using a RI-101 differential refractive index detector (Thermo Scientific, USA) based on the Analytica-EBC method 8.7 - Fermentable carbohydrates in wort by HPLC.

[0220] result:

[0221] The saccharification experiments with 45% barley malt plus 55% crushed rice confirmed the observations in the previous sorghum saccharification examples. In this experiment, the saccharification temperature for simultaneous liquefaction and saccharification was tested between 78°C and 87°C (the maximum temperature for sorghum saccharification was 85°C), and within this temperature range, it was observed that the combination of the thermostable glucoamylase JPO-172 and the thermostable α-amylase continued to improve with increasing temperature.

[0222] Glucose formation by AMG JPO-172 increased continuously from 189.1 g / L to 206.6 g / L as the final saccharification temperature increased, while the concentration obtained in the wort using AMG NL decreased significantly from 184.7 g / L to 116.4 g / L (see Tables 12 and 13) when the final saccharification temperature increased from 78°C to 85°C, resulting in a delta difference ranging from 2.4% to 76.3%. Increasing the final saccharification temperature to 87°C did not result in a further decrease in glucose concentration, indicating that glucose was formed during the heating phase before the final temperature reached 87°C.

[0223] Table 12: Comparison of glucose concentration in wort produced in four different mashing schedules AD using 45% malt and 55% ground rice, AMG NL or AMG JPO-172.

[0224]

[0225] Table 13: Effect of mashing protocols AD on glucose concentration in wort produced from 45% malt and 55% crushed rice with AMG NL or AMG JPO-172

[0226]

[0227] Example 9. Starch saccharification experiments with 45% malt and 55% crushed rice and other thermostable JPO variants

[0228] Table 14: Moisture content of malt and rice used in the experiment

[0229] Moisture (%) Barley malt 7.6 Crushed Rice 12.2

[0230] Barley malt and ground rice were ground using a Lab mill 3100 (Perkin Elmer, Sweden) and a 0.8 mm (mm nominal diameter) retention screen.

[0231] 100 ppm Ca was achieved by adding CaCl2 to deionized water. 2+ -ions to prepare the starch saccharification water.

[0232] The enzyme dilution was prepared in the mash water, with the goal of adding 0.2 g of enzyme dilution to 17.8 g of mash water. 3.3 g of rice and 2.7 g of ground malt were added to an RVA aluminum can and manually mixed with 17.8 g of preheated (55°C) mash water (containing calcium ions and enzymes) and immediately placed in a rapid viscoanalyzer RVA 4500 (PerkinElmer, Sweden).

[0233] Table 15: Enzyme dosage used at the beginning of saccharification

[0234]

[0235]

[0236] Therefore, starch saccharification was performed according to the following starch saccharification scheme:

[0237] Starch saccharification scheme A: Stand at 55°C for 20 minutes; heat from 55°C to 78°C at a heating rate of 1°C per minute; stand at 78°C for 127 minutes; cool to room temperature in 6 minutes. Total time without cooling = 170 minutes.

[0238] Starch saccharification scheme D: Stand at 55°C for 20 minutes; heat from 55°C to 87°C at a heating rate of 1°C per minute; stand at 87°C for 118 minutes; cool to room temperature in 6 minutes. Total time without cooling = 170 minutes.

[0239] After saccharification, all mashes were adjusted to an original weight of 24.0 g at the start of saccharification for evaporation losses. The mashes were then centrifuged at 4000 rpm for 10 min at 8°C using a Heraeus 3S-R benchtop centrifuge (Heraeus, Germany), and the supernatant was immediately frozen and stored until subsequent analysis.

[0240] analyze:

[0241] Finely ground malt and rice malt were obtained by grinding using a laboratory hammer mill Lab mill 3100 (Perkin Elmer, Sweden) and a 0.8 mm (mm nominal diameter) retention screen, and their moisture content was determined using a HR73 halogen moisture analyzer (Mettler Toledo, Switzerland), and the drying temperature was set at 105°C.

[0242] Glucose in wort was analyzed with a Dionex HPLC system ICS-5000 using a RI-101 differential refractive index detector (Thermo Scientific, USA) based on the Analytica-EBC method 8.7 - Fermentable carbohydrates in wort by HPLC.

[0243] result:

[0244] When the main residue in saccharification was kept at 87°C, glucose formation was increased by 7.4% to 9.3% for different glucoamylase variants (JPO168, JPO169 or JPO-172) with melting points > 87°C compared to 78°C.

[0245] On the other hand, when increasing the main residue from 78°C to 87°C, a decrease in glucose formation of 19.6% to 36.6% was observed for variants with a melting point <= 85°C (JPO048 or JPO081) (see Table 15 below).

[0246] These observations demonstrate that the thermostable glucoamylase variants according to the present invention, which can function at a temperature that achieves sufficient gelatinization and liquefaction of the thermostable α-amylase, i.e., in the range of 87° C., enable an efficient and simple starch saccharification process.

[0247] Table 16: Comparison of glucose concentration in wort produced by amyloglucosidase variants with different thermostabilities using 45% malt and 55% ground rice in two different mashing protocols.

[0248]

[0249] Example 10. Starch saccharification experiment with 100% malt

[0250] Table 17: Moisture content of malt used in the experiment

[0251]

[0252]

[0253] The malt was ground using a laboratory disc mill DLFU (Bühler AG, Switzerland) with a gap distance of 1.3 mm and the obtained ground malt was stored dry in a closed container until further use.

[0254] The saccharification water was prepared by adding CaCl2 to deionized water to achieve a final concentration of 100 ppm Ca2+-ions. The saccharification water was dispensed into a saccharification beaker in an amount of 192 g (corresponding to a water: ground malt ratio of 3:1) and placed in an LP electronic saccharification apparatus (Lochner GmbH, Germany), followed by preheating to a starting saccharification temperature of 55°C.

[0255] The ground malt was added to the saccharification water in an amount of 64 g / beaker. The water and ground malt were homogenized by stirring at 150 rpm for 5 minutes. The pH was adjusted to 5.2 using lactic acid (concentration 20%). The stirring speed was then reduced to 100 ppm, and after this step, enzymes were added according to Table 18 below, and the saccharification process was started. The following enzymes were added:

[0256] - ANTI-BASE® Pro (Novozymes) is a commercial product for so-called high attenuation in brewing, comprising a glucoamylase from Talaromyces emersonii / Rossella emersonii and a pullulanase (PulC) from Bacillus acidopullulyticus.

[0257] - Core (Novozymes) is a commercial product for so-called high attenuation in brewing, comprising a glucoamylase from Talaromyces emersonii / Rossata emersonii (AMG-T).

[0258] - ANTI-GLUCOAMYLASE 87 (International Flavors and Fragrances Inc., IFF) is a commercial product that claims to be an effective blend of glucoamylases that maximizes starch conversion and that, according to the manufacturer, can be added during saccharification at temperatures below 66°C.

[0259] Table 18. Enzyme dosages used at the start of mashing of 100% malt at 52°C.

[0260]

[0261]

[0262] The starch saccharification was carried out according to the following starch saccharification scheme:

[0263] Starch saccharification scheme A : 20 minutes at 52°C; heat from 52°C to 64°C at a heating rate of 1°C per minute; 60 minutes at 64°C; heat from 64°C to 72°C at a heating rate of 1°C per minute; 15 minutes at 72°C; heat from 72°C to 78°C at a heating rate of 1°C per minute; 30 minutes at 78°C. Total time until end of 78°C cooking = 151 minutes. After saccharification, all mashes were adjusted for evaporation losses to the original weight of 256 g at the start of saccharification.

[0264] At the end of the mashing process A, the complete sample was transferred to a 600 ml centrifuge cup and centrifuged at 4600 rpm (4566 g) for 5 minutes in a Heraeus Multifuge 3S-R benchtop centrifuge equipped with a Sorvall 75006445 rotor to separate the sweet wort from the crushed malt. 100 g of sweet wort was poured into 50 g of mashing water (preheated to 78° C.) to start mashing process B.

[0265] Starch saccharification scheme B : Stand at 78°C for 30 minutes; heat from 78°C to 96°C at a heating rate of 0.6°C per minute; cool to room temperature in 10 minutes. Total time without cooling = 60 minutes.

[0266] After mashing, all mashes were adjusted to an original weight of 150 g at the start of mashing protocol B for evaporation losses. The mash was filtered through a filter funnel with 240 mm (Macherey-Nagel, Germany) filter paper. The wort obtained was immediately frozen and stored until subsequent analysis.

[0267] Starch saccharification scheme C : 20 minutes at 52°C; heat from 52°C to 64°C at a heating rate of 1°C per minute; 15 minutes at 64°C; heat from 64°C to 72°C at a heating rate of 1°C per minute; 15 minutes at 72°C; heat from 72°C to 78°C at a heating rate of 1°C per minute; 30 minutes at 78°C. Total time until end of 78°C cooking = 106 minutes. After saccharification, all mashes were adjusted for evaporation losses to the original weight of 256 g at the start of saccharification.

[0268] At the end of the mashing scheme C, the complete sample was transferred to a 600 ml centrifuge cup and centrifuged at 4600 rpm (4566 g) for 5 minutes in a Heraeus Multifuge 3S-R benchtop centrifuge equipped with a Sorvall 75006445 rotor to separate the sweet wort from the crushed malt. 100 g of sweet wort was poured into 50 g of mashing water (preheated to 78° C.) to start mashing method B.

[0269] analyze:

[0270] The ground malt was obtained by grinding with a laboratory disc mill DLFU (Bühler AG, Switzerland) at a gap distance of 1.3 mm, and its moisture content was determined using a HR73 halogen moisture analyzer (Mettler Toledo, Switzerland), and the drying temperature was set at 105°C.

[0271] Glucose in wort was analyzed with a Dionex HPLC system ICS-5000 using a RI-101 differential refractive index detector (Thermo Scientific, USA) based on the Analytica-EBC method 8.7 - Fermentable carbohydrates in wort by HPLC.

[0272] result:

[0273] The final glucose concentrations produced by saccharification of starch with glucoamylase JPO-172 (SEQ ID NO: 6) were significantly higher than those obtained with the following three commercial glucoamylases tested: Pro, Core and 87; see Tables 19 and 20.

[0274] The final glucose concentration of the wort produced with AMG JPO-172 (SEQ ID NO: 6) was increased by 146.4% compared to the composition of the wort produced in the 151-minute mashing protocol (A).

[0275] The thermostable amyloglucosidase JPO-172 (SEQ ID NO: 6) was observed to release higher levels of glucose than the commercial product tested in the saccharification protocol (extended saccharification at 64°C for 60 minutes followed by rest at 72°C for 15 minutes), so a shorter saccharification procedure was adopted.

[0276] A shorter mashing protocol C was designed to shorten the mashing between 64°C and 78°C by 50% (44.5 min vs. 89 min) by reducing the mashing step at 64°C to 15 min. Ultimately the overall process was shortened by 22% (166 min vs. 211 min). Thermostable amyloglucosidase JPO-172 was tested in this mashing protocol (Table 19). The final glucose concentration of wort produced with AMG JPO-172 (SEQ ID NO: 6) using the shorter mashing protocol C was higher than that previously observed with the commercial enzyme tested in longer mashing. The final glucose concentration of wort produced with AMG JPO-172 (SEQ ID NO: 6) using the shorter mashing protocol C was higher than that observed with the commercial enzyme tested in longer mashing. The final glucose concentration of wort produced with AMG JPO-172 (SEQ ID NO: 6) ... of the commercial enzyme tested in longer mashing. The final glucose concentration of wort produced with AMG JPO-172 (SEQ ID NO: 6) was higher than that of the commercial enzyme tested in longer mashing. The final glucose concentration of wort produced with AMG JPO-172 (SEQ ID NO: 6) was higher than that of the commercial enzyme tested in longer mashing. The final glucose concentration of wort produced with AMG J Pro (used as a benchmark), a glucose yield of 123.6% was obtained with the wort produced with JPO-172 in the shorter process (Table 19).

[0277] Table 19: Comparison of glucose concentration in wort produced in different mashing protocols using 100% malt, commercial products containing amyloglucosidase variants with different thermostabilities. *) Determined in quadruplicate - means are given in the table.

[0278]

[0279] After the mashing at the end of schedules A and C, the sweet wort was collected and mixed with foaming water (in a 2:1 ratio) to simulate foaming and transferred to mashing schedule B for another 60 minutes of processing.

[0280] Relative to Attenuzyme (used as a benchmark), the glucose yield obtained with the cold wort produced with JPO-172 in a 211 minute process (saccharification A+B) was 176.7% (Table 20).

[0281] The final glucose concentration of wort produced with AMG JPO-172 (SEQ ID NO: 6) using the shorter saccharification protocol (C+B) was higher than that previously observed with other commercial enzymes in longer saccharifications. (used as a benchmark), the glucose yield obtained with the cold wort produced with AMG JPO-172 during 166 minutes (saccharification C+B) was 160.5% (Table 20).

[0282] These observations demonstrate that the thermostable glucoamylase JPO-172 (SEQ ID NO: 6), which can function at temperatures above 78°C, enables an efficient and simple starch saccharification process and can shorten saccharification to achieve higher fermentable sugars and higher glucose composition of wort.

[0283] Table 20: Comparison of glucose concentration in wort produced in different mashing protocols using 100% malt, commercial products containing amyloglucosidase variants with different thermostabilities. *) Determined in quadruplicate - means are given in the table.

[0284]

Claims

1. A method for producing beer wort, the method comprising adding to the mash before, during or after starch liquefaction a mature thermostable variant of a parent glucoamylase having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO:

10.

2. The method according to claim 1, wherein the parent glucoamylase is from a species of the genus Penicillium, preferably from Penicillium oxalicum, Penicillium miekii, Penicillium roqueforti or Penicillium luminosporum.

3. The method of any one of claims 1-2, wherein the mature variant comprises at least one amino acid modification at one or more or all of the positions corresponding to positions 1, 2, 4, 6, 7, 11, 31, 34, 50, 65, 79, 103, 132, 327, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595 in SEQ ID NO:

1.

4. The method according to claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 2, 4, 11, 65, 79 and 327 in SEQ ID NO: 1, preferably, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, P2N, P4S, P11F, T65A, K79V and Q327F in SEQ ID NO:

1.

5. The method of claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 79, 103, 132, 445, 447, 481, 566, 568, 594 and 595 in SEQ ID NO: 1, preferably, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, K79V, S103N, A132P, D445N, V447S, S481P, D566T, T568V, Q594R and F595S in SEQ ID NO:

1.

6. The method of claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 50, 103, 132, 445, 447, 481, 501, 539, 566, 568, 594 and 595 in SEQ ID NO: 1, preferably, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, S103N, A132P, D445N, V447S, S481P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO:

1.

7. The method of claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 50, 103, 132, 445, 447, 481, 501, 539, 566, 568, 594 and 595 in SEQ ID NO: 1, preferably, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, S103N, A132P, D445N, V447S, S481P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO:

1.

8. The method according to any one of claims 1 to 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595 in SEQ ID NO: 1, preferably, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO:

1.

9. The method according to any one of claims 1 to 8, wherein the mature thermostable variant has an improvement in thermostability (Td) of at least 5°C, preferably at least 6°C, 7°C or 8°C relative to its parent.

10. The method according to any one of claims 1 to 9, wherein the mature thermostable variant has a relative activity of at least 150, preferably at least 200, more preferably at least 250, most preferably at least 300 at 91°C compared to its parent.

11. The method according to any one of claims 1 to 10, wherein saccharification comprises an incubation step at 65°C or higher for at least 20 minutes; preferably at 67°C or higher for at least 20 minutes; even more preferably at 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C or most preferably at 87°C or higher for at least 20 minutes.

12. The method of any one of claims 1-11, wherein the pH of the mash is about 4.6 to about 6.

4.

13. The method according to any one of claims 1 to 12, wherein the target glucose concentration is achieved in a shorter saccharification time compared to adding an AMG enzyme having a lower thermal stability than that of claim 1.

14. The method according to any one of claims 1 to 13, comprising further adding to the mash one or more additional enzymes selected from the group consisting of alpha amylase, maltogenic amylase, raw starch degrading alpha amylase, beta amylase, aminopeptidase, carboxypeptidase, catalase, cellobiose oxidase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, dextran 1,4-alpha-maltotetraose hydrolyzate, hydrolase, glucanase, beta-glucanase, galactanase, alpha-galactosidase, beta-galactosidase, glucose oxidase, a-glucosidase, beta-glucosidase, haloperoxidase, hemicellulolytic enzyme, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinase, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, protease, pullulanase, ribonuclease, transglutaminase and xylanase.

15. The method according to any one of claims 1-14, wherein the wort has higher than 80% glucose compared to the total carbohydrate content of the wort.

16. A mashing or brewing composition comprising a mature thermostable variant of a parent glucoamylase as defined in any one of claims 1 to 10.

17. The starch saccharification or brewing composition of claim 16, further comprising one or more additional enzymes selected from the group consisting of: alpha amylase, maltogenic amylase, raw starch degrading alpha amylase, beta amylase, aminopeptidase, carboxypeptidase, catalase, cellobiose oxidase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1,4-alpha-maltotetraose hydrolyzing enzyme. Enzymes, glucanases, beta-glucanases, galactanases, alpha-galactosidases, beta-galactosidases, glucose oxidases, a-glucosidases, beta-glucosidases, haloperoxidases, hemicellulolytic enzymes, invertases, laccases, lipases, mannanases, mannosidases, oxidases, pectinolytic enzymes, peptidylglutaminases, peroxidases, phospholipases, phytases, polyphenol oxidases, proteases, pullulanases, ribonucleases, transglutaminases, and xylanases.

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