Brewing with thermostable AMG manifolds

Abstract

The present invention relates to a method for producing a brewery's wort comprising adding a mature thermostable variant of a parent glucoamylase that is at least 70% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:10 to a mash before, during or after starch liquefaction, and to a mashing or brewing composition comprising said mature thermostable variant of a parent glucoamylase.

Description

[Technical field]

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

[0002] The present invention relates to a method for producing a brewery wort, said method comprising adding to a mash a mature thermostable variant of a parent glucoamylase that is at least 70% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10, and a mashing or brewing composition comprising said variant. [Background technology]

[0003] Grain starch is composed of glucose polymers in which glucose residues are linked by either alpha-1,4 or alpha-1,6 bonds. The extraction of the grain components in brewing is called mashing. During this stage, water is adsorbed by the starch granules and their intermolecular bonds are progressively broken until the starch is irreversibly dissolved in water. The temperature required to start this process (called swelling) varies with the type of starch. Some cereal grain starches, such as barley or wheat starch, start swelling at 58-65°C, while others, such as corn, rice and sorghum starches, start swelling at 60-85°C. For brewing with grains that have a high pasting temperature, a parallel process, the so-called liquefaction or cooking of the grain, is typically applied, in which the solubilization and liquefaction of the starch (typically achieved with thermostable alpha-amylases) is ensured by subjecting the starch to temperatures of 85-100°C for a defined time. After this step, the liquefied starch is brought to a temperature (by cooling or mixing with cold malt mash) where the saccharification enzymes can hydrolyze the non-fermentable dextrins of the invention into fermentable sugars. The main fermentable sugars are glucose, maltose, maltotriose, but also traces of sucrose and fructose.

[0004] Wort produced without exogenous enzyme supplementation typically consists of 65-80% fermentable sugars and dextrins ranging from 20-35%. The degree of fermentable sugars depends on the amylase amylase activity of the endogenous malt and the mashing regime applied. Due to the natural limit of the endogenous amylase activity of malt (i.e. β-amylase), there will always be residual non-fermentable dextrins that cannot be converted to ethanol by the brewer's yeast.

[0005] Breweries aiming to increase the ethanol yield per unit raw material without significantly changing the sensory perception of the final beer can overcome this limitation by using commercially available pullulanases, amyloglucosidases, alpha-amylases, beta-amylases, maltogenic amylases, and combinations thereof in the saccharification stage of liquefied starch. Furthermore, low-calorie beers and low-residual carbohydrate beers are very popular in the US beer market and have recently attracted worldwide attention. Such beers are obtained by the same principle of using commercially available saccharification enzymes, but with the goal of maximum possible hydrolysis of dextrins.

[0006] Another application field of commercial saccharification enzymes is the production of wort using 100% unmalted raw materials such as barley, wheat, sorghum, corn, rice, cassava, etc. Such raw materials lack or contain insufficient endogenous amylolytic enzymes for brewing purposes, making the use of commercial amylolytic enzymes essential. For mashing with malted barley, the mashing regime design needs to ensure that the grain starch is fully gelatinized and liquefied and that the subsequent saccharification has progressed to such an extent that the fermentable sugars in the wort can provide the targeted substantial degree of fermentation by yeast.

[0007] For raw materials that exhibit high gelatinization temperatures (e.g., rice, corn, sorghum), the mashing regime typically involves a liquefaction stage at temperatures between 85 and 100 °C, followed by saccharification at lower temperatures of 62-70 °C. Such processes typically take more time and potentially require additional equipment.

[0008] Improved and more thermostable amylolytic enzymes are needed to simplify and shorten the mashing process without affecting process efficiency.

[0009] US Pat. No. 3,379,534 describes the preparation of low dextrin beer by using amyloglucosidase.

[0010] US Pat. No. 4,536,477 describes a thermostable glucoamylase that is particularly useful for preparing glucose-containing syrups from starch.

[0011] WO 2009 / 075682 describes the use of certain pullulanases to produce brewery wort in which mashing is accomplished using less enzyme protein.

[0012] Matthews et al., 2001, Journal of Institute of Brewing 107(3) pp185-194, discloses the preparation of low carbohydrate beer by mashing at high temperature using glucoamylase derived from Aspergillus niger.

[0013] WO 2012 / 140075 (Novozymes A / S, Denmark) discloses a method for refining brewery wort by adding a specific glucoamylase. Summary of the Invention [Means for solving the problem]

[0014] The inventors have found that certain thermostable variants of glucoamylases show significantly improved performance in mashing, i.e., brewing. Another improved performance of the thermostable variants is that they increase the sweetness or sweet taste of the product, which allows a reduction in the amount of added sugar in traditional recipes.

[0015] Thus, in a first aspect, the present invention relates to a method for producing a brewery's wort comprising adding a mature thermostable variant of a parent glucoamylase that is at least 70% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10 to a mash before, during or after starch liquefaction.

[0016] A second aspect of the present invention relates to a mashing or brewing composition comprising a mature thermostable variant of a parent glucoamylase as defined in the first aspect, preferably also comprising an alpha-amylase, a maltogenic amylase, a raw starch degrading alpha-amylase, a beta amylase, an aminopeptidase, a carboxypeptidase, a catalase, a cellobiose oxidase, a cellulase, a chitinase, a cutinase, a cyclodextrin glycosyltransferase, a deoxyribonuclease, an esterase, a glucan 1,4-alpha-maltotetrahydrolase, a glucanase, a beta glucanase, a galactose-binding enzyme, a glyceryl esterase ... and one or more additional enzymes selected from the group consisting of tannase, alpha-galactosidase, beta-galactosidase, cellobiose oxidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hemicellulolytic enzymes, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinolytic enzymes, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, protease, pullulanase, ribonuclease, transglutaminase, and xylanase. [Brief description of the drawings]

[0017] [Figure 1-1] 1 shows a multiple alignment of the amino acid sequences of the following mature proteins: - wild type AMG from Penicillium oxalicum (PoAMG) according to SEQ ID NO: 1 - the PoAMG variant designated "AMG NL" according to SEQ ID NO: 2 - the PoAMG variant designated "AMG anPAV498" according to SEQ ID NO: 3 - the PoAMG variant designated "AMG JPO001" according to SEQ ID NO: 4 - the PoAMG variant designated "AMG JPO124" according to SEQ ID NO: 5 - the PoAMG variant designated "AMG JPO-172" according to SEQ ID NO: 6 - the PoAMG variant designated "AMG JPO-172" according to SEQ ID NO: 7 - the PoAMG variant designated "AMG JPO-172" according to SEQ ID NO: 8 - the PoAMG variant designated "AMG JPO-172" according to SEQ ID NO: 9 - the PoAMG variant designated "AMG JPO-172" according to SEQ ID NO: 10 - the PoAMG variant designated "AMG JPO-172" according to SEQ ID NO: 11 Wild-type AMG (PoAMG) from P. glabrum [Figure 1-2] 1 shows a multiple alignment of the amino acid sequences of the following mature proteins: - wild type AMG from Penicillium oxalicum (PoAMG) according to SEQ ID NO: 1 - the PoAMG variant designated "AMG NL" according to SEQ ID NO: 2 - the PoAMG variant designated "AMG anPAV498" according to SEQ ID NO: 3 - the PoAMG variant designated "AMG JPO001" according to SEQ ID NO: 4 - the PoAMG variant designated "AMG JPO124" according to SEQ ID NO: 5 - the PoAMG variant designated "AMG JPO-172" according to SEQ ID NO: 6 - the PoAMG variant designated "AMG JPO-172" according to SEQ ID NO: 7 - the PoAMG variant designated "AMG JPO-172" according to SEQ ID NO: 8 - the PoAMG variant designated "AMG JPO-172" according to SEQ ID NO: 9 - the PoAMG variant designated "AMG JPO-172" according to SEQ ID NO: 10 - the PoAMG variant designated "AMG JPO-172" according to SEQ ID NO: 11 Wild-type AMG (PoAMG) from P. glabrum DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] definition Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0019] For the purposes of the present invention, sequence identity between two amino acid sequences is preferably determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48; 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), 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 value of Needle, labeled "longest identity" (obtained using the -no brief option), is used as the percent identity, calculated as follows: (identical residues × 100) / (length of alignment – ​​total number of gaps in alignment)

[0020] Variant: The term "variant" refers to a polypeptide that includes modifications, i.e., substitutions, insertions, and / or deletions at one or more (e.g., several) positions. A substitution means that an amino acid at a position is replaced with another amino acid, a deletion means that an amino acid at a position is removed, and an insertion means that one or more amino acids are added immediately adjacent to an amino acid at a position. The amino acid changes can be minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect folding and / or activity of the protein; small deletions, typically 1-30 amino acids; small amino- or carboxyl-terminal extensions such as an amino-terminal methionine residue; small linker peptides of 20-25 residues or less; or small extensions that facilitate purification by altering net charge or another function, such as a polyhistidine tract, an antigenic epitope, or a binding domain. Examples of conservative substitutions are in 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 low molecular weight amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not change the specific activity are known in the art and are described, for example, in 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.

[0021] Improved Thermostability: Improved thermostability (Td) in ° C. is a measure of how much variants have improved thermostability over their parent glucoamylase under the same conditions, determined as exemplified herein. The term "grist" as used herein is understood as the starch or sugar-containing material on which beer is made, e.g., barley malt and additives. Generally, grist does not contain any added water.

[0022] The term "malt" is to be understood as any malted cereal grain, in particular barley.

[0023] The term "adjunct" is understood to be a portion of grist that is not barley malt. Adjuncts can be any starch-rich plant material, such as, but not limited to, unmalted grains such as barley, corn, rice, sorghum, wheat, etc., including easily fermentable sugars and / or syrups. Some adjunct starches have a relatively low gelatinization temperature that allows them to be mashed in together with the malt, while other adjuncts, such as rice, corn, sorghum, etc., have a higher gelatinization temperature, and such adjuncts are typically cooked and liquefied separately with alpha-amylase before being added to the mash.

[0024] The term "mash" is understood as the steeping of a starch-containing slurry containing crushed malted barley, crushed unmalted grain, other starch-containing materials, or combinations thereof, in water to produce wort.

[0025] The term "wort" is understood as the unfermented liquor released after extraction of the grist during mashing.

[0026] The term "spent grain" is to be understood as the discharged solids remaining when the grist has been extracted and the wort separated.

[0027] The term "beer" is understood here as an alcoholic beverage brewed from fermented wort, i.e. barley malt, optionally with additives and hops. The term "beer" as used here is intended to include at least beers prepared from mashes prepared from unmalted grains as well as all mashes prepared from malted grains and all mashes prepared from a mixture of malted and unmalted grains. The term "beer" also includes beers prepared with additives and beers with the highest possible alcohol content.

[0028] 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) allows the determination of the onset and peak temperatures of starch gelatinization (T o and T p The gelatinization onset temperature (T) is one technique that can be used to study the gradual process of starch gelatinization. o The term "gelatinization peak temperature (T )" is understood as the temperature at which gelatinization begins. p The term "gelatinization end temperature (T c ) is understood as the temperature at which gelatinization is complete.

[0029] wort production A first aspect of the invention relates to a method for producing a brewery's wort comprising adding a mature thermostable variant of a parent glucoamylase that is at least 70% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:10 to a mash before, during or after starch liquefaction.

[0030] Preferably, the method of the first aspect achieves an affinity for SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10 prior to, during or after liquefaction of the starch, such as at least 71%, such as at least 72%, for example at least 73%, such as at least 74%, for example at least 75%, such as at least 76%, for example at least 77%, such as at least 78%, for example at least 79%, such as at least 80%, for example at least 81%, such as at least 82%, for example at least 83%, such as at least The present invention relates to a method for the preparation of a glucoamylase comprising the steps of: adding to the mash a mature thermostable variant of a parent glucoamylase which is at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99% identical to the parent glucoamylase;

[0031] The mashing process generally applies a controlled stepwise increase in temperature, with each step prioritizing the action of one enzyme over the other, ultimately degrading proteins, cell walls, and starch. Mashing temperature profiles are generally known in the art. In the present invention, the saccharification (amylolysis) stage in the mashing process is preferably carried out at 60°C-66°C, more preferably 61°C-65°C, even more preferably 62°C-64°C, and most preferably 63°C-64°C. In a particular embodiment of the present invention, the saccharification temperature is 64°C.

[0032] In one embodiment, the mashing process of the present invention includes, but is not limited to, a mashing-off step. In one embodiment, the mashing-off 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 embodiment, the mashing-off step includes, but is not limited to, 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 Incubation for 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 65 minutes, at least 70 minutes, at least 75 minutes, at least 80 minutes, at least 85 minutes, at least 90 minutes, at least 95 minutes, at least 100 minutes, at least 105 minutes, at least 110 minutes, at least 115 minutes, at least 120 minutes, at least 125 minutes, at least 130 minutes, at least 135 minutes, at least 140 minutes, at least 145 minutes, for example, at least 150 minutes. In a particular embodiment, the mashing off is performed at 75° C. for 120 minutes.

[0033] In one embodiment, the invention relates to a method of producing a brewery wort comprising adding to a mash a mature thermostable variant of a parent glucoamylase that is active during mashing conditions at a temperature of at least 65° C. In another aspect, 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.

[0034] Preferably, the mature thermostable variant of the parent glucoamylase exhibits activity during lautering, mashing and / or mash filtration.

[0035] According to a first aspect, the mash is obtained by grinding grist containing malt and / or additives. Preferably, the mash can be preheated by adding water to the grist in order to reach the desired mash temperature at the moment of mash formation. If the temperature of the formed mash is below the desired mashing temperature, preferably additional heat is provided in order to reach the desired process temperature. Preferably, the desired mashing temperature is reached within 15 minutes after mash formation, 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 at the time of mash formation. The temperature profile of the mashing process can be from a conventional mashing process, where the temperature is set to achieve optimal degradation of the grist dry matter by the malt enzymes.

[0036] The mashing process generally applies a controlled stepwise increase in temperature, with each step prioritizing the action of one enzyme over the other, ultimately degrading proteins, cell walls, and starch. Mashing temperature profiles are generally known in the art. In the present invention, the saccharification (amylolysis) stage in the mashing process is preferably carried out at 60°C-66°C, more preferably 61°C-65°C, even more preferably 62°C-64°C, and most preferably 63°C-64°C. In a particular embodiment of the present invention, the saccharification temperature is 64°C.

[0037] In a preferred embodiment of the first aspect, the mashing comprises an incubation step at a temperature of 65°C or higher for at least 20 minutes, preferably at a temperature of 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 a temperature of 87°C or higher for at least 20 minutes.

[0038] In one embodiment, the pH of the mash is in the range of about 4.6 to about 6.4. In another embodiment, the pH is in the range of about 4.6 to 6.2, for example, in the range of about pH 4.8 to 6.0, preferably in the range of about pH 5.0 to about 6.0, more preferably in the range of about pH 5.0 to about 5.6, and even more preferably in the range of about pH 5.0 to about 5.4.

[0039] In another preferred embodiment of the first aspect, the pH of the mash is from about 4.6 to about 6.4.

[0040] The malt is preferably derived from one or more grains selected from the list consisting of corn, barley, wheat, rye, sorghum, millet and rice. Preferably, the malt is barley malt. The grist preferably comprises 0.5% to 99%, preferably 1% to 95%, more preferably 5% to 90%, even more preferably 10% to 80% malt.

[0041] Preferably the wort of the first aspect has more than 80% glucose compared to the total carbohydrate content of the wort.

[0042] In addition to the malted grains, the grist may preferably contain additives such as raw starch and / or refined starch and / or sugar-containing materials derived from unmalted corn, such as barley, wheat, rye, oats, corn, rice, milo, millet and / or sorghum, or plants such as unmalted grains, wheat, rye, oats, corn, rice, milo, millet, sorghum, potato, sweet potato, cassava, tapioca, sago, banana, sugar beet and / or sugar cane. In the present invention, the additives may be obtained from tubers, roots, stems, leaves, legumes, cereals and / or whole grains. Preferred are additives obtained from corn and / or rice, more preferably the additives are rice starch, corn starch and / or corn grits. The mash preferably contains between 1% and 60%, preferably between 5% and 45%, more preferably between 10% and 40% additive starch. The additives may also easily include fermentable carbohydrates such as sugars or syrups and may be added to the malt mash before, during or after the mashing process of the present invention, but are preferably added after the mashing process. Prior to forming the mash, the malt and / or additives are preferably milled, most preferably dry or wet milled. In one aspect, the additives have a high gelatinization temperature, more particularly the higher gelatinization onset temperature of, for example, corn, rice, and sorghum. In one aspect, the additives are gelatinized before mashing. In another aspect, the additives are not gelatinized before mashing.

[0043] In one embodiment, the mash is comprised of at least 20% of the adjunct having a starch gelatinization temperature, preferably a gelatinization onset temperature of at least 65° C. In another aspect, the mash is comprised 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 65% of the adjunct having a starch gelatinization temperature, preferably a gelatinization onset temperature of at least 65° C.

[0044] In one embodiment, the mash comprises at least 10% unmalted grain compared to the total grist, hi another aspect, the mash comprises at least 15%, e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, e.g., at least 60% unmalted grain.

[0045] Preferably, the additive comprises corn. In another embodiment of the invention, the mash comprises at least 20% corn additive. In one embodiment, the mash comprises at least 25%, e.g., at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, e.g., at least 60% corn additive, preferably the corn additive is not gelatinized when added to the mash.

[0046] In another preferred embodiment, the additive comprises rice. In another aspect of the invention, the mash comprises at least 20% rice additive. 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 additive, preferably the rice additive is not gelatinized when added to the mash.

[0047] In a preferred embodiment, practicing the method of the invention leads to additional glucose formation 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%, e.g., 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%, when compared to wort produced in the absence of such glucoamylase.

[0048] Preferably, practicing the method of the invention leads to a reduction in the concentration of maltose in the wort, hi one embodiment, 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%, when compared to wort produced in the absence of such glucoamylase.

[0049] In a preferred embodiment, the thermostable variant glucoamylase is exogenously provided and / or present in the mash. In one aspect, the glucoamylase is introduced at the beginning of mashing. In another aspect, the glucoamylase is introduced during mashing. In another aspect, the glucoamylase is introduced under lautering.

[0050] As will be evident from the examples below, when the thermostable glucoamylase variants of the first aspect of the invention are utilized, the saccharification or mashing stage in brewing can be shortened, which is of clear commercial interest since it reduces both capacity and energy costs, which in turn allows the brewer to reach a predefined desired target glucose concentration faster.

[0051] Therefore, a preferred embodiment of the present invention relates to a method according to the first aspect, which reaches the target glucose concentration in a shorter saccharification time compared to when a less thermostable AMG enzyme than that of claim 1 is added.

[0052] Preferably, the method of the present invention leads to shorter mashing times, more preferably, the method leads to a reduction in mashing times of at least 5 minutes, such as at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, more preferably at least 30 minutes, compared to a method performed without the addition of a thermostable glucoamylase variant of the present invention.

[0053] Glucoamylase Glucoamylases are also called amyloglucosidases and glucan 1,4-alpha-glucosidases (EC 3.2.1.3), more commonly they are referred to as AMG.

[0054] According to the present invention, different types of amyloglucosidases may be used as parents for the production of thermostable amyloglucosidase variants, for example, amyloglucosidases derived from DNA sequences found in fungal strains of the genus Aspergillus, Rhizopusor, Talaromyces (Rasamsonia) or Penicillium, preferably DNA sequences found in fungal strains of the genus Penicillium, even more preferably Penicillium oxysporum, Penicillium oxalicum, Penicillium miczynskii, Penicillium russellii, Penicillium oxysporum, Penicillium oxalicum, Penicillium miczynskii, Penicillium russellii, Penicillium oxysporum, Penicillium oxalicum, Penicillium miczynskii, Penicillium russellii, Penicillium oxysporum, Penicillium oxalicum, Penicillium miczynskii, Penicillium oxysporum, Penicillium oxalicum ... The parent glucoamylase may be a polypeptide encoded by a DNA sequence found in a fungal strain of Penicillium russellii, or Penicillium glabrum. Preferably, the parent glucoamylase is derived from a species of Penicillium, preferably Penicillium oxicalum, Penicillium miczynskii, Penicillium russellii, or Penicillium glabrum.

[0055] Other examples of suitable fungi include Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Rhizopus delemar, Rhizopus niveus, Rhizopus oryzae, and Talaromyces emersonii (Rasamsonia emersonii).

[0056] Below is shown the percent identity between the AMG amino acid sequences aligned in Figure 1 and also provided in the sequence listing.

[0057] [Table 1]

[0058] Thermostable variants of PoAMG have been produced (see Table 2 below). In a preferred embodiment, the mature thermostable glucoamylase variants of the invention comprise one or more or all of the combinations of amino acid substitutions listed in Table 2 below.

[0059] In a preferred embodiment, the mature variant of the invention 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, preferably 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 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, 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, 79, 103, 132, 445, 447, 481, 566, 568, 594 and 595 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 R1A, G6S, G7T, R31F, K34Y, K79V, S103N, A Preferably, the at least one amino acid modification comprises a substitution at one or more of the positions corresponding to 132P, D445N, V447S, S481P, D566T, T568V, Q594R and F595S, or preferably, the at least one amino acid modification comprises a substitution at one or more of the positions corresponding to 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, or preferably, the at least one amino acid modification comprises a substitution at one or more of the positions corresponding to R1A, G6S, G7T, R3S, G4S, G5S, G6S, G7T, G8S, G9S, G10S, G11S, G12S, G13S, G14S, G15S, G16S, G17S, G18S, G19S, G20S, G21S, G22S, G23S, G24S, G25S, G26S, G27S, G28S, G30S, G31S, G32S, G33S, G34S, G35S, G36S, G37S, G38S, G39S, G40S, G41S, G42S, G43S, G44S, G45S, G56S, G57S, G58S, G65S, G665, G675, G685, G795, G875, G885, G995, G995, G100, G110, G120, G132, G140, G150, G 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 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519 , 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 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.

[0060] The thermostability improvement (Td) of the variants in Table 2 are listed in Table 3, where the Td of the PoAMG variant designated "anPAV498" (parent) was set to zero. In a preferred embodiment, a mature thermostable variant of the invention has a thermostability improvement (Td) over its parent of at least 5°C, preferably at least 6°C, 7°C or 8°C, preferably determined as exemplified herein.

[0061] In another preferred embodiment, a mature thermostable variant of the invention has a relative activity at 91° C. of at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300, compared to its parent.

[0062] Additional enzymes In a preferred embodiment of the first aspect, one or more additional enzymes are added to the mash, said additional enzymes being 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-maltotetrahydrolase, glucanase, beta glucanase, galactanase, alpha amylase, beta ... The enzyme may be selected from the group consisting of a-galactosidase, beta-galactosidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hemicellulolytic enzymes, invertase, laccase, lipase, mannase, mannosidase, oxidase, pectolytic enzymes, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, protease, pullulanase, raw starch degrading alpha-amylase, ribonuclease, transglutaminase, and xylanase.

[0063] Raw starch-digesting alpha-amylase As used herein, "raw starch degrading alpha-amylase" refers to an enzyme that can directly degrade raw granular starch below the gelatinization temperature of starch.

[0064] Examples of raw starch degrading alpha-amylases include those disclosed in WO 2005 / 003311, US 2005 / 0054071, and U.S. Patent No. 7,326,548. Examples also include the enzymes disclosed in Tables 1-5 of the Examples in U.S. Patent No. 7,326,548, U.S. Patent No. 2005 / 0054071 (Table 3 on page 15), and the enzymes disclosed in WO 2004 / 020499, WO 2006 / 06929, and WO 2006 / 066579.

[0065] In one embodiment, the raw starch degrading alpha-amylase is a GH13_1 amylase.

[0066] In one embodiment, the raw starch degrading alpha-amylase enzyme is selected from the group consisting of those described in EP 2981170 (Novozymes A / S) or to the raw starch degrading alpha-amylase as set forth in SEQ ID NO: 11 herein.

[0067] amylase Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, EC.3.2.1.1) comprise a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glucoside oligosaccharides and polysaccharides.

[0068] A number of alpha-amylases are referred to as Termamyl™, Termamyl™ SC and "Termamyl™-like alpha-amylases" and are known, for example from WO 90 / 11352, WO 95 / 10603, WO 95 / 26397, WO 96 / 23873 and WO 96 / 23874.

[0069] Another group of alpha-amylases are referred to as Fungamyl™ and “Fungamyl™-like alpha-amylases,” which are alpha-amylases related to the alpha-amylase derived from Aspergillus oryzae disclosed in WO 01 / 34784.

[0070] Proteases Suitable proteases include microbial proteases, such as fungal and bacterial proteases. Preferred proteases are acidic proteases, i.e. proteases characterized by their ability to hydrolyze proteins under acidic conditions below pH 7. Proteases are responsible for reducing the full length of high molecular weight proteins in the mash to low molecular weight proteins. Low molecular weight proteins are necessary for yeast nutrition, while high molecular weight proteins ensure foam stability. It is therefore well known to those skilled in the art that proteases should be added in a balanced amount that simultaneously provides yeast with abundant free amino acids and leaves enough high molecular weight proteins to stabilize the foam. In one aspect, the protease activity is provided by a proteolytic enzyme system with 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%, most preferably at least 99% or even 100% identity to the amino acid sequence shown in SEQ ID NO: 6 described in WO 9967370. In another embodiment, the protease is Neutrase® available from Novozymes A / S. The protease may be added in an amount of 0.0001-1000 AU / kg DS, preferably 1-100 AU / kg DS, most preferably 5-25 AU / kg dry weight of grain. The proteolytic activity may be determined by using modified hemoglobin as a substrate. In the Anson hemoglobin method for the determination of 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 that gives 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 such that under standard conditions (i.e., 25°C, pH 7.5, reaction time 10 min) the amount of TCA-soluble products per minute that give the same color with phenol reagent as one milliequivalent of tyrosine is liberated.

[0071] Enzyme Composition The mature thermostable variant glucoamylase of the invention as well as any additional enzymes may be added in any suitable form, such as, for example, in the form of a liquid, particularly a stabilized liquid, or it may be added as a substantially dry powder or granules.

[0072] Granules can be produced, for example, as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452. Liquid enzyme preparations can be stabilized, for example, by adding sugars or sugar alcohols, or lactic acid, according to established methods. Other enzyme stabilizers are well known in the art.

[0073] The enzymes may be added in any suitable manner, such as as individual components (separate or sequential addition of enzymes) or by adding the enzymes together in one step or composition.

[0074] Granules and agglomerated powders can be prepared by conventional methods, for example by spraying the enzyme onto a carrier in a fluid bed granulator. The carrier can consist of a particle core with a suitable particle size. The carrier can 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 grits, or soy.

[0075] One aspect of the present invention relates to a mashing or brewing composition comprising a mature thermostable variant of a parent glucoamylase as defined in the first aspect of the invention and preferred embodiments thereof. Preferably, the mashing or brewing composition also comprises an alpha-amylase, a maltogenic amylase, a raw starch degrading alpha-amylase, a beta amylase, an aminopeptidase, a carboxypeptidase, a catalase, a cellobiose oxidase, a cellulase, a chitinase, a cutinase, a cyclodextrin glycosyltransferase, a deoxyribonuclease, an esterase, a glucan 1,4-alpha-maltotetrahydrolase, a glucanase, a beta glucanase, a galactanase, an alpha-galactosidase, a galactanase, a galactosidase ... , beta-galactosidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hemicellulolytic enzymes, invertase, laccase, lipase, mannase, mannosidase, oxidase, pectolytic enzymes, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, protease, pullulanase, ribonuclease, transglutaminase, and xylanase. EXAMPLES

[0076] Example 1: Construction of a PoAMG library The PoAMG library was constructed as follows: forward or reverse primers with NNK or desired mutations at the target site with 15 bp overlap were designed. Inverse PCR, which means the amplification of the entire plasmid DNA sequence by the reverse primer, was carried out using an appropriate template plasmid DNA (e.g., plasmid DNA containing the JPO-0001 gene) under the following conditions. The resulting PCR fragments were purified by QIAquick Gel extraction kit [QIAGEN] and then introduced into Escherichia coli ECOS competent E. coli DH5α [NIPPON GENE CO., LTD.]. Plasmid DNA was extracted from E. coli transformants by MagExtractor plasmid extraction kit [TOYOBO] and then introduced into A. niger competent cells.

[0077] PCR reaction mix: PrimeSTAR Max DNA polymerase [TaKaRa] Total volume: 25 μl 1.0 μl template DNA (1 ng / μl) 9.5 μl HO 12.5μl 2x PrimeSTAR Max Premix 1.0 μl forward primer (5 μM) 1.0 μl reverse primer (5 μM)

[0078] PCR program: 98℃ / 2 minutes 25×(98℃ / 10 seconds, 60℃ / 15 seconds, 72℃ / 2 minutes) 10℃ / hold

[0079] Example 2: Screening for better thermostability The B. subtilis library constructed as in Example 1 was fermented for 3 days at 32° C. in either 96-well or 24-well MTPs containing COVE liquid medium (2.0 g / L sucrose, 2.0 g / L iso-maltose, 2.0 g / L maltose, 4.9 mg / L, 0.2 ml / L 5N NaOH, 10 ml / L COVE salts, 10 ml / L 1M acetamide). Then, AMG activity in the culture supernatant was measured at several temperatures by pNPG assay described as follows.

[0080] pNPG Thermostability Assay: The culture supernatant containing the desired enzyme was mixed with the same volume of pH 5.0 200 mM NaOAc buffer. 20 microliters of this mixture was dispensed into either a 96-well plate or an 8-strip PCR tube, and then heated by a thermal cycler at various temperatures for 30 minutes. These samples were mixed with 10 μl of substrate solution containing 0.1% (w / v) pNPG[wako] in pH 5.0 200 mM NaOAc buffer, and incubated at 70°C for 20 minutes for enzyme reaction. After the reaction, 60 μl of 0.1 M Borax buffer was added to stop the reaction. 80 microliters of the reaction supernatant was taken and its OD 405 The values ​​were read photometrically to assess the enzyme activity.

[0081] [Table 2]

[0082] [Table 3]

[0083] [Table 4]

[0084] [Table 5]

[0085] [Table 6]

[0086] [Table 7]

[0087] [Table 8]

[0088] [Table 9]

[0089] [Table 10]

[0090] [Table 11]

[0091] [Table 12]

[0092] Example 3: Fermentation of Aspergillus niger Aspergillus niger strains were fermented in 500 ml baffled flasks containing 100 ml MU1 with 4 ml 50% urea on a rotary 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 sediment.

[0093] Example 4: Purification of PoAMG (JPO-001) variants The PoAMG variants were purified by cation exchange chromatography. Each peak fraction was individually pooled and dialyzed against 20 mM sodium acetate buffer pH 5.0, and then the sample was concentrated using a centrifugal filter unit (Vivaspin Turbo 15, Sartorius). The enzyme concentration was determined by A280 value.

[0094] Example 5: Thermal Stability Determination (TSA) The purified enzyme was diluted to 0.5 mg / ml in 50 mM sodium acetate buffer, pH 5.0, and mixed with an equal volume of SYPRO Orange (Invitrogen) diluted in Milli-Q water. 18 ul of the mixture was transferred to a LightCycler 480 Multiwell Plate 384 (Roche Diagnostics) and the plate was sealed.

[0095] TSA instrument parameters: Equipment: LightCycler 480 real-time PCR system (Roche Applied Science) Scanning speed: 0.02°C / sec Scanning range: 37~96℃ Integration time: 1.0 seconds Excitation wavelength: 465nm Emission wavelength: 580nm

[0096] The resulting fluorescent signals were normalized to a 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, with the Td of PoAMG variants listed as 0 for anPAV498.

[0097] Example 6: PoAMG activity assay Maltodextrin (DE11) assay by GOD-POD method substrate solution 30g maltodextrin (pindex#2 from MATSUTANI chemical industry Co., Ltd.) 100 ml of 120 mM sodium acetate buffer, pH 5.0 Glucose CII test kit (Wako Pure Chemical Industries, Ltd.)

[0098] 20ul of enzyme sample was mixed with 100ul of substrate solution and incubated at set temperature for 2 hours. Sample was cooled on aluminum block for 3 minutes and then 10ul of reaction solution was mixed with 590ul of 1M Tris-HCl pH 8.0 to stop the reaction. 10ul of solution was mixed with 200ul of working solution of test kit and then left at room temperature for 15 minutes. Absorbance was read at A505. Activity is listed in Table 3 as relative activity of PoAMG variant denoted as anPAV498.

[0099] [Table 13]

[0100] [Table 14]

[0101] [Table 15]

[0102] Example 7: Mashing Experiments with 100% Unmalted Sorghum

[0103] [Table 16]

[0104] Sorghum was ground using a laboratory disc mill DLFU (Buehler AG, Switzerland) at a gap distance of 0.6 mm and the resulting grist was stored dry in a closed container until further use.

[0105] Add CaCl2 to deionized water to obtain 100 ppm Ca 2+Mashing water was prepared to achieve a final concentration of ions: Mashing water was distributed into mashing beakers in 125 g portions and placed in an LP Electronic Mashing Device (Lochner GmbH, Germany) and subsequently preheated to a mash-in temperature of 55°C.

[0106] Sorghum grist was added to the mashing water at 50 (low tannin sorghum) or 12.5 (low tannin sorghum) + 37.5 (high tannin sorghum) grams per beaker, respectively. The water and grist were homogenized by stirring at 150 rpm for 5 minutes. The stirring speed was then reduced to 100 ppm, after which enzymes were added according to Table 5 below and the mashing process was started.

[0107] [Table 17]

[0108] The following mashing regimes were applied: Mashing regime A: 20 min dwell at 55°C, heat from 55 to 78°C at a heating rate of 1°C per minute, 60 min dwell at 78°C, heat from 78 to 85°C at a heating rate of 1°C per minute, 30 min dwell at 85°C, cool to 80°C, 15 min dwell at 80°C, cool to room temperature within 10 min. Total time without cooling = 170 min. Mashing regime B: 20 ​​min dwell at 55 °C, heat from 55 to 85 °C at a heating rate of 1 °C per minute, 90 min dwell at 85 °C, cool to 80 °C, 15 min dwell at 80 °C, cool to room temperature within 10 min. Total time without cooling = 170 min.

[0109] After mashing, all mashes were adjusted for evaporation loss and brought to an original weight of 175 g at the start of mashing. The mashes were then filtered at room temperature using a filtering funnel equipped with MN614 φ 240 mm (Macherey-Nagel, Germany) filter paper. The resulting wort was immediately frozen and stored until further analysis.

[0110] analysis: The moisture of finely ground sorghum grist obtained by milling in a laboratory hammer mill Lab mill 3100 (Perkin Elmer, Sweden) with a retention sieve of 0.8 mm (nominal diameter in mm) was determined using a HR73 Halogen Moisture Analyser (Mettler Toledo, Switzerland) set at a drying temperature of 105 °C.

[0111] The concentration of condensed tannins in sorghum was analyzed by the colorimetric vanillin HCL assay as 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.

[0112] Glucose in the wort was analyzed on a Dionex HPLC system ICS-5000 with a RI-101 refractive index detector (Thermo Scientific, USA) based on fermentable carbohydrates in wort by Analytica-EBC method 8.7-HPLC.

[0113] The density was analyzed by a DMA™ 4500M density analyzer (Anton Paar, Austria) to determine the concentration of the extract.

[0114] Extract yields (given as a % of dry matter grist added to the mash) were calculated based on the total moisture in the mash (including moisture from the sorghum grist), the main extract in the wort (expressed in °Plateau), and the dry matter charged to the mash.

[0115] result: Mashing with glucoamylase JPO-172 (SEQ ID NO: 6) resulted in a significantly higher final glucose concentration than that achieved with glucoamylase AMG NL (SEQ ID NO: 2) (see Tables 5 and 6). This is very clear when comparing the glucose concentrations obtained with mashing regimes A and B with the individual glucoamylases.

[0116] Glucose concentrations increased by 6.1-7.4% in mashing regime B, whereas a drop of 24-27.5% was observed in AMG NL (SEQ ID NO:2) when AMG JPO-172 (SEQ ID NO:6) was applied in the mash (see Tables 8 and 9).

[0117] For both AMGs (SEQ ID NO:2 and SEQ ID NO:6), the overall increase in extract yield when mashing regime B was applied can be attributed to the effect of higher temperature favouring starch solubilisation and liquefaction by the added raw starch degrading amylase JA126PE096 (SEQ ID NO:11) and thermostable alpha-amylase (SEQ ID NO:12).

[0118] Thus, it has been found that increasing the thermostability of a saccharifying glucoamylase to a temperature that benefits the solubilization and liquefaction of starches with high gelatinization temperatures allows for simultaneous liquefaction of starch (by a thermostable alpha-amylase) and saccharification (by a thermostable glucoamylase), making the overall process simpler and more efficient.

[0119] The combination of a thermostable alpha-amylase and the thermostable glucoamylase variant JPO-172 significantly improved the simultaneous liquefaction and saccharification of starch from unmalted sorghum in high temperature infusion mashing compared to the combination of a thermostable alpha-amylase and the thermostable glucoamylase variant AMG NL.

[0120] In mashing with unmalted sorghum, glucose formation with AMG JPO-172 increased by 10.8-43.6% compared to AMG NL, and higher mashing temperatures increased the delta improvement rate. When AMG NL was used, increasing mashing temperature decreased glucose formation by 14.0-17.5%, whereas when AMG JPO-172 was used, an increase of 6.1-7.4% was observed with increasing mashing temperature.

[0121] [Table 18]

[0122] [Table 19]

[0123] [Table 20]

[0124] [Table 21]

[0125] Example 8. Mashing Experiments with 45% Malt and 55% Broken Rice

[0126] [Table 22]

[0127] Malted barley and broken rice were milled using a Lab mill 3100 (Perkin Elmer, Sweden) and a retention sieve of 0.8 mm (nominal diameter mm).

[0128] Add CaCl2 to deionized water to obtain 100 ppm Ca 2+ Mashing water was prepared to achieve a final concentration of ions. An enzyme dilution was prepared in the mashing water with the goal of adding 0.2g of enzyme dilution to 17.8g of mashing water. 3.3g of rice and 2.7g of malt grist were added to an RVA aluminium can and mixed manually with 17.8g of pre-heated (55°C) mashing water containing calcium ions and enzymes according to Table 11 below and immediately placed in a Rapid Visco Analyser RVA4500 (Perkin Elmer, Sweden).

[0129] [Table 23]

[0130] The mashing was carried out according to the following mashing regime: Mashing regime A: 20 min dwell at 55°C, heat from 55 to 78°C at a heating rate of 1°C per minute, 127 min dwell at 78°C, cool to room temperature within 6 min. Total time without cooling = 170 min. Mashing regime B: 20 ​​min dwell at 55°C, heat from 55 to 80°C at a heating rate of 1°C per minute, 125 min dwell at 80°C, cool to room temperature within 6 min. Total time without cooling = 170 min. Mashing regime C: 20 min dwell at 55°C, heat from 55 to 85°C at a heating rate of 1°C per minute, 120 min dwell at 85°C, cool to room temperature within 6 min. Total time without cooling = 170 min. Mashing regime D: 20 min dwell at 55°C, heat from 55 to 87°C at a heating rate of 1°C per minute, 118 min dwell at 87°C, cool to room temperature within 6 min. Total time without cooling = 170 min.

[0131] After mashing, all mashes were adjusted for evaporation losses to an original weight of 24.0 g at the start of mashing, then centrifuged at 4000 rpm for 10 min at 8 °C in a Heraeus 3 SR benchtop centrifuge (Heraeus, Germany) and the supernatant was immediately frozen and stored until further analysis.

[0132] analysis: The moisture of finely ground malt and rice grist obtained by grinding in a laboratory hammer mill Lab mill 3100 (Perkin Elmer, Sweden) with a retention sieve of 0.8 mm (nominal diameter in mm) was determined using a HR73 halogen moisture analyzer (Mettler Toledo, Switzerland) set at a drying temperature of 105 °C.

[0133] Glucose in the wort was analyzed on a Dionex HPLC system ICS-5000 with a RI-101 refractive index detector (Thermo Scientific, USA) based on fermentable carbohydrates in wort by Analytica-EBC method 8.7-HPLC.

[0134] result: Mashing trials using 45% malted barley and 55% broken rice confirmed the observations made in the previous sorghum mashing experiment, where simultaneous liquefaction and saccharification mashing temperatures were tested between 78 and 87°C (maximum temperature for sorghum mashing was 85°C) and within this temperature range, continuous improvement with increasing temperature was observed for the combination of thermostable glucoamylase JPO-172 and thermostable alpha-amylase.

[0135] Glucose formation with AMG JPO-172 increased steadily as the final mashing temperature increased from 189.1 to 206.6 g / L, whereas the concentration obtained in the wort with AMG NL application decreased significantly from 184.7 g / L to 116.4 g / L when increasing the final mashing temperature from 78 to 85°C (see Tables 12 and 13), resulting in a delta difference ranging from 2.4 to 76.3%. Increasing the final mashing temperature to 87°C did not result in a further decrease in glucose concentration, indicating that glucose was already formed in the heating phase prior to the final temperature of 87°C.

[0136] [Table 24]

[0137] [Table 25]

[0138] Example 9. Mashing experiments with 45% malt and 55% cracked rice and other heat stable JPO varieties

[0139] [Table 26]

[0140] Malted barley and broken rice were milled using a Lab mill 3100 (Perkin Elmer, Sweden) and a retention sieve of 0.8 mm (nominal diameter mm).

[0141] Add CaCl2 to deionized water to obtain 100 ppm Ca 2+ Mashing water was adjusted to achieve the final concentrations of ions.

[0142] An enzyme dilution was prepared in the mashing water with the goal of adding 0.2 g of enzyme dilution to 17.8 g of mashing water. 3.3 g of rice and 2.7 g of malt grist were added to an RVA aluminium can and mixed manually with 17.8 g of pre-heated (55°C) mashing water containing calcium ions and enzymes and immediately placed in a Rapid Visco Analyser RVA4500 (Perkin Elmer, Sweden).

[0143] [Table 27]

[0144] Consequently, the mashing was carried out according to the following mashing regime: Mashing regime A: 20 min dwell at 55°C, heat from 55 to 78°C at a heating rate of 1°C per minute, 127 min dwell at 78°C, cool to room temperature within 6 min. Total time without cooling = 170 min. Mashing regime D: 20 min dwell at 55°C, heat from 55 to 87°C at a heating rate of 1°C per minute, 118 min dwell at 87°C, cool to room temperature within 6 min. Total time without cooling = 170 min.

[0145] After mashing, all mashes were adjusted for evaporation losses to an original weight of 24.0 g at the start of mashing, then centrifuged at 4000 rpm for 10 min at 8 °C in a Heraeus 3 SR benchtop centrifuge (Heraeus, Germany) and the supernatant was immediately frozen and stored until further analysis.

[0146] analysis: The moisture of finely ground malt and rice grist obtained by grinding in a laboratory hammer mill Lab mill 3100 (Perkin Elmer, Sweden) with a retention sieve of 0.8 mm (nominal diameter in mm) was determined using a HR73 halogen moisture analyzer (Mettler Toledo, Switzerland) set at a drying temperature of 105 °C.

[0147] Glucose in the wort was analyzed on a Dionex HPLC system ICS-5000 with a RI-101 refractive index detector (Thermo Scientific, USA) based on fermentable carbohydrates in wort by Analytica-EBC method 8.7-HPLC.

[0148] result: Glucose formation by different glucoamylase variants with melting points >87°C: JPO168, JPO169 or JPO-172 increased by 7.4-9.3% when the main rest in the mash was held at 87°C compared to 78°C.

[0149] On the other hand, for variants with a melting point of ≦85° C.: JPO048 or JPO081, a decrease in glucose formation of 19.6% to 36.6% was observed when increasing the main pause from 78° C. to 87° C. (see Table 15 below).

[0150] These observations support that the thermostable glucoamylase variants according to the invention, capable of acting at temperatures where sufficient gelatinization and liquefaction by thermostable alpha-amylases is obtained, i.e. in the range of 87°C, enable an efficient and simple mashing process.

[0151] [Table 28]

[0152] Example 10. Mashing experiment with 100% malt

[0153] [Table 29]

[0154] The malt was ground using a laboratory disc mill DLFU (Buehler AG, Switzerland) with a gap distance of 1.3 mm and the resulting grist was stored dry in a closed container until further use.

[0155] Mashing water was prepared by adding CaCl2 to deionized water to achieve a final concentration of 100 ppm Ca2+ ions. The mashing water was distributed into mashing beakers in portions of 192 g (corresponding to a water:grist ratio of 3:1) and placed in an LP Electronic Mashing Device (Lochner GmbH, Germany), which was then preheated to a mash-in temperature of 55 °C.

[0156] Malt grist was added to the mashing water at 64 grams per beaker. The water and grist were homogenized by stirring at 150 rpm for 5 minutes. The pH was adjusted to 5.2 by using lactic acid (20% concentration). The stirring speed was then reduced to 100 ppm, after which enzymes were added according to Table 18 below and the mashing process started. The following enzymes were added: - Attenuzyme® Pro (Novozymes A / S) is a commercial product for so-called high attenuation in brewing, containing glucoamylase from Talaromyces / Rasamsonia emersonii and pullulanase (PulC) from Bacillus acidopullulyticus. - Attenuzyme® Core (Novozymes A / S) is a commercial product containing a glucoamylase from Talaromyces / Rasamsonia emersonii (AMG-T) for so-called high attenuation in brewing. - Diazyme® 87 (International Flavors and Fragrances Inc., IFF) is a commercial product advertised as an efficient blend of glucoamylases that maximizes starch conversion and is added during saccharification at temperatures below 66°C according to the manufacturer.

[0157] [Table 30]

[0158] The mashing was carried out according to the following mashing regime: Mashing regime A: 20 min rest at 52°C, heat from 52 to 64°C at a heating rate of 1°C per minute, 60 min rest at 64°C, heat from 64 to 72°C at a heating rate of 1°C per minute, 15 min rest at 72°C, heat from 72 to 78°C at a heating rate of 1°C per minute, 30 min rest at 78°C. Total time to mash off at 78°C = 151 min. After mashing, all mashes were adjusted for evaporation losses to an original weight of 256g at the start of mashing.

[0159] At the end of mashing regime A, the entire sample is transferred into a 600 ml centrifuge cup and centrifuged for 5 minutes at 4600 rpm (4566 g) in a Heraeus Multifuge 3 SR benchtop centrifuge fitted with a Sorvall 75006445 rotor to separate the sweet wort from the grist. Decant 100 grams of sweet wort into 50 grams of mashing water (preheated to 78°C) and start mashing process B.

[0160] Mashing regime B: 30 min rest at 78 °C, heat from 78 to 96 °C with a heating rate of 0.6 °C per minute, cool to room temperature within 10 min. Total time without cooling = 60 min.

[0161] After mashing, all mashes were adjusted for evaporation losses to an original weight of 150 g at the start of mashing regime B. The mash was then filtered at room temperature using a filtering funnel fitted with MN614 φ 240 mm (Macherey-Nagel, Germany) filter paper. The resulting wort was immediately frozen and stored until further analysis.

[0162] Mashing regime C: 20 min rest at 52°C, heat from 52 to 64°C at a heating rate of 1°C per minute, 15 min rest at 64°C, heat from 64 to 72°C at a heating rate of 1°C per minute, 15 min rest at 72°C, heat from 72 to 78°C at a heating rate of 1°C per minute, 30 min rest at 78°C. Total time to mash off at 78°C = 106 min. After mashing, all mashes were adjusted for evaporation losses to an original weight of 256g at the start of mashing.

[0163] At the end of mashing regime C, the entire sample is transferred to a 600 ml centrifuge cup and centrifuged for 5 minutes at 4600 rpm (4566 g) in a Heraeus Multifuge 3 SR benchtop centrifuge fitted with a Sorvall 75006445 rotor to separate the sweet wort from the grist. Decant 100 grams of sweet wort into 50 grams of mashing water (preheated to 78°C) and start mashing process B.

[0164] analysis: The moisture content of the malt grist obtained after grinding with a laboratory disc mill DLFU (Buehler AG, Switzerland) at a gap distance of 1.3 mm was determined using a HR73 halogen moisture analyzer (Mettler Toledo, Switzerland) set at a drying temperature of 105 °C.

[0165] Glucose in the wort was analyzed on a Dionex HPLC system ICS-5000 with a RI-101 refractive index detector (Thermo Scientific, USA) based on fermentable carbohydrates in wort by Analytica-EBC method 8.7-HPLC.

[0166] result: Mashing with glucoamylase JPO-172 (SEQ ID NO: 6) resulted in a final glucose concentration significantly higher than that achieved with the three commercially available glucoamylases tested: Attenuzyme® Pro, Attenuzyme® Core and Diazyme® 87 (see Tables 19 and 20).

[0167] Comparing the composition of sweet wort produced with the 151 min mash regime (A), wort produced with AMG JPO-172 (SEQ ID NO: 6) had a 146.4% higher final glucose concentration.

[0168] Following the observation that the thermostable amyloglucosidase JPO-172 (SEQ ID NO: 6) was able to release higher levels of glucose than the commercial products tested in a mashing regime with extended saccharification at 64°C for 60 min followed by a 15 min rest at 72°C, a shorter mashing program was applied.

[0169] The shorter mashing regime C was designed to shorten the saccharification from 64°C to 78°C by 50% (44.5 min compared to 89 min), which was achieved by reducing the saccharification step at 64°C to 15 min. The resulting total process was shortened by 22% (166 min compared to 211 min). The thermostable amyloglucosidase JPO-172 was tested in this mashing regime (Table 19). The wort produced with AMG JPO-172 (SEQ ID NO: 6) showed a higher final glucose concentration due to the application of the shorter saccharification mashing regime C than previously observed with the commercial enzymes tested and with longer mashing times. The glucose yield obtained with the wort produced with JPO-172 in the shorter process was 123.6% compared to Attenuzyme® Pro in the 45 min longer mashing used as a benchmark (Table 19).

[0170] [Table 31]

[0171] After the final mash-off of regime A and regime C, the sweet wort is collected, mixed with sparging water (2:1 ratio) to simulate sparging, and transferred to mashing regime B for a further 60 minutes of processing.

[0172] Compared to Attenuzyme Pro® used as a benchmark, the glucose yield obtained in cold wort produced with JPO-172 in the 211 minute process (Mashing A+B) was 176.7% (Table 20).

[0173] By applying the shorter mashing regime (C+B) in the wort produced with AMG JPO-172 (SEQ ID NO: 6), the final glucose concentration was higher than previously observed with other commercial enzymes and longer mashing times. The glucose yield obtained in the cold wort produced with AMG JPO-172 in the 166 min process (mashing C+B) was 160.5% compared to Attenuzyme Pro® in the 45 min long mashing used as a benchmark (Table 20).

[0174] These observations support that the thermostable glucoamylase JPO-172 (SEQ ID NO: 6), which can function at temperatures higher than 78°C, allows for an efficient and simple mashing process with shorter saccharification to achieve higher fermentable sugars and higher wort composition in glucose.

[0175] [Table 32]

Claims

1. 1. A method for producing wort for a brewery, comprising adding a mature thermostable variant of a parent glucoamylase that is at least 70% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:10 to a mash before, during, or after starch liquefaction.

2. 2. The method of claim 1, wherein the parent glucoamylase is derived from a species of the genus Penicillium, preferably from Penicillium oxycalum, Penicillium miczynskii, Penicillium russellii, or Penicillium glabrum.

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

1.

5. 4. The method of claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to 1, 6, 7, 31, 34, 79, 103, 132, 445, 447, 481, 566, 568, 594 and 595 in SEQ ID NO:1, preferably wherein 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.

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

1.

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

1.

8. 4. The method of claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to 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 wherein 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.

9. 2. The method of claim 1, wherein the mature thermostable variant has a thermostability improvement (Td) over its parent of at least 5°C, preferably at least 6°C, 7°C, or 8°C.

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

11. 2. The method of claim 1, wherein the mashing comprises an incubation step at a temperature of 65°C or higher for at least 20 minutes, preferably at a temperature of 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 a temperature of 87°C or higher for at least 20 minutes.

12. 10. The method of claim 1, wherein the pH of the mash is from about 4.6 to about 6.

4.

13. 10. The method of claim 1, wherein the target glucose concentration is reached in a shorter saccharification time than when a less thermostable AMG enzyme than that of claim 1 is added.

14. One or more additional enzymes may also be added to the mash, the additional enzymes being 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-maltotetrahydrolase, glucanase, beta glucanase, galactanase, alpha- ...

2. The method of claim 1, wherein the enzyme is selected from the group consisting of lactosidase, beta-galactosidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hemicellulolytic enzymes, invertase, laccase, lipase, mannase, mannosidase, oxidase, pectolytic enzymes, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, protease, pullulanase, ribonuclease, transglutaminase, and xylanase.

15. 10. The method of claim 1, wherein the wort has more 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 claim 1.

17. 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-maltotetrahydrolase, glucanase, beta-glucanase, galactanase, alpha-galactosidase, beta-galactosidase, glucose oxyesterase 17. The mashing or brewing composition of claim 16, also comprising one or more additional enzymes selected from the group consisting of: enzymes such as maltose, maltodextrin, cellulose acetate, cellulose acetate esters, maltodextrin ...