Protease for beer turbidity reduction

Abstract

The present invention relates to endoproteases. More particularly, the present invention relates to the use of an endoprotease for reducing or eliminating beer turbidity.

Description

[0001] This application is a divisional application of Chinese patent application 202280043433.8 (PCT / US2022 / 034013), filed on June 17, 2022, entitled "Protein for Reducing Turbidity in Beer". Technical Field

[0002] This invention relates to endopeptides. More particularly, this invention relates to the use of endopeptides in reducing or eliminating beer haze and stabilizing beer foam. Background Technology

[0003] Beer turbidity (the cloudy appearance of beer) is caused by the aggregation of hydrophobic proteins (such as gliadin in barley) and polyphenols, resulting in an undesirable cloudy or hazy appearance. Turbidity in beer is most common when beer is stored in the refrigerator. This phenomenon is sometimes called chill-haze. Turbidity can also form in wine and fruit juice.

[0004] Acidic proteases, such as papain, are known to be used to proteolytically hydrolyze hydrophobic proteins and thus prevent turbidity. However, broad-spectrum proteases, such as papain, have been found to impair beer foam formation. More selective proteases, such as proline-specific endopeptides, have also been used to reduce beer turbidity. However, current commercial products are too expensive and do not completely remove beer turbidity. In this respect, current beer turbidity proteases are not specific enough to achieve complete hydrolysis and require additional steps, such as filtration assisted by PVPP (polyvinylpyrrolidone) or silica gel. Therefore, there is a continued need for proteases that can be used at a reasonable cost to more thoroughly prevent cooling turbidity, have increased specificity, and cannot survive the brewing process.

[0005] In addition, endopeptides are needed that can reduce beer turbidity without adversely affecting foam formation and stability. Summary of the Invention

[0006] According to one aspect of the invention, a method for reducing or preventing turbidity in a beverage is provided, comprising the step of adding an endopeptidase to the beverage, wherein the endopeptidase is an enzyme or an endopeptidase fragment thereof having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:16, such as a mature protein.

[0007] Optionally, the endopeptide active fragment is a mature protein. Optionally, the beverage contains protein. Optionally, the beverage contains polyphenols. Optionally, the beverage is beer. Optionally, the beverage is wine. Optionally, the beverage is fruit juice.

[0008] Optionally, the endoprotein is added to the wort. Optionally, the endoprotein is added to the beer after turbidity has formed. Optionally, the endoprotein is added to the beer before turbidity has formed.

[0009] Optionally, the beer exhibits increased relative foam stability and increased relative turbidity reduction. Optionally, the increased relative foam stability is greater than 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 110%. Optionally, the increased relative turbidity reduction, as measured by 90° scattering, is greater than 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%.

[0010] Optionally, the amount of endopeptidase in the wort is 4 to 300 mg protease / hL wort, optionally 10 to 250 mg protease / hL wort, optionally 20 to 200 mg protease / hL wort, optionally 30 to 150 mg protease / hL wort, or optionally 40 to 100 mg protease / hL wort.

[0011] Optionally, the method includes an additional step of adding one or more of ALDC enzyme, glucosylamylase, maltose α-amylase, amylopectinase, catalase, or transglucosidase.

[0012] Optionally, the ALDC enzyme is acetolactate decarboxylase as described in EC 4.1.1.5. Optionally, the glucosylamylase is 1,4-α-glucosidase as described in EC 3.2.1.3. Optionally, the maltose α-amylase is dextran 1,4-α-maltose hydrolase as described in EC 3.3.1.133. Optionally, the amylopectinase is α-dextrin endoglucosidase, restriction dextrinase, amylopectin 6-glucan hydrolase, or debranching enzyme as described in EC 3.2.1.41. Optionally, the transglucosidase is 1,4-α-glucan-branching enzyme or oligoglucan-branched glucosyltransferase as described in EC 2.4.1.24.

[0013] In another aspect of the invention, a method for increasing the relative foam stability in beer is presented, the method comprising the step of adding an endopeptidase to the beverage, wherein the endopeptidase is an enzyme or an endopeptidase fragment thereof having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:16, such as a mature protein.

[0014] Optionally, the increased relative foam stability is greater than 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 110%. Optionally, the endopeptide active fragment is a mature protein.

[0015] Optionally, the endoprotein is added to the wort. Optionally, the endoprotein is added to the beer after turbidity has formed. Optionally, the endoprotein is added to the beer before turbidity has formed.

[0016] Optionally, the amount of endopeptidase in the wort is 4 to 300 mg protease / hL wort, optionally 10 to 250 mg protease / hL wort, optionally 20 to 200 mg protease / hL wort, optionally 30 to 150 mg protease / hL wort, or optionally 40 to 100 mg protease / hL wort.

[0017] Optionally, the method includes an additional step of adding one or more of ALDC enzyme, glucosylamylase, maltose α-amylase, amylopectinase, catalase, or transglucosidase.

[0018] Optionally, the ALDC enzyme is acetolactate decarboxylase as described in EC 4.1.1.5. Optionally, the glucosylamylase is 1,4-α-glucosidase as described in EC 3.2.1.3. Optionally, the maltose α-amylase is dextran 1,4-α-maltose hydrolase as described in EC 3.3.1.133. Optionally, the amylopectinase is α-dextrin endoglucosidase, restriction dextrinase, amylopectin 6-glucan hydrolase, or debranching enzyme as described in EC 3.2.1.41. Optionally, the transglucosidase is 1,4-α-glucan-branching enzyme or oligoglucan-branched glucosyltransferase as described in EC 2.4.1.24.

[0019] Optionally, the beer exhibits increased relative foam stability and increased relative turbidity reduction. Optionally, as measured by 90° scattering, the increased relative turbidity reduction is greater than 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%.

[0020] In another aspect of the invention, isolated polypeptides comprising endopeptides are presented, wherein the endopeptide is an enzyme or an endopeptide active fragment thereof having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:16.

[0021] In another aspect of the invention, isolated polynucleotides are presented having nucleic acid sequences encoding the aforementioned polypeptides.

[0022] In another aspect of the invention, a nucleic acid construct is presented having the aforementioned polynucleotide operably linked to one or more control sequences that direct the production of the polypeptide in a suitable expression host.

[0023] In another aspect of the present invention, a recombinant expression vector having the above-described nucleic acid construct is presented.

[0024] In another aspect of the invention, recombinant host cells having the above-described nucleic acid constructs or vectors are presented.

[0025] In another aspect of the invention, a method for producing the aforementioned polypeptide is presented, the method comprising the steps of: culturing the aforementioned recombinant host cells to produce a supernatant and / or cells containing the polypeptide; and recovering the polypeptide.

[0026] In another aspect of the present invention, a polypeptide produced by the above method is presented.

[0027] In another aspect of the invention, the use of the filtrate obtained from the fermentation broth obtained by the above method in preventing or reducing turbidity in beverages is presented.

[0028] In another aspect of the invention, compositions having the polypeptide, isolated polynucleotide, nucleic acid construct, recombinant expression vector, or recombinant host cell as described above are presented.

[0029] In another aspect of the invention, the use of the polypeptides, isolated polynucleotides, nucleic acid constructs, recombinant expression vectors, or recombinant host cells described above for increasing the relative foam stability in beverages is presented.

[0030] In another aspect of the invention, the use of the polypeptides, isolated polynucleotides, nucleic acid constructs, recombinant expression vectors, or recombinant host cells described above for increasing the relative reduction of turbidity in beverages is presented.

[0031] In another aspect of the invention, a method for reducing turbidity in a beverage is presented, the method comprising the steps of: adding an endopeptidase to the beverage, wherein the beverage contains a protein or peptide having glutamine residues, the glutamine residues being cleaved by the protease thereby reducing turbidity. Optionally, the endopeptidase also cleaves proline residues. Optionally, the beverage is fruit juice, wine, or beer. Optionally, the beverage is beer.

[0032] Optionally, the glutamine endopeptide comprises a polypeptide or its endopeptide active fragment having at least 80%, 85%, 90%, 95%, 98%, or 99% homology to SEQ ID NO:2 or SEQ ID NO:16, such as a mature protein lacking a signal sequence. Optionally, the glutamine endopeptide comprises the polypeptide according to SEQ ID NO:16.

[0033] Biological sequence description

[0034] SEQ ID NO:1 is the AbePro2 precursor protein.

[0035] SEQ ID NO:2 is the AniPro_2 precursor protein.

[0036] SEQ ID NO:3 is the AtrPro1 precursor protein.

[0037] SEQ ID NO:4 is the AhoPro3 precursor protein.

[0038] SEQ ID NO:5 is the ApsPro1 precursor protein.

[0039] SEQ ID NO:6 is the AnePro2 precursor protein.

[0040] SEQ ID NO:7 is the AalPro2 precursor protein.

[0041] SEQ ID NO:8 is the AcoPro2 precursor protein.

[0042] SEQ ID NO:9 is the AwePro2 precursor protein.

[0043] SEQ ID NO:10 is the AbrPro1 precursor protein.

[0044] SEQ ID NO:11 is the AscPro5 precursor protein.

[0045] SEQ ID NO:12 is the full-length MorPro1 DNA.

[0046] SEQ ID NO:13 is a full-length MorPro1 precursor.

[0047] SEQ ID NO:14 is the maturation enzyme predicted by MorPro1.

[0048] SEQ ID NO:15 is a synthetic nucleotide sequence encoding the full-length MorPro1.

[0049] SEQ ID NO:16 is the mature protein of AniPro_2. Attached Figure Description

[0050] Figure 1 It is the AbePro2 precursor protein.

[0051] Figure 2 It is the AniPro_2 precursor protein.

[0052] Figure 3 It is the precursor protein of AtrPro1.

[0053] Figure 4 It is the AhoPro3 precursor protein.

[0054] Figure 5 The turbidity reduction performance of purified AhoPro3 and AnPro in response to enzyme concentrations from 3 to 190 ppm is shown, as OD600.

[0055] Figure 6 The turbidity reduction performance of purified AtrPro1, AbePro2, and AnPro in response to enzyme concentrations from 3 to 190 ppm is shown as OD600.

[0056] Figure 7 The turbidity reduction performance of purified AniPro_2 and AnPro in response to enzyme concentrations from 0.625 to 20 ppm is shown, as OD600.

[0057] Figure 8 It is the ApsPro1 precursor protein.

[0058] Figure 9 It is the AnePro2 precursor protein.

[0059] Figure 10 It is the AalPro2 precursor protein.

[0060] Figure 11 It is the AcoPro2 precursor protein.

[0061] Figure 12 It is the AwePro2 precursor protein.

[0062] Figure 13 It is the AbrPro1 precursor protein.

[0063] Figure 14 It is the AscPro5 precursor protein.

[0064] Figure 15 The turbidity reduction performance of purified ApsPro1, AnePro2, AalPro2, AcoPro2, AwePro2, AbrPro1, AscPro5, and AnPro in response to enzyme concentrations from 3 to 200 ppm is shown as A600.

[0065] Figure 16 The protease activities of AhoPro3, ApsPro1, AnePro2, AalPro2, AcoPro2, AwePro2, AbrPro1, AscPro5, and AnPro in response to enzyme concentrations from 0 to 10 ppm are shown as A405.

[0066] Figure 17 The full-length MorPro1 DNA was displayed.

[0067] Figure 18 The full-length MorPro1 precursor is shown.

[0068] Figure 19 The MorPro1-predicted maturation enzyme is shown.

[0069] Figure 20 The synthetic nucleotide sequence encoding the full-length MorPro1 is shown.

[0070] Figure 21A This demonstrates a substrate-specific nomenclature for the protease. The amino acid residues in the substrate are numbered from the protease cleavage site outwards as: P3, P2, P1, P1', P2', P3', with the cleavage site highlighted by a black arrow between P1 and P1'.

[0071] Figure 21B Peptides produced on a 1 hL scale from malt beer with or without endopeptidase were depicted, along with the relative amino acid content at the peptide terminus corresponding to P3'-P3 via protease cleavage. The relative amino acid content of the peptide at the P1 position is shown in the left-hand plot, representing the control (without enzyme), MorPro1, AnPro, and AniPro2. The right-hand plot shows the relative amino acid content at the peptide positions corresponding to P3-P3', with the graph displaying the cumulative content of each amino acid at a given position, thus revealing a detailed protease-specific preference.

[0072] Figure 22The images show peptides from malt beers produced at a 1 hL scale with or without endopeptides, analyzed by LC-MS, and the average peptide length (number of amino acid residues) was calculated from over 300 unique peptides identified as described in Example 31. The average peptide lengths were calculated for beers produced with AniPro2, AnPro, MorPro1, and a control (without the enzyme).

[0073] Figure 23 This shows the mature AniPro_2 protein. Detailed Implementation

[0074] Unless otherwise stated, the practice of the teachings of this invention will utilize conventional techniques within the scope of the art, including those in molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry. Such techniques are well explained in the following documents, for example, Molecular Cloning: A Laboratory Manual Second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (Edited by MJ Gait, 1984;) Current Protocols in Molecular Biology (FM Ausubel et al., editors, 1994); PCR: The Polymerase Chain Reaction [PCR: Polymerization [Enzyme chain reaction] (Mullis et al., editors, 1994); Gene Transfer and Expression: A Laboratory Manual [Gene Transfer and Expression: A Laboratory Manual] (Kriegler, 1990), and The Alcohol Textbook (Ingledew et al., editors, 5th edition, 2009), and Essentials of Carbohydrate Chemistry and Biochemistry: Fundamentals (Lindhorste, 2007).

[0075] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the teachings of this invention pertain. Singleton et al. Dictionary of Microbiology and Molecular Biology Dictionary , Second Edition, John Wiley and Sons, New York (1994), and Hale and Markham, The Harper HarperCollins Dictionary of Biology Harper Perennial, New York (1991), provides a general dictionary of many terms used in this invention for those skilled in the art. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the teachings of this invention.

[0076] The numerical ranges provided in this article include those that limit the range.

[0077] Definitions and abbreviations

[0078] Regarding polypeptides, the terms "wild-type," "parent," or "reference" refer to naturally occurring polypeptides that do not contain artificial substitutions, insertions, or deletions at one or more amino acid positions. Similarly, regarding polynucleotides, the terms "wild-type," "parent," or "reference" refer to naturally occurring polynucleotides that do not include artificial nucleoside changes. However, note that polynucleotides encoding wild-type, parent, or reference polypeptides are not limited to naturally occurring polynucleotides and encompass any polynucleotide encoding wild-type, parent, or reference polypeptides.

[0079] Regarding peptides, the term "variant" refers to a peptide that differs from a specified wild-type, parent, or reference peptide because it includes one or more naturally occurring or artificial amino acid substitutions, insertions, or deletions. Similarly, regarding polynucleotides, the term "variant" refers to a polynucleotide that differs from a specified wild-type, parent, or reference polynucleotide in terms of its nucleotide sequence. The characteristics of the wild-type, parent, or reference peptide or polynucleotide will become apparent from the context.

[0080] The term "recombinant" when used to refer to subject cells, nucleic acids, proteins, or vectors indicates that the subject has been modified from its natural state. Thus, for example, recombinant cells express genes not found in their natural (non-recombinant) form, or express natural genes at levels different from those found in nature, or under conditions different from those found in nature. Recombinant nucleic acids differ from their natural sequence by one or more nucleotides and / or are operatively linked to a heterologous sequence, such as a heterologous promoter in an expression vector. Recombinant proteins may differ from their natural sequence by one or more amino acids and / or be fused to a heterologous sequence. A vector containing a nucleic acid encoding an endonuclease is a recombinant vector.

[0081] The terms “recycled,” “isolated,” and “separate” refer to compounds, proteins (peptides), cells, nucleic acids, amino acids, or other specified materials or components removed from at least one other material or component found in nature and naturally associated with it. “Isolated” peptides include, but are not limited to, culture media containing secreted peptides expressed in heterologous host cells.

[0082] The term "purified" refers to material in a relatively pure state (e.g., isolated polypeptides or polynucleotides), for example, at least about 90% pure, at least about 95% pure, at least about

[0083] The term "amino acid sequence" is synonymous with and used interchangeably with the terms "polypeptide," "protein," and "peptide." When such amino acid sequences exhibit activity, they can be called "enzymes." A standard single-letter or three-letter code is used for the amino acid residues, where the amino acid sequence is presented in a standard N-terminal to C-terminal orientation (i.e., N→C).

[0084] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding polypeptides. Nucleic acids can be single-stranded or double-stranded and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the compositions and methods of the present invention cover nucleotide sequences encoding specific amino acid sequences. Unless otherwise stated, nucleic acid sequences are presented in a 5′ to -3′ orientation.

[0085] The terms “transformation,” “stable transformation,” and “transgenic” used in relation to cells refer to cells containing non-natural (e.g., heterologous) nucleic acid sequences that are integrated into their genome or carried as an appendage maintained through multiple generations.

[0086] In the context of inserting nucleic acid sequences into cells, the term “introduction” refers to what is known in the art as “transfection,” “conversion,” or “transduction.”

[0087] A “host strain” or “host cell” is an organism in which an expression vector, bacteriophage, virus, or other DNA construct has been introduced, including a polynucleotide encoding a target polypeptide (e.g., an endonuclease). An exemplary host strain is a microbial cell (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the target polypeptide. The term “host cell” includes protoplasts derived from cells.

[0088] The term "heterogeneous" in relation to polynucleotides or proteins refers to polynucleotides or proteins that are not naturally present in host cells.

[0089] The term "endogenous" in relation to polynucleotides or proteins refers to polynucleotides or proteins that are naturally present in host cells.

[0090] The term "expression" refers to the process of producing polypeptides based on nucleic acid sequences. This process includes both transcription and translation.

[0091] A "vector" is a multinucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, etc.

[0092] An "expression vector" is a DNA construct containing a DNA sequence encoding a target polypeptide, which is operatively linked to a suitable control sequence that enables DNA expression in a suitable host. Such control sequences may include promoters that enable transcription, optional operon sequences that control transcription, sequences encoding suitable ribosome binding sites on mRNA, enhancers, and sequences that control the termination of transcription and translation.

[0093] As used in this article, "sequence identity percentage" refers to the percentage of amino acid residues in a given sequence that are identical to those in a specified reference sequence when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. [Nucleic Acid Research] 22:4673-4680. The default parameters for the CLUSTAL W algorithm are:

[0094] Open shot penalty: 10.0

[0095] Extended penalty for open looks: 0.05

[0096] Protein weight matrix: BLOSUM series

[0097] DNA weight matrix: IUB

[0098] Delayed divergence sequence %: 40

[0099] Empty space separation distance: 8

[0100] DNA conversion weight: 0.50

[0101] List of hydrophilic residues: GPSNDQEKR

[0102] Using a negative matrix: [closed]

[0103] Switching special residue penalty: On

[0104] Switch to hydrophilic penalty: On

[0105] Switch to end the empty space separation penalty level.

[0106] Deletions are considered distinct residues compared to a reference sequence. This includes deletions occurring at either end.

[0107] The term "about" refers to ±5% of the reference value.

[0108] As used herein, the term "beer" traditionally refers to an alcoholic beverage derived from malt (which is derived from barley) and optional adjuncts (such as grains) and flavored with hops. However, "beer" can also be derived from adjuncts such as rice and sorghum. Beer can be made from a variety of grains using essentially the same process. All grain starches are glucose homopolymers in which glucose residues are linked by either α-1,4- or α-1,6- bonds, with the former being dominant. The process of making fermented malt beverages is generally called brewing. The main ingredients used to make these beverages are water, hops, and malt. In addition, adjuncts such as common corn grits, refined corn grits, rice, sorghum, refined corn starch, barley, barley starch, hulled barley, wheat, wheat starch, baked cereals, cereal flakes, rye, oats, potatoes, cassava, and syrups such as corn syrup, cane syrup, invert sugar syrup, barley and / or wheat syrup can be used as sources of starch or fermentable sugar types. Starch is eventually converted into dextrins and fermentable sugars. For several reasons, malt, primarily produced from selected barley varieties, has the greatest impact on the overall characteristics and quality of beer. First, malt is the main flavoring agent in beer. Second, malt provides the main component of fermentable sugars. Third, malt provides proteins, which contribute to the body and foam characteristics of beer. Fourth, malt provides the enzyme activity necessary during starch saccharification.

[0109] The “beer-making process” is a well-known process in the field, but in short, it involves five steps: (a) adjunct cooking and / or starch saccharification, (b) wort separation and extraction, (c) wort boiling and hopping, (d) cooling, fermentation, and storage, and (e) maturation, processing, and packaging. In the first step, milled or crushed malt is mixed with water and kept at a controlled temperature for a period of time, for example, to allow enzymes present in the malt to convert the starch present in the malt into fermentable sugars. In the second step, the mash is transferred to a “lauter tun” or wort filter, where the liquid is separated from the grain residue. This sweet liquid is called “wort,” and the remaining grain residue is called “spent grain.” During wort separation, the wort is typically extracted, which involves adding water to the wort to recover residual soluble extracts from the spent grain. In the third step, the wort is vigorously boiled. This sterilizes the wort and helps develop color, flavor, and aroma. Hops are added at some point during boiling. In the fourth step, the wort is cooled and transferred to a fermentation tank containing yeast or to which yeast has been added. The yeast converts sugars into alcohols and carbon dioxide gas through fermentation; the fermentation tank is cooled at the end of fermentation, or it can be cooled to stop fermentation. Yeast flocculants are removed. In the final step, the beer is cooled and stored for a period of time, during which time the beer becomes clear and develops flavor, and any materials that might impair the beer's appearance, flavor, and shelf life precipitate out. Before bottling, the beer is carbon dioxide-puffed and optionally filtered and pasteurized. After fermentation, a beverage is obtained that typically contains about 2% to about 10% alcohols. Non-fermentable carbohydrates are not converted during fermentation and form most of the dissolved solids in the final beer. This residue remains because maltase cannot hydrolyze the α-1,6-bonds of starch and completely degrade non-starch polysaccharides.

[0110] As used in this article, "turbidity" refers to the presence of insoluble substances in beverages such as juice, wine, and beer. Turbidity is known to occur when proteins (typically those rich in proline and glutamine) interact with polyphenols to form insoluble complexes. These complexes are well-known in beer brewing, particularly during refrigerated beer storage. This phenomenon is sometimes referred to as chilling turbidity.

[0111] The term "glutamine endopeptidase" refers to an endopeptidase that preferentially cleaves a substrate protein or peptide at one or more glutamine residues.

[0112] As used in this article, “increased relative foam stability” means the increased foam stability of the final beer achieved by fermenting the same wort in the presence of endopeptides compared to fermented wort without endopeptides.

[0113] Foam stability can be measured using the NIBEM foam stability test as described in Example 15. Relative foam stability can be calculated by measuring the NIBEM foam collapse time (30 mm) of beer produced from fermented wort in the presence of PEP enzyme and by measuring the NIBEM foam collapse time (30 mm) of beer produced from fermented wort without PEP enzyme. The relative foam stability can be calculated using the following formula: (collapse time NIBEM 30 mm with PEP enzyme) / (collapse time NIBEM 30 mm without PEP enzyme) x 100%.

[0114] Preferably, the increased relative foam stability is higher than 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, and 110%.

[0115] As used in this article, “increased relative turbidity reduction” means an increase in the reduction of turbidity in the final beer achieved by fermenting the same wort in the presence of endopeptides compared to fermented wort without endopeptides.

[0116] Relative turbidity reduction can be measured using the EBC TOHA forced haze method as described in Example 13. The relative turbidity reduction can be calculated by measuring the 90° EBC scattering from EBC TOHA forced haze of beer produced in the presence of endopeptides in fermented wort and by measuring the 90° EBC scattering from EBC TOHA forced haze of beer produced by fermenting the same wort but without endopeptides. The relative turbidity reduction using endopeptides can be calculated for beer without any PEP enzymes as follows: (EBC...) 浊度90°没有酶 - EBC 浊度90°具有PEP酶 ) / EBC 浊度90°没有酶 x 100%. Similarly, the relative turbidity reduction can be calculated by measuring the EBC TOHA forced turbidity of beer produced in fermented wort in the presence of endopeptides using 25° EBC scattering and measuring the EBC TOHA forced turbidity of beer produced by fermenting the same wort but without endopeptides using 25° EBC scattering. The relative turbidity reduction using endopeptides can be calculated for beer without any PEP enzymes as follows: (EBC 浊度25°没有酶 -EBC 浊度25°具有PEP酶 ) / EBC 浊度25°没有酶 x 100%.

[0117] The preferred increase in relative turbidity reduction (90°) is above 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, or 83%.

[0118] The endonucleases of the present invention can be “precursors,” “immature,” or “full-length,” in which case they contain a signal sequence; or “mature,” in which case they lack a signal sequence. Mature peptides are generally the most useful. The endonuclease peptides of the present invention can also be truncated to remove the N-terminus or C-terminus, provided that the resulting peptide retains endonuclease activity. Furthermore, the endonuclease can be an active fragment derived from a longer amino acid sequence. An active fragment is characterized by retaining some or all of the activity of the full-length enzyme, but having deletions from the N-terminus, from the C-terminus, or within or in combination thereof. Mature proteins can be considered as active fragments of precursors, immature, or full-length proteins.

[0119] The endonuclease of the present invention may be a "chimeric" or "hybrid" polypeptide because it comprises at least a portion of a first endonuclease polypeptide and at least a portion of a second endonuclease polypeptide. The endonuclease of the present invention may further comprise a heterologous signal sequence, i.e., an epitope that allows for tracking or purification, etc. Exemplary heterologous signal sequences are derived from Bacillus licheniformis amylase (LAT), Bacillus subtilis (AmyE or AprE), and Streptomyces CelA.

[0120] Production of endopeptides

[0121] The endonuclease of the present invention can be produced in host cells, for example, by secretion, by surface display, or by intracellular expression. After secreting the endonuclease into a cell culture medium, cultured cell material containing the endonuclease (e.g., whole-cell culture medium) can be obtained. Optionally, the endonuclease can be isolated from host cells, or even from the cell culture medium, depending on the desired purity of the final endonuclease. Alternatively, the endonuclease can be expressed on the surface of host cells.

[0122] Genes encoding endonucleases can be cloned and expressed using methods well-known in the art. Suitable host cells include bacteria, fungi (including yeasts and filamentous fungi), and plant cells (including algae). Particularly useful host cells include *Saccharomyces cerevisiae*, *Saccharomyces pastorianus*, *Brettanomyces*, *Aspergillus niger*, *Aspergillus oryzae*, or *Trichoderma reesei*. Other host cells include bacterial cells such as *Bacillus subtilis* or *Bacillus licheniformis*, as well as *Streptomyces* and *Escherichia coli*.

[0123] In one aspect of the invention, yeast cells expressing endopeptides can be directly used for fermentation in beer production. In this aspect of the invention, it is not necessary to add exogenous endopeptides. The endopeptides expressed by yeast provide turbidity reduction.

[0124] The host cell can also express nucleic acids encoding homologous or heterologous endonucleases (i.e., endonucleases from a different species than the host cell) or one or more other enzymes. Endonucleases can be variant endonucleases. Additionally, the host can express one or more coenzymes, proteins, or peptides.

[0125] carrier

[0126] DNA constructs containing nucleic acids encoding endonucleases can be constructed for expression in host cells. Due to the well-known degeneracy of the genetic code, variant polynucleotides encoding the same amino acid sequence can be designed and prepared using conventional techniques. Optimizing codon usage for specific host cells is also well-known in the art. The nucleic acid encoding the endonuclease can be incorporated into a vector. The vector can be transferred into host cells using well-known transformation techniques, such as those disclosed below.

[0127] The vector can be any vector that can be transformed into and replicated in a host cell. For example, a vector containing a nucleic acid encoding an endonuclease can be transformed and replicated in a bacterial host cell as a means of propagating and amplifying the vector. The vector can also be transformed into an expression host so that the nucleic acid encoding the endonuclease can be expressed. Host cells used as expression hosts can include, for example, filamentous fungi. The strain catalogue of the Center for Fungal Genetics, USA (FGSC) lists vectors suitable for expression in fungal host cells. See FGSC, Strain Catalogue, University of Missouri, www.fgsc.net (last updated January 17, 2007). A representative vector is pJG153, a promoterless Cre expression vector that can replicate in a bacterial host. See Harrison et al., (June 2011) Applied Environ. Microbiol [Applied and Environmental Microbiology] 77: 3916-22. pJG153 can be modified using conventional techniques to contain and express nucleic acids encoding endonucleases.

[0128] Nucleic acid encoding an endonuclease can be operatively linked to a suitable promoter, thereby allowing transcription to occur in a host cell. The promoter can be any DNA sequence that exhibits transcriptional activity in the selected host cell and can be derived from a gene encoding a protein homologous or heterologous to that of the host cell. Exemplary promoters used to direct transcription of DNA sequences encoding endonucleases (especially in bacterial hosts) include the promoter of the *lac* operon in *E. coli*, the promoter of the *dagA* or *celA* agarase gene in *Streptomyces coelicolor*, the promoter of the α-amylase gene (*amyL*) in *Bacillus licheniformis*, the promoter of the raw maltose amylase gene (*amyM*) in *Bacillus stearothermophilus*, the promoter of the α-amylase gene (*amyQ*) in *Bacillus amyloliquefaciens*, and the promoters of the *xylA* and *xylB* genes in *Bacillus subtilis*, among others. For transcription in fungal hosts, examples of useful promoters are those derived from genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic protease, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger glucosylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae trisaccharide isomerase, or Aspergillus nidulans acetamase. When genes encoding endonucleases are expressed in bacterial species (such as Escherichia coli), suitable promoters can be selected from, for example, phage promoters including the T7 promoter and the phage λ promoter. Examples of suitable promoters for expression in yeast species include, but are not limited to, the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the AOX1 or AOX2 promoters of Pichia pastoris. cbh1 is an endogenously inducible promoter from Trichoderma reesei. See Liu et al. (2008) “Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbh1) promoter optimization,” Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.

[0129] The coding sequence can be operatively linked to the signal sequence. The DNA encoding the signal sequence can be a DNA sequence naturally associated with the gene for which the endonuclease to be expressed or derived from a different genus or species. The signal sequence and promoter sequence, comprising a DNA construct or vector, can be introduced into a fungal host cell, and they can be derived from the same source. For example, the signal sequence could be the cbh1 signal sequence operatively linked to the cbh1 promoter.

[0130] The expression vector may also contain a suitable transcription terminator, and, in eukaryotes, a polyadenylated sequence operatively linked to a DNA sequence encoding a variant endonuclease. The terminator and polyadenylated sequence may be appropriately derived from the same source as the promoter.

[0131] The vector may further contain DNA sequences that enable it to replicate in the host cell. Examples of such sequences are the origins of replication for plasmids pUC19, pACYC177, pUB110, pE194, pAMB1, and pIJ702.

[0132] The vector may also contain selectable markers, such as genes whose products compensate for defects in isolated host cells, like the dal gene from Bacillus subtilis or Bacillus licheniformis, or genes conferring antibiotic resistance (e.g., resistance to ampicillin, kanamycin, chloramphenicol, or tetracycline). Furthermore, the vector may contain Aspergillus selectable markers, such as amdS, argB, niaD, and xxsC, markers that induce hygromycin resistance, or selection may be achieved through co-transformation (as known in the art).

[0133] Transformation and culture of host cells

[0134] Isolated cells containing DNA constructs or expression vectors are advantageously used as host cells for recombinant production of endonucleases. Cells can be conveniently transformed with DNA constructs encoding enzymes by integrating the DNA construct (in one or more copies) into the host chromosome. This integration is generally considered advantageous because the DNA sequence is more likely to remain stable in the cell. The DNA construct can be integrated into the host chromosome using conventional methods, for example, through homologous or heterologous recombination. Alternatively, cells can be transformed with expression vectors associated with different types of host cells as described above.

[0135] Suitable yeast host organisms can be selected from biotechnology-related yeast species, such as, but not limited to, species of the genera *Pichia*, *Hansenula*, *Kluyveromyces*, *Yarrowinia*, *Schizosaccharomyces*, or *Saccharomyces*, including *Saccharomyces cerevisiae*, or species belonging to the genus *Schizosaccharomyces*, such as, for example, *Schizosaccharomyces pombe*. Methyltrophic yeast strains such as *Pichia* can be used as host organisms. Alternatively, host organisms can be species of the genus *Hansenula*. Suitable host organisms among filamentous fungi include species of the genus *Aspergillus*, such as *Aspergillus niger*, *Aspergillus oryzae*, *Aspergillus tubigensis*, *Aspergillus awamori*, or *Aspergillus nidus*. Alternatively, strains of *Fusarium* species (e.g., *Fusarium oxysporum*) or strains of *Rhizomucor* species (e.g., *Rhizomucor miltiorrhiza*) can be used as host organisms. Other suitable strains include species of *Thermomyces* and *Mucor*. Additionally, species of *Trichoderma* sp. can be used as hosts. Suitable procedures for transforming *Aspergillus* host cells include, for example, the procedures described in EP238023. Endonucleases expressed by the fungal host cells can be glycosylated, i.e., containing a glycosyl moiety. The glycosylation pattern can be the same as or different from that present in wild-type endonucleases. The type and / or extent of glycosylation may alter the enzyme and / or biochemical properties.

[0136] It is advantageous to express a gene missing from the host, where the gene defect can be cured by the transformed expression vector. Known methods are available for obtaining fungal host cells with one or more inactivated genes. Gene inactivation can be accomplished by complete or partial deletion, by insertional inactivation, or by any other means that renders the gene ineffective for its intended purpose, thereby preventing the expression of a functional protein. Any cloned gene from Trichoderma species or other filamentous fungal hosts can be deleted, for example, the cbh1, cbh2, egl1, and egl2 genes. Gene deletion can be accomplished by methods known in the art by inserting the desired gene, in its original form, into a plasmid.

[0137] Introducing DNA constructs or vectors into host cells includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, such as lipid transfection-mediated and DEAE-dextrin-mediated transfection; incubation with calcium phosphate DNA precipitation; high-speed bombardment with DNA-coated microparticles; and protoplast fusion. Common transformation techniques are known in the art. See, for example, Sambrook et al. (2001), ibid. Expression of heterologous proteins in *Trichoderma* is described, for example, in U.S. Patent No. 6,022,725. For transformation of *Aspergillus* strains, also refer to Cao et al. (2000) Science [Science] 9:991-1001. Genetically stable transformants can be constructed using vector systems, thereby stably integrating nucleic acids encoding endonucleases into the host cell chromosome. Transformants are then selected and purified using known techniques.

[0138] The preparation of Trichoderma species for transformation can, for example, involve the preparation of protoplasts from fungal mycelium. See Campbell et al. (1989) Curr. Genet. [Contemporary Genetics] 16: 53-56. Mycelium can be obtained from germinating vegetative spores. Protoplasts are produced by treating the mycelium with enzymes that digest the cell wall. The protoplasts are protected by the presence of osmotic stabilizers in the suspension medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, etc. Typically, the concentration of these stabilizers varies between 0.8 M and 1.2 M; for example, a 1.2 M solution of sorbitol can be used in the suspension medium.

[0139] The uptake of DNA into the host Trichoderma species depends on the calcium ion concentration. Typically, CaCl2 at approximately 10 mM to 50 mM is used in the uptake solution. Additional suitable compounds include buffer systems such as TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS (pH 6.0) and polyethylene glycol. PEG is believed to induce cell membrane fusion, thereby allowing the contents of the culture medium to be delivered into the cytoplasm of the Trichoderma species. This fusion often leaves multiple copies of plasmid DNA integrated into the host chromosome.

[0140] Typically, protoplasts or cells that have undergone permeation treatment are used to transform Trichoderma species, usually at a ratio of 10. 5 Up to 10 7 / mL, especially 2 x 10 6The process is carried out at a density of / mL. 100 μL of these protoplasts or cells in a suitable solution (e.g., 1.2 M sorbitol and 50 mM CaCl2) can be mixed with the desired DNA. Typically, a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension; however, adding approximately 0.25 volumes is useful. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, etc., can also be added to the uptake solution to promote transformation. Similar procedures can be used for other fungal host cells. See, for example, U.S. Patent No. 6,022,725.

[0141] Express

[0142] Methods for producing endopeptidase may include culturing host cells as described above under conditions favorable for the production of the enzyme, and recovering the enzyme from the cells and / or culture medium.

[0143] The culture medium used to culture cells can be any conventional medium suitable for the growth of the host cells under consideration and for obtaining expression of endopeptide proteases. Suitable media and media components can be obtained from commercial suppliers or can be prepared according to publicly available formulations, such as those described in the catalogue of the American Type Culture Collection.

[0144] Enzymes secreted from host cells can be used in whole culture preparations. In the method of this invention, any culture method known in the art can be used to prepare used whole fermentation broth of recombinant microorganisms, resulting in the expression of endopeptides. Therefore, fermentation can be understood as including shake-flask cultures, small-scale or large-scale fermentations (including continuous fermentation, batch fermentation, fed-batch fermentation, or solid-state fermentation) carried out in a laboratory or industrial fermenter under suitable culture media and conditions allowing for the expression or isolation of endopeptides. The term "used whole fermentation broth" is defined herein as the ungraded contents of fermentation material comprising culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It should be understood that the term "used whole fermentation broth" also encompasses cellular biomass that has been lysed or permeated using methods well known in the art.

[0145] Enzymes secreted from host cells can be readily recovered from the culture medium using well-known procedures, which include separating cells from the medium by centrifugation or filtration, precipitating the protein components of the medium with the aid of salts (e.g., ammonium sulfate), and then using chromatographic procedures such as ion exchange chromatography, affinity chromatography, etc.

[0146] A polynucleotide encoding an endonuclease in a vector can be operatively linked to a control sequence that can provide expression of the coding sequence through the host cell; that is, the vector is an expression vector. The control sequence can be modified, for example, by adding other transcriptional regulatory elements, to make the transcriptional level directed by the control sequence more responsive to transcriptional regulators. The control sequence may specifically contain a promoter.

[0147] Host cells can be cultured under suitable conditions that allow for the expression of endopeptides. The expression of these enzymes can be constitutive, enabling their continuous production, or inducible, requiring stimulation to initiate expression. In the case of inducible expression, protein production can be initiated when needed, for example, by adding an inducing agent such as dexamethasone, IPTG, or sophorose to the culture medium. Peptides can also be recombinantly produced in vitro in cell-free systems, such as the TNT™ (Promega) rabbit reticulocyte system.

[0148] The expression host can also be cultured under aerobic conditions in a medium suitable for the host. A combination of shaking or agitation and aeration can be provided, with production occurring at a temperature suitable for the host (e.g., from about 25°C to about 75°C (e.g., 30°C to 45°C), depending on the host's needs and the desired production of the endopeptide). Culture can occur for a duration of about 12 to about 100 hours or longer (and any hourly values ​​in between, e.g., from 24 to 72 hours). Typically, the pH of the culture medium is from about 4.0 to about 8.0, again depending on the culture conditions required by the host for the production of the endopeptide.

[0149] Methods for enriching and purifying endopeptides

[0150] Fermentation, separation, and concentration techniques are well known in the field, and solutions containing endopeptide peptides can be prepared using conventional methods.

[0151] After fermentation, the fermentation broth is obtained. Microbial cells and various suspended solids (including the remaining crude fermentation material) are removed using conventional separation techniques to obtain an endopeptide solution. Commonly used methods include filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, post-centrifugation ultrafiltration, extraction, or chromatography.

[0152] The aim is to concentrate solutions containing endopeptide peptides to optimize recovery. Using undiluted solutions requires increased incubation time to collect the enriched or purified enzyme precipitate.

[0153] The enzyme-containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme-containing solution can be achieved using any of the techniques discussed herein. Exemplary methods for enrichment and purification include, but are not limited to, rotary vacuum filtration and / or ultrafiltration.

[0154] The enzyme solution is concentrated until the enzyme activity of the concentrated solution containing endopeptide peptides reaches the desired level.

[0155] Concentration can be achieved using precipitants, such as metal halide precipitants. Metal halide precipitants include, but are not limited to, alkali metal chlorides, alkali metal bromides, and blends of two or more of these metal halides. Exemplary metal halides include sodium chloride, potassium chloride, sodium bromide, potassium bromide, and blends of two or more of these metal halides. Sodium chloride, a metal halide precipitant, can also be used as a preservative.

[0156] Metal halide precipitants are used in amounts that effectively precipitate endopeptides. After routine testing, the selection of at least effective and optimal amounts of metal halides to effectively induce enzyme precipitation, as well as the precipitation conditions used to maximize recovery, including incubation time, pH, temperature, and enzyme concentration, will be readily apparent to those skilled in the art.

[0157] Typically, a metal halide is added to concentrated enzyme solutions at a rate of at least about 5% w / v (weight / volume) to about 25% w / v, and usually at least 8% w / v. Generally, no more than about 25% w / v of metal halide is added to concentrated enzyme solutions, and usually no more than about 20% w / v. The optimal concentration of the metal halide precipitant will, among other things, depend on the nature of the specific endopeptide peptide and its concentration in the concentrated enzyme solution.

[0158] Another alternative to precipitating enzymes is the use of organic compounds. Exemplary organic compound precipitants include: 4-hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid, alkyl esters of 4-hydroxybenzoic acid, and blends of two or more of these organic compounds. The addition of the organic compound precipitant can be performed before, simultaneously with, or after the addition of the metal halide precipitant, and the addition of both precipitants, the organic compound, and the metal halide can be performed sequentially or simultaneously.

[0159] Typically, organic precipitants are selected from the group consisting of alkali metal salts (such as sodium or potassium salts) of 4-hydroxybenzoic acid, and straight-chain or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains 1 to 12 carbon atoms, and blends of two or more of these organic compounds. The organic compound precipitant can be, for example, a straight-chain or branched alkyl ester of 4-hydroxybenzoic acid, wherein the alkyl group contains 1 to 10 carbon atoms, and blends of two or more of these organic compounds. An exemplary organic compound is a straight-chain alkyl ester of 4-hydroxybenzoic acid, wherein the alkyl group contains 1 to 6 carbon atoms, and blends of two or more of these organic compounds. Methyl esters, propyl esters, butyl esters, ethyl esters of 4-hydroxybenzoic acid, and blends of two or more of these organic compounds can also be used. Additional organic compounds include, but are not limited to, methyl 4-hydroxybenzoate (called methylparaben) and propyl 4-hydroxybenzoate (called propylparaben), which are also preservatives. For further description, see, for example, U.S. Patent No. 5,281,526.

[0160] The addition of organic compound precipitants provides the advantage of high flexibility in precipitation conditions regarding pH, temperature, endopeptide concentration, precipitant concentration, and incubation time.

[0161] Organic compound precipitants are used to effectively improve the amount of enzyme precipitation by means of metal halide precipitants. After routine testing, the selection of at least an effective and optimal amount of organic compound precipitant, along with conditions for maximizing precipitate recovery, including incubation time, pH, temperature, and enzyme concentration, will be apparent to those skilled in the art.

[0162] Generally, at least about 0.01% w / v of an organic compound precipitant is added to the concentrated enzyme solution, and typically at least about 0.02% w / v. Generally, no more than about 0.3% w / v of an organic compound precipitant is added to the concentrated enzyme solution, and typically no more than about 0.2% w / v.

[0163] Concentrated peptide solutions containing metal halide precipitants and organic compound precipitants can be adjusted to a specific pH, which will depend on the enzyme to be enriched or purified. Generally, the pH is adjusted to a level close to the isoelectric point of the endopeptide. The pH can be adjusted within a range from approximately 2.5 pH units below the isoelectric point (pI) to approximately 2.5 pH units above the isoelectric point.

[0164] The incubation time required to obtain an enriched or purified enzyme precipitate depends on the nature of the specific enzyme, its concentration, and one or more specific precipitants and their concentrations. Generally, the effective precipitation time for enzymes is between about 1 and 30 hours; typically not exceeding about 25 hours. In the presence of organic compound precipitants, the incubation time can still be reduced to less than about 10 hours, and in most cases even to about 6 hours.

[0165] Typically, the temperature during incubation is between approximately 4°C and approximately 50°C. The method is usually carried out at temperatures between approximately 10°C and approximately 45°C (e.g., between approximately 20°C and approximately 40°C). The optimal temperature for inducing precipitation varies depending on the solution conditions and the enzyme or one or more precipitants used.

[0166] The overall recovery rate of the enriched or purified enzyme precipitate and the efficiency of the process are improved by stirring a solution containing the enzyme, the added metal halide, and the added organic compound. The stirring step is performed during the addition of the metal halide and the organic compound, and during the subsequent incubation. Suitable stirring methods include mechanical stirring or shaking, vigorous aeration, or any similar technique.

[0167] After the incubation period, the enriched or purified enzyme is then separated from the dissociated pigments and other impurities and collected using conventional separation techniques such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, pressure filtration, cross-flow membrane microfiltration, and cross-current membrane microfiltration. Further enrichment or purification of the enzyme precipitate can be achieved by washing the precipitate with water. For example, the enriched or purified enzyme precipitate can be washed with water containing a metal halide precipitant, or with water containing both metal halides and organic compounds as precipitants.

[0168] During fermentation, endopeptide peptides accumulate in the culture medium. To isolate, enrich, or purify the desired endopeptide, the culture medium is centrifuged or filtered to remove cells, and the resulting cell-free liquid is used for enzyme enrichment or purification. In one embodiment, the cell-free culture medium is salted out using ammonium sulfate to approximately 70% saturation; the 70% saturated precipitate fraction is then dissolved in buffer and applied to a column (such as a Sephadex G-100 column), and eluted to recover the enzyme activity fraction. For further enrichment or purification, conventional procedures, such as ion-exchange chromatography, can be used. The enriched or purified enzyme can be prepared as a liquid (solution, slurry) or solid (granules, powder) final product.

[0169] Detailed description of preferred embodiments

[0170] According to one aspect of the invention, a method for reducing or preventing turbidity in a beverage is provided, comprising the step of adding an endopeptidase to the beverage, wherein the endopeptidase is an enzyme or an endopeptidase fragment thereof having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:16, such as a mature protein.

[0171] Preferably, the endopeptide active fragment is a mature protein. Preferably, the beverage contains protein. Preferably, the beverage contains polyphenols. Preferably, the beverage is beer. Preferably, the beverage is wine. Preferably, the beverage is fruit juice.

[0172] Preferably, the endopeptide is added to the wort. Preferably, the endopeptide is added to the beer after turbidity has formed. Preferably, the endopeptide is added to the beer before turbidity has formed.

[0173] Preferably, the beer exhibits increased relative foam stability and increased relative turbidity reduction. Preferably, the increased relative foam stability is greater than 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 110%. Preferably, as measured by 90° scattering, the increased relative turbidity reduction is greater than 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%.

[0174] Preferably, the amount of endopeptidase in the wort is 4 to 300 mg protease / hL wort, preferably 10 to 250 mg protease / hL wort, preferably 20 to 200 mg protease / hL wort, preferably 30 to 150 mg protease / hL wort, or preferably 40 to 100 mg protease / hL wort.

[0175] Preferably, the method includes an additional step of adding one or more of ALDC enzyme, glucosylamylase, maltose α-amylase, amylopectinase, catalase, or transglucosidase.

[0176] Preferably, the ALDC enzyme is acetolactate decarboxylase as described in EC 4.1.1.5. Preferably, the glucosylamylase is 1,4-α-glucosidase as described in EC 3.2.1.3. Preferably, the maltose α-amylase is dextran 1,4-α-maltose hydrolase as described in EC 3.3.1.133. Preferably, the amylopectin enzyme is α-dextrin endoglucosidase, restriction dextrinase, amylopectin 6-glucan hydrolase, or debranching enzyme as described in EC 3.2.1.41. Preferably, the transglucosidase is 1,4-α-glucan-branching enzyme or oligoglucan-branched glucosyltransferase as described in EC 2.4.1.24.

[0177] In another aspect of the invention, a method for increasing the relative foam stability in beer is presented, the method comprising the step of adding an endopeptidase to the beverage, wherein the endopeptidase is an enzyme or an endopeptidase fragment thereof having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:16, such as a mature protein.

[0178] Preferably, the increased relative foam stability is higher than 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 110%. Preferably, the endopeptide active fragment is a mature protein.

[0179] Preferably, the endopeptide is added to the wort. Preferably, the endopeptide is added to the beer after turbidity has formed. Preferably, the endopeptide is added to the beer before turbidity has formed.

[0180] Preferably, the amount of endopeptidase in the wort is 4 to 300 mg protease / hL wort, preferably 10 to 250 mg protease / hL wort, preferably 20 to 200 mg protease / hL wort, preferably 30 to 150 mg protease / hL wort, or preferably 40 to 100 mg protease / hL wort.

[0181] Preferably, the method includes an additional step of adding one or more of ALDC enzyme, glucosylamylase, maltose α-amylase, amylopectinase, catalase, or transglucosidase.

[0182] Preferably, the ALDC enzyme is acetolactate decarboxylase as described in EC 4.1.1.5. Preferably, the glucosylamylase is 1,4-α-glucosidase as described in EC 3.2.1.3. Preferably, the maltose α-amylase is dextran 1,4-α-maltose hydrolase as described in EC 3.3.1.133. Preferably, the amylopectin enzyme is α-dextrin endoglucosidase, restriction dextrinase, amylopectin 6-glucan hydrolase, or debranching enzyme as described in EC 3.2.1.41. Preferably, the transglucosidase is 1,4-α-glucan-branching enzyme or oligoglucan-branched glucosyltransferase as described in EC 2.4.1.24.

[0183] Preferably, the beer exhibits increased relative foam stability and increased relative turbidity reduction. Preferably, as measured by 90° scattering, the increased relative turbidity reduction is greater than 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%.

[0184] In another aspect of the invention, isolated polypeptides comprising endopeptides are presented, wherein the endopeptide is an enzyme or an endopeptide active fragment thereof having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:16.

[0185] In another aspect of the invention, isolated polynucleotides are presented having nucleic acid sequences encoding the aforementioned polypeptides.

[0186] In another aspect of the invention, a nucleic acid construct is presented having the aforementioned polynucleotide operably linked to one or more control sequences that direct the production of the polypeptide in a suitable expression host.

[0187] In another aspect of the present invention, a recombinant expression vector having the above-described nucleic acid construct is presented.

[0188] In another aspect of the invention, recombinant host cells having the above-described nucleic acid constructs or vectors are presented.

[0189] In another aspect of the invention, a method for producing the aforementioned polypeptide is presented, the method comprising the steps of: culturing the aforementioned recombinant host cells to produce a supernatant and / or cells containing the polypeptide; and recovering the polypeptide.

[0190] In another aspect of the present invention, a polypeptide produced by the above method is presented.

[0191] In another aspect of the invention, the use of the filtrate obtained from the fermentation broth obtained by the above method in preventing or reducing turbidity in beverages is presented.

[0192] In another aspect of the invention, compositions having the polypeptide, isolated polynucleotide, nucleic acid construct, recombinant expression vector, or recombinant host cell as described above are presented.

[0193] In another aspect of the invention, the use of the polypeptides, isolated polynucleotides, nucleic acid constructs, recombinant expression vectors, or recombinant host cells described above for increasing the relative foam stability in beverages is presented.

[0194] In another aspect of the invention, the use of the polypeptides, isolated polynucleotides, nucleic acid constructs, recombinant expression vectors, or recombinant host cells described above for increasing the relative reduction of turbidity in beverages is presented.

[0195] In another aspect of the invention, a method for reducing turbidity in a beverage is presented, the method comprising the steps of: adding an endopeptidase to the beverage, wherein the beverage contains a protein or peptide having glutamine residues, the glutamine residues being cleaved by the protease thereby reducing turbidity. Preferably, the endopeptidase also cleaves proline residues. Preferably, the beverage is fruit juice, wine, or beer. More preferably, the beverage is beer.

[0196] Preferably, the glutamine endopeptide comprises a polypeptide or its endopeptide active fragment having at least 80%, 85%, 90%, 95%, 98%, or 99% homology to SEQ ID NO:2 or SEQ ID NO:16, such as a mature protein lacking a signal sequence. More preferably, the glutamine endopeptide comprises the polypeptide according to SEQ ID NO:16.

[0197] Example

[0198] This disclosure is further described in detail in the following examples, which are not intended to limit the scope of the protection claimed in any way. The accompanying drawings are intended to be considered an integral part of the specification and description of this disclosure. The following examples are provided to illustrate, but not to limit, the content of the claimed disclosure.

[0199] Example 1

[0200] As an example of a commercial sample of proline-specific endonuclease from Aspergillus niger (AnPro), Brewers Clarex™ (5 PPU / g product) from DSM was used. The activity of the proline-specific endonuclease (PEP) was determined based on the hydrolysis of the synthetic peptide Z-Gly-Pro-pNA at 37°C in citrate / disodium phosphate buffer (pH 4.6). The reaction products were monitored spectrophotometrically at 405 nm, and one unit (1 PPU) was defined as the amount of enzyme releasing 1 mmol of p-nitroaniline per minute under these test conditions.

[0201] Example 2

[0202] Cloning of Aspergillus homologus CBS 101889 protease AhoPro3 (CRC21077-WT)

[0203] Aspergillus homomorphus CBS 101889 was selected as a potential source of enzymes for various industrial applications. One of the genes identified in Aspergillus homomorphus CBS 101889 (named AhoPro3 (CRC21077-WT)) encodes a protein homologous to a protease identified from a BLAST search (Altschul et al., J Mol Biol, 215: 403–410, 1990). The protein encoded by the AhoPro3 gene is shown in SEQ ID NO:4 (NCBI reference sequence: XP_025547970.1). At the N-terminus, the protein has a signal peptide of 22 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen et al. (2011) Nature Methods, 8:785-786). The presence of the signal sequence indicates that AhoPro3 is a secreted enzyme.

[0204] Example 3

[0205] Cloning of Aspergillus transarticularis CBS 130015 protease AtrPro1 (CRC21068-WT)

[0206] Aspergillus transmontanensis CBS 130015 was selected as a potential source of enzymes for various industrial applications. One of the genes identified in Aspergillus transmontanensis CBS 130015 (named AtrPro1 (CRC21068-WT)) encodes a protein homologous to a protease identified from a BLAST search (Altschul et al., J Mol Biol, 215: 403–410, 1990). The protein encoded by the AtrPro1 gene is shown in SEQ ID NO:3 (JGI reference sequence: Asptra1_564990). At the N-terminus, the protein has a signal peptide of 22 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen et al. (2011) Nature Methods, 8:785-786). The presence of the signal sequence indicates that AtrPro1 is a secreted enzyme.

[0207] Example 4

[0208] Cloning of Aspergillus brasiliensis IBT 29228 protease AbePro2 (CRC21079-WT)

[0209] Aspergillus bertholletius IBT 29228 was selected as a potential source of enzymes for various industrial applications. One of the genes identified in Aspergillus bertholletius IBT 29228 (named AbePro2 (CRC21079-WT)) encodes a protein homologous to a protease identified from a BLAST search (Altschul et al., J Mol Biol, 215: 403–410, 1990). The protein encoded by the AbePro2 gene is shown in SEQ ID NO:1 (JGI reference sequence: Aspber1_278881). At the N-terminus, the protein has a signal peptide of 22 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen et al. (2011) Nature Methods, 8:785-786). The presence of the signal sequence indicates that AbePro2 is a secreted enzyme.

[0210] Example 5

[0211] Cloning of the Aspergillus niger ATCC 1015 protease AniPro_2 (CRC02753-WT)

[0212] Aspergillus niger ATCC 1015 was selected as a potential source of enzymes for various industrial applications. One of the genes identified in Aspergillus niger ATCC 1015 (named AniPro_2 (CRC02753-WT)) encodes a protein homologous to a protease identified from a BLAST search (Altschul et al., J Mol Biol [Journal of Molecular Biology], 215: 403–410, 1990). The protein encoded by the AniPro_2 gene is shown in SEQ ID NO:2 (JGI reference sequence: Aspni5_52703). At the N-terminus, the protease protein has a signal peptide of 21 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen et al. (2011) Nature Methods [Nature Methods] [Nature Methods], 8:785-786) and is derived from the aspartic protease of *Carya rubrum* (uniport id. G0R8T0). The presence of the signal sequence indicates that AniPro_2 is a secreted enzyme.

[0213] Example 6

[0214] Expression, fermentation, purification, and identification of AhoPro3, AtrPro1, AbePro2, and AniPro_2

[0215] DNA sequences encoding AhoPro3, AtrPro1, AbePro2, or AniPro_2 were chemically synthesized and inserted into the *Trichoderma reesei* expression vector pGXT (identical to the pTTTpyr2 vector described in published PCT application WO 2015 / 017256, which is incorporated herein by reference) from Generay Bio-Medical Electronics Co., Ltd. (Shanghai, China). The resulting plasmids were labeled as pGXT-AhoPro3, pGXT-AtrPro1, pGXT-AbePro2, or pGXT-AniPro2, respectively.

[0216] Each individual expression plasmid was then transformed into a suitable *Trichoderma reesei* strain using protoplast transformation (Te'o et al. (2002) J. Microbiol. Methods [Microbial Methods Journal] 51:393-99) (described in published PCT application WO 05 / 001036). Transformants were selected on a medium containing acetamide (as the sole nitrogen source). After 5 days of growth on acetamide plates, the transformants were collected and fermented via DASGIP (Eppendorf, Jülich, Germany).

[0217] To initiate fermentation of AhoPro3, AtrPro1, AbePro2, or AniPro_2, seed cultures were grown in 1 L shake flasks, each containing 100 mL of defined medium (pH 5.5 before sterilization). This medium consisted of 50 g / L glucose monohydrate, 6 g / L glycine, 5 g / L (NH4)2SO4, 4.5 g / L KH2PO4, 1 g / L CaCl2·2H2O, 1 g / L MgSO4·7H2O, 2 g / L Mazu 6000K, and 2.5 mL of 400× Trichoderma reesei trace metals (400× trace metal stock solution (approximately pH 1) containing 175 g / L C6H8O7·H2O, 200 g / L FeSO4·7H2O, 16 g / L ZnSO4·7H2O, 3.2 g / L CuSO4·5H2O, and 1.4 g / L...). The solution consisted of MnSO4·H2O and 0.8 g / L H3BO3. The seed culture was shaken at 250 rpm and 30°C for 48 hours. After incubation, 200 mL of the seed culture was transferred to a 2 L bioreactor (DASGIP).

[0218] The fermentation medium in the 2 L bioreactor (DASGIP) contained 60 g / L dextrose, 6 g / L glycine, 1 g / L CaCl2·2H2O, 4.5 g / L KH2PO4, 4 g / L (NH4)2SO4, 1 g / L MgSO4·7H2O, 1.2 g / L Mazu 6000K, and 2.5 ml of 400× Trichoderma reesei trace metals. An induction solution containing 250 g glucose / sophorose per kg was prepared and sterilized.

[0219] After inoculation, batch fermentation was initiated with a working volume of 1 L, maintained at pH 3.5 and 34°C. Dissolved oxygen levels were maintained above 35% throughout the fermentation process by adjusting airflow rate, oxygen supply, and agitation. After 22 hours of fermentation, the glucose in the fermentation broth was depleted, at which point a feed of 250 g (glucose / sophorose) / kg solution was introduced. Gradual feed rates of 4 mL / h and 6 mL / h were applied at intervals of 22–46 hours and 46–72 hours, respectively. With the start of the fed-batch phase, the pH was linearly adjusted to 4.0, and the temperature was adjusted to 28°C. Fermentation was completed after 72 hours of operation. The fermentation broth was harvested by centrifugation, filtered, and subsequently concentrated.

[0220] To purify AhoPro3, the crude product from a 1 L Dasgip fermenter was concentrated using a VivaFlow 200 ultrafiltration system (Sartorius Steyr) and ammonium sulfate was added to a final concentration of 1 M. The solution was then loaded into HiPrep pre-equilibrated with 20 mM NaAc (pH 5.0). TM The target protein was eluted from the column using a Phenyl FF column supplemented with 1 M ammonium sulfate. The resulting active protein fractions were then pooled, concentrated using a 10K Amicon Ultra device, and stored at -20°C in 40% glycerol until use.

[0221] To purify AtrPro1 and AbePro2, the crude product from a 1 L Dasgip fermenter was concentrated and ammonium sulfate was added to a final concentration of 1 M. The solution was then loaded into HiPrep. TM On a Phenyl FF 16 / 10 column, the column was pre-equilibrated with 20 mM NaAc (pH 5.0) supplemented with an additional 1 M ammonium sulfate. The target protein was eluted from the column with 0.5 M ammonium sulfate. The resulting active protein fractions were then pooled, concentrated, and buffer-exchanged to 20 mM NaAc (pH 5.0), 150 mM NaCl via a 10K Amicon Ultra device, and stored at -20°C in 40% glycerol until use.

[0222] To purify AniPro_2, the crude product from a 1 L Dasgip fermenter was concentrated, and ammonium sulfate was added to a final concentration of 1 M. The solution was then loaded into HiPrep pre-equilibrated with 20 mM NaAc (pH 5.0). TM A Phenyl FF 16 / 10 column was used, supplemented with 1 M ammonium sulfate (Buffer A). The target protein was eluted from the column with 0.75 M ammonium sulfate. The fractions were combined, concentrated, and the buffer was exchanged for 20 mM NaPi (pH 7.0) using a VivaFlow 200 ultrafiltration system (Sartorius Steady Systems) (Buffer B). The resulting solution was applied to a HiLoad filter pre-equilibrated with Buffer B. TM Q FF 16 / 10 column. Elute the target protein from the column with 0.3 M NaCl. Combine the fractions containing the active protein, concentrate them, and exchange the buffer for 20 mM NaAc (pH 5.0) and 150 mM NaCl via a 10K Amicon Ultra device, and store in 40% glycerol at -20°C until use.

[0223] Surprisingly, AniPro_2 was found to be proteolytically modified in the final fermentation broth at the end of fermentation, compared to the precursor variant shown in SEQ ID NO:2 and the predicted mature form of this sequence. The generated variant was verified using mass spectrometry in detail below. Therefore, compared to the 21-amino acid cleavage predicted by SignalP version 4.0, the final expressed variant was further processed at the N-terminus, resulting in a final N-terminal truncation of 36 amino acids, corresponding to the mature version of AniPro_2 with the polypeptide sequence of SEQ ID NO:16. Figure 23 As shown. This mature variant of AniPro_2 has been used in all the instances discussed in this article.

[0224] To obtain the precise peptide sequence of the mature variant of AniPro_2, protein bands were excised from SDS-PAGE gels and digested using three different enzymes (trypsin, A-chymotrypsin, and Glu-C) to prepare samples for mass spectrometry analysis. Trypsin specifically hydrolyzes the peptide bonds on the carboxyl side of arginine I and lysine (K) residues, except when proline (P) is located on the carboxyl side. A-chymotrypsin specifically hydrolyzes the peptide bonds on the carboxyl side of tyrosine (Y), phenylalanine (F), tryptophan (W), and leucine (L), except when proline (P) is located on the carboxyl side. In ammonium bicarbonate buffer at pH 8, Glu-C preferentially cleaves on the carboxyl side of glutamyl (E), but if hydrolyzed in phosphate buffer at pH 8, it will also cleave on the carboxyl side of aspartic (D).

[0225] To accurately detect the C-terminus, protein characterization was performed using the IFF procedure (A2963) to prepare the target protein for analysis, with one modification being the use of 40% of the digestion buffer. 18 O-water. Protein hydrolysis and cleavage will therefore... 18 O-water and 16 O-water merges into the resulting peptide, thus appearing as a double peak in the MS spectrum. However, the C-terminus of the protein will only show a peak with a... 16 A single peptide containing O-water appears because it has not been cleaved, but is simply the "last peptide" left behind by the protein. In this way, MS / MS analysis is used to map the C-terminus. To detect the precise N-terminus of the protein, the N-terminus of the intact protein is acetylated prior to proteolytic digestion (IFF A manual 3448). Guanidinization of lysine converts lysine to homoarginine and protects lysine (side chain) from acetylation. Only peptides originating from the N-terminus of the protein are acetylated and thus definitively identified.

[0226] Example 7

[0227] Proteolytic activity of AhoPro3, AtrPro1, AbePro2, and AniPro_2

[0228] The proteolytic activities of purified AhoPro3, AtrPro1, AbePro2, or AniPro_2 were measured in 25 mM citrate / phosphate buffer (pH 5) using Ala-Ala-Ala-Pro-p-nitroaniline (AAAP-pNA) (synthesized by GL Biochem, Shanghai, China) as substrate. Prior to the reaction, the enzymes were diluted with water to specific concentrations. The AAAP-pNA substrate was dissolved in 100% dimethyl sulfoxide (DMSO) to a final concentration of 10 mM. To initiate the reaction, 5 μL of substrate was mixed with 85 μL of citrate / phosphate buffer in a non-bound 96-well microtiter plate (96-MTP) (Corning Life Sciences, #3641) and pre-incubated at 37°C for 5 min in an Eppendorf mixer. Then, 10 μL of appropriately diluted purified enzyme (or water as a blank) was added. After sealing with 96-MTP, the reaction was carried out in a thermostat at 37°C and 650 rpm for 10 min, and the absorbance of the resulting solution was measured at 405 nm using a SpectraMax 190 (A). 405 ). By from enzyme A 405 Subtract blank control A from the middle 405 To calculate net A 405 And then plotted a graph relative to different protein concentrations. Each value is the average of two repeated measurements. Proteolytic activity is shown as net A. 405 Proteolytic assays using AAAP-pNA as a substrate showed that AhoPro3, AtrPro1, AbePro2, and AniPro2 are all active proteases.

[0229] Example 8

[0230] pH curves of AhoPro3, AtrPro1, AbePro2 and AniPro_2

[0231] Using AAAP-pNA as a substrate, pH profiles of AhoPro3, AtrPro1, AbePro2, or AniPro_2 were investigated in 25 mM NaAc / glycine / HEPES buffer (pH range 3 to 10). To begin the assay, 85 μl of NaAc / glycine / HEPES buffer at a specific pH was first mixed with 5 μl of 10 mM AAAP-pNA in 96-MTP and pre-incubated at 37°C for 5 min. Then, 10 μl of water (25 ppm for AhoPro3 and AtrPro1; 100 ppm for AbePro2 and AniPro_2) or water (blank control) was added to dilute the enzyme. The reaction was performed and analyzed as described in Example 7. Enzyme activity at each pH is reported as relative activity, with the optimal pH activity being 100%. The pH values ​​tested were 3, 4, 5, 6, 7, 8, 9, and 10. Each value is the average of three replicate assays. AhoPro3, AtrPro1, AbePro2, and AniPro_2 were identified as acidic proteases.

[0232] Example 9

[0233] Temperature profiles of AhoPro3, AtrPro1, AbePro2, and AniPro_2

[0234] Using AAAP-pNA as a substrate, temperature profiles for AhoPro3, AtrPro1, AbePro2, or AniPro_2 were analyzed in 25 mM citrate / phosphate buffer (pH 5). Enzyme samples and AAAP-pNA substrates were prepared as in Example 7. Before the reaction, 85 μl of citrate / phosphate buffer and 5 μl of 10 mM AAAP-pNA were mixed in a 200 μl PCR tube and incubated for 5 min in a Peltier thermal cycler (BioRad) at the desired temperature (30°C to 80°C). After incubation, 10 μl of diluted enzyme (25 ppm for AhoPro3 and AtrPro1; 100 ppm for AbePro2 and AniPro_2) or water (blank control) was added to the solution, and the reaction was performed in a Peltier thermal cycler for 10 min at different temperatures. Subsequent absorbance measurements were performed as in Example 7. Reported activities are relative activities, with the activity at the optimal temperature set to 100%. Each value is the average of three repeated measurements. The data indicate that the optimal temperatures for AhoPro3, AtrPro1, AbePro2, and AniPro_2 are 60°C, 57°C, 53°C, and 64°C, respectively.

[0235] Example 10

[0236] Thermal stability of AhoPro3, AtrPro1, AbePro2, and AniPro_2

[0237] Thermal stability analysis of AhoPro3, AtrPro1, AbePro2, or AniPro_2 was performed using 50 mM acetate / phosphate buffer (pH 4.5) as the incubation buffer and AAAP-pNA as the substrate for residual activity measurement. Purified AhoPro3, AtrPro1, AbePro2, or AniPro_2 (or purified AnPro (Brewer's Clarex)) were used. TM The enzyme-buffer mixture (using the reference value) was diluted to a final concentration of 1 mg / mL in 1 mL of incubation buffer and then incubated at 65°C for 0, 10, 20, 30, 45, or 60 min. At the end of each incubation period, 100 μL of the enzyme-buffer mixture was transferred to 96-MTP and placed on ice. After the entire incubation was completed, the enzyme-buffer mixture was further diluted with buffer to achieve the specific enzyme concentration for downstream activity assays (25 ppm for AhoPro3 and AtrPro1; 100 ppm for AbePro2 and AniPro_2). Activity was measured as in Example 7. The reported activities are relative activities, where the activity at 0 min incubation time was set to 100%; and each value is the average of three replicate assays. As shown in Table 2, AhoPro3, AtrPro1, and AbePro2 completely lost activity after 10 min incubation at 65°C, while AniPro_2 retained approximately 64% activity after 1 hr incubation.

[0238] Table 2 Thermal stability of AhoPro3, AtrPro1, AbePro2, or AniPro_2 at 65°C

[0239]

[0240] Example 11

[0241] Turbidity reduction performance of AhoPro3, AtrPro1, AbePro2, and AniPro_2

[0242] The turbidity reduction performance of AhoPro3, AtrPro1, AbePro2, or AniPro_2 was calculated using a gliadin-catechin assay. Before the reaction, the enzymes were diluted with water to specific concentrations. Gliadin substrate (Sigma, catalog number G3375) was dissolved in 20 mM acetate / phosphate buffer (pH 4.5), supplemented with an additional 0.2% ethanol to a final concentration of 2 mg / mL, and catechin substrate (Sigma, catalog number C1251) was dissolved in 20 mM citrate / phosphate buffer (pH 4.5), supplemented with an additional 0.2% ethanol to a final concentration of 2 mg / mL. To begin the assay, 100 μL of gliadin solution was mixed with 5 μL of appropriately diluted AhoPro3, AtrPro1, AbePro2, or AniPro_2 (or purified AnPro (Brewer's Clarex)) in 96-MTP. TM (Using 96-MTP as a reference) was mixed; after incubation at 45°C for 90 min in a thermostat, the resulting 96-MTP was placed on ice for 5 min, followed by the addition of 100 μl of catechin solution. Turbidity formed at room temperature for 30 min. The absorbance (A) of the turbidity formed at 600 nm was measured using a SpectraMax 190. 600 Then, graphs were plotted for different enzyme concentrations. Each value is the average of three replicate measurements. For example... Figure 5 , 6 As shown in Figures 7 and 8, AhoPro3, AtrPro1, AbePro2, or AniPro_2 are more effective in reducing gliadin-catechin turbidity compared to the baseline.

[0243] Example 12

[0244] Protein assay methods

[0245] Protein determination using a standard stain-free imager criterion.

[0246] Protein quantification was performed using an SDS-PAGE gel and densitometric assay with a Gel Doc™ EZ imaging system. Reagents used in the assay included: concentrated (2x) Laemmli sample buffer (Bio-Rad, catalog 161-0737); 26-well XT 4-12% Bis-Tris gel (Bio-Rad, catalog 345-0125); protein marker "Precision Plus Protein Standard" (Bio-Rad, catalog 161-0363); protein standard BSA (Thermo Scientific, catalog 23208); and SimplyBlue Safestain (Ingenium, catalog LC 6060). The assay was performed as follows: 50 µL of diluted enzyme sample was mixed with 50 µL of sample buffer containing 2.7 mg DTT in a 96-well PCR plate. The plate was sealed with a Microseal 'B' membrane from Bio-Rad Laboratories and heated to 70°C for 10 minutes in a PCR machine. Afterward, the chamber was filled with run buffer to set the gel cassette. Then, 10 μL of each sample and standard (0.125–1.00 mg / mL BSA) were loaded onto the gel, along with 5 μL of marker. Electrophoresis was then run at 200 V for 45 min. After electrophoresis, the gel was washed three times in water for 5 min each time, stained overnight in Safe-stain, and finally destained in water. The gel was then transferred to an imager. The intensity of each band was calculated using Image Lab software. A calibration curve was constructed using BSA (Thermo Fisher Scientific, catalog number 23208), and the amount of target protein was determined by the band intensity and the calibration curve. Enzyme samples for subsequent examples were prepared using protein quantification methods. Protein concentrations of proteases: 8.1 mg / ml for purified AhoPro3, 2.3 mg / ml for purified AbePro2, 8.2 mg / ml for purified AtrPro1, 2.4 mg / ml for purified AniPro2, and 41 mg / ml for AnPro.

[0247] Example 13

[0248] The turbidity reduction performance of proteases from Aspergillus homologa in turbidity-sensitive beer.

[0249] Cloudy sensitive beer substrate

[0250] To test the performance of the protease, bottled beer was used as the substrate. Beer brewed at the 2 hL pilot brewery was filtered (without silica or PVPP), but the unstabilized all-malt Pilsner had approximately 66% RDF and an alcohol content of 4.7% (v / v). As described in Table 3, the beer was filtered using an 8-plate diatomaceous earth filter with a pre-coated and main feed. After diatomaceous earth filtration, the beer passed first through a 1.2 µm membrane filter and then through a 0.45 µm membrane filter.

[0251] Table 3 Preparation of diatomaceous earth filtration with a flow rate of 160 l / hr.

[0252]

[0253] Incubation and pasteurization

[0254] Enzymes were applied to bottled beer by opening capsules, adding enzyme solution, and immediately resealing the bottles with new capsules. Control beer samples were prepared similarly, with an equal volume of milliQ water (ddH2O) added to the enzyme solution. Enzymes were applied at low and high doses based on ppm protease, as shown in Table 4 below.

[0255] Table 4. Addition of protease in turbidity-sensitive beer.

[0256]

[0257] Beer samples were stored at 14°C for 5 days to allow the enzymes to activate. Subsequently, the samples were pasteurized in a water bath to approximately 30 PU by heating to 63°C (60 minutes) and maintaining the temperature at 63°C for 60 minutes, then the heat source was turned off and the temperature was lowered to room temperature (approximately 20°C).

[0258] Assessment of the likelihood of turbidity in beer samples

[0259] The prediction of turbidity development in beer samples was assessed using a forced turbidity method based on EBC Analytica method 9.30 "Prediction of beer shelf life," hereinafter referred to as the EBC TOHA forced turbidity method. Instrument calibration was performed according to the supplier's instructions, and turbidity measurements are expressed in EBC units.

[0260] Forced Turbidity EBC TOHA Method

[0261] Turbidity in beer was measured using a Sigris Labscat2. Turbidity was measured at a 90° scattering angle (S90 / S0 EBC) to detect the presence of small particles, and turbidity measured at a 25° scattering angle was used as additional information about larger particles. Turbidity was measured at 20°C prior to alternating cooling and heating cycles on the bottled sample; this measurement is called the blind value.

[0262] The sample was then placed in a constant temperature water bath (Julabo, Germany) and the temperature was lowered to 0°C and maintained for 24 hours. The turbidity was measured at 0°C and is referred to as the initial total turbidity.

[0263] Beer samples were placed in a constant-temperature water bath and kept at 60°C for 48 hours, then cooled to 0°C and kept there for 24 hours. Turbidity was measured at 0°C and termed final total turbidity. The results are shown in Table 5.

[0264] The measurements of initial and final total turbidity clearly show that both endopeptides significantly reduced beer turbidity compared to a reference without proteases. It is equally clear that low and high doses of AhoPro3, compared to AnPro, resulted in a surprisingly significant increase in final total turbidity. This was observed in turbidity measurements taken at 90° and 25° scattering (small and large particles).

[0265] Table 5. Turbidity of beer with and without added protease (EBC 90° and 25°). Forced turbidity was measured according to the EBC TOHA method, and blind values, initial total turbidity, and final total turbidity are shown. Standard deviation was determined from two measurements.

[0266]

[0267] Example 14

[0268] Turbidity formation in beer produced by various endopeptides during fermentation

[0269] In a 2 hL semi-industrial pilot brewery, the same mashing protocol was used to produce pure wort. Pilsner malt (Fuglsang, Denmark, batch 21.08.2020) was used and milled prior to mashing under standard settings, with an initial water-to-malt ratio of 2.8:1. The CaCl2 content in the water was adjusted to 40 ppm with 90% lactic acid and the pH was adjusted to 5.5. LAMINEX® MaxFlow 4G (DuPont Nutrition Bioscience, Denmark) was added at a dosage of 0.10 kg / t malt. The following mashing protocol was applied: mash at 63°C for 25 minutes; maintain at 63°C for 45 minutes; increase to 72°C for 9 minutes (1°C / min); maintain at 72°C for 20 minutes; during mashing, increase the temperature to 78°C for 6 minutes (1°C / min) and maintain at 78°C for 10 minutes. An iodine-negative test was performed at 72°C to ensure iodine negativity. Water was added during the transfer of the malt mash from the mashing pot to the filtering tank to maintain a final water-to-maltose ratio of 3.2:1.

[0270] The conditions for the added water, the volume collected, and the flow rate of the HGB used during filtration are shown in Table 6 below.

[0271] Table 6. Filtering conditions.

[0272]

[0273] Wort boiling: The wort is boiled for 80 minutes, evaporating 10% per hour to reach 16.0°P. The final brewed beer contains 20 BU of hops, consisting of 50% CO2-extracted hops (St. Johann, Hallertauer, Germany) and 50% standard polyphenol hops (P90 hops) (St. Johann, Hallertauer, Germany). At the end of boiling, the wort pH is adjusted to 5.2 with 90% lactic acid. The wort is then divided into two 100 L fermentation tanks and treated with 0.10 ppm ZnCl2. 2+ Add to vortex. The original extract (OE) of the wort samples after mashing was measured using an Anton Paar (DMA5000) according to DuPont Standard Brewing Instruction 23.8580-B09, and all experiments showed similar results. Furthermore, the content of free α-amino nitrogen (mg / L) in the wort was measured according to DuPont Standard Brewing Instruction 23.8580-B15, and all experiments showed similar results.

[0274] Fermentation: All experiments were conducted at a standard oxygen concentration of 13–16 ppm in the wort. The pH of the pitching wort was adjusted to 5.00 ± 0.05 with acetic acid. Brewer's yeast was used. Pitching rate: 25–30 milled cells / ml. Cold ripening / stabilization and subsequent filtration were performed after fermentation. The primary fermentation temperature was set at 12°C, the ripening temperature at 15°C, and the temperature was cooled to 1°C at the end of fermentation. Enzyme application was based on mg of protease (Pro) per hL, as shown in Table 7 below.

[0275] Table 7. Enzyme addition in fermenters.

[0276]

[0277] Beer filtration: Use 8 plates at a flow rate of 160 l / hr, and maintain with the following amounts of filter aid given in Table 8. Perform yeast cell counting before filtration.

[0278] Table 8. Preparation of diatomaceous earth filtration with a flow rate of 160 l / hr.

[0279]

[0280] The entire volume of filtered beer was bottled into 33 cL bottles, and all bottles were pasteurized at 62°C for 20 minutes (approximately 30 PU). Turbidity of the beer samples was measured using a Sigris Labscat2. Turbidity was measured at a 90° scattering angle (S90 / S0 EBC) to detect the presence of small particles, and turbidity measured at a 25° scattering angle was used as additional information regarding larger particles. Turbidity was measured at 20°C prior to alternating cooling and heating cycles of the bottled samples; this is referred to as the blind value.

[0281] The samples were then placed in a constant-temperature water bath (Julabo, Germany) and the temperature was lowered to 0°C and maintained for 24 hours. Turbidity was measured at 0°C and referred to as initial total turbidity. Beer samples were then placed in a constant-temperature water bath and the temperature was raised to 60°C and maintained for 48 hours, then lowered to 0°C and maintained for 24 hours. Turbidity was measured at 0°C and referred to as final total turbidity. The results are shown in Table 9.

[0282] Table 9. Turbidity of beers with various protease additions (EBC 90° and 25°). Forced turbidity was measured according to the EBC TOHA method, and blind values, initial total turbidity, and final total turbidity are shown. Standard deviation (Std.) was determined from two measurements.

[0283]

[0284]

[0285]

[0286] It is clear from all turbidity measurements that all proteases significantly reduced beer turbidity in both blind values, initial turbidity, and final total turbidity compared to a beer reference without proteases. It is equally clear that the addition of both AhoPro3 and AniPro_2 resulted in a significant reduction in turbidity compared to AnPro (determined by all turbidity values, including final total turbidity). This was observed in turbidity measurements taken at 90° and 25° scattering (small and large particles).

[0287] The relative reduction in turbidity can be calculated for beer without any enzymes as follows: (EBC) 浊度90 / 25°没有酶 -EBC 浊度90 / 25°具有酶 ) / EBC 浊度90 / 25°没有酶 x 100%, and the results for AnPro, AhoPro3, and AniPro_2 are shown in Table 10 below. It was observed that, compared to AnPro and MorPro1, both AhoPro3 and AniPro_2 achieved relative turbidity reductions of over 37% to 67% (measured via 90° EBC) and over 84% to 94% (measured via 25° EBC).

[0288] Table 10. Relative turbidity reduction is calculated based on turbidity without added enzymes (EBC 90° and 25°) and given as a percentage.

[0289]

[0290] Furthermore, following the MEBAK-Analytica (Method 2.14.2.1. Forciermethode, Mitteleuropäische Brautechnische Analysen Methoden) commonly used in the beer industry, bottled beer samples were subjected to accelerated aging by storage at high temperatures. Turbidity (S90 / S0 EBC) was measured at a 90° scattering angle to detect the presence of small particles, and turbidity measured at a 25° scattering angle was used as additional information regarding larger particles (using Sigrist Labscat2). Turbidity, referred to as the blind value, was measured at 20°C prior to alternating cooling and heating cycles of the bottled samples.

[0291] The sample was then placed in a constant-temperature water bath (Julabo, Germany), and the temperature was raised to 40°C and maintained for 24 hours, followed by a drop to 0°C and maintenance for 24 hours. Turbidity was measured at 0°C and termed total turbidity. This cycle was repeated, and Δturbidity was calculated as the turbidity after cycle (n), e.g., (total turbidity) - (blind value).

[0292] The results are shown in Table 11. It is clear from all turbidity measurements that all proteases significantly reduced beer turbidity compared to the beer reference without proteases, through blind values ​​and total turbidity for cycles 1 and 2. Again, it is equally clear that the addition of AhoPro3 or AniPro_2 surprisingly resulted in significantly greater turbidity reductions compared to AnPro (measured by all turbidity values, including the final total turbidity).

[0293] Table 11. Turbidity of beers with various protease additions (EBC 90° and 25°). Forced turbidity was measured according to the MEBAK method, and blind values ​​and total turbidity were calculated from cycles 1 and 2. Standard deviation (Std.) was determined from two measurements.

[0294]

[0295] Similar to the EBC TOHA test, the relative turbidity reduction can be calculated from the MEBAK test for beer without any enzymes (cycle 2 value) as follows: (EBC 浊度90 / 25°没有酶 - EBC 浊度90 / 25°具有酶 ) / EBC 浊度90 / 25°没有酶 x 100%, and the results for AnPro, AhoPro3, and AniPro_2 are shown in Table 12 below. It was observed that, compared to AnPro, both AhoPro3 and AniPro_2 achieved relative turbidity reductions of over 73% (measured via 90° EBC) and over 86% (measured via 90° EBC), respectively.

[0296] Table 12. Relative turbidity reduction is calculated based on turbidity without added enzymes (EBC 90° and 25°) and given as a percentage.

[0297]

[0298] Example 15

[0299] Foam stability of beer produced by endopeptidase during fermentation

[0300] As described in Example 14, beer was produced in a semi-industrial pilot brewery, and proteases AnPro, AniPro_2, and AhoPro3 were added at the start of the beer fermentation process as described in Example 14. Depending on the substrate specificity of a given protease, it may most undesirably degrade various foam-stabilizing proteins during beer production. Therefore, the foam stability of beer produced with proteases AnPro and AhoPro3 was compared with that of similar beer without the addition of any enzymes during fermentation. The foam stability of the beer was determined using a NIBEM-T meter (using a Haffmans Nibem foam stability tester from Pentair) and according to EBC Analytica Method 9.42.1 “Foam stability of beer using a NIBEM-T meter – 2004”.

[0301] Bottled beer is temperature-controlled to 20°C ± 0.5°C in a water bath. The beer is then dispensed via a flash foaming device, where it is forced through an orifice at approximately 2 bar of carbon dioxide pressure into a thoroughly clean standard glass with an inner diameter of 60 ± 1.2 mm and an internal height of 120 ± 1.9 mm. This produces a standard glass of beer / foam.

[0302] Place a standard beer / foam glass under the needle electrode system of the calibrated NIBEM-T meter so that the reference position corresponds to the full rim of the glass.

[0303] The electrode was moved downwards until one of the four external needles contacted the foam surface. As the foam collapsed, the contact was broken, and the electrode system was moved downwards again until one of the four external needles contacted the foam surface once more. Timing was started when the foam collapsed below a reference position, and the time taken to collapse to a further set distance of 30 mm was measured. The collapse time (in seconds) was determined and is shown in Table 13 below. Clearly, the addition of AhoPro3 or AniPro_2 improved beer foam stability compared to AnPro, showing similar stability to the control beer without the enzyme. Surprisingly, the addition of AhoPro3 or AniPro_2 significantly improved foam stability compared to the control beer without the enzyme.

[0304] Table 13. NIBEM foam stability of beers with various protease additions.

[0305]

[0306] Relative foam stability can be calculated for beer without any enzymes from the NIBEM foam test (30 mm) as follows: (collapse time) NIBEM 30mm具有酶 (collapse time) NIBEM 30mm没有酶The values ​​are calculated as follows: x 100%, and the values ​​for AnPro, AhoPro3, and AniPro_2 are shown in Table 14 below. It was observed that, compared to AnPro, both AhoPro3 and AniPro_2 improved foam stability, with foam stability exceeding 99.5%.

[0307] Table 14. Relative foam stability (NIBEM-30 mm) calculated for the absence of enzymes and given as a percentage.

[0308]

[0309] Example 16

[0310] The turbidity reduction performance of proteases from Aspergillus brasiliensis, Aspergillus simonii, and Aspergillus niger in turbidity-sensitive beer.

[0311] As described in Example 13, bottled turbidity-sensitive beer was used to test the performance of the protease. The enzyme was applied to the bottled beer by opening the capsule, adding the enzyme solution, and immediately resealing the bottle with a new capsule. A control beer sample was prepared similarly, with an equal volume of milliQ water (ddH2O) added to the enzyme solution. Enzymes from *Aspergillus brasiliensis* (AbePro2), *Aspergillus transartinidia* (AtrPro1), *Aspergillus niger* (AniPro_2), or AnPro were applied at low and high doses based on ppm protease, as given in Table 15 below.

[0312] Table 15. Enzyme addition in turbidity-sensitive beer.

[0313]

[0314] Beer samples were stored at 14°C for 5 days to allow the enzymes to activate. Thereafter, the samples were pasteurized in a water bath to approximately 30 PU by heating to 63°C (60 minutes) and maintaining that temperature for 60 minutes, followed by turning off the heat source and allowing the temperature to drop to approximately 20°C. The turbidity potential in the beer after enzyme treatment was evaluated according to the EBC TOHA method described in Example 13. The results are shown in Table 16.

[0315] Table 16. Turbidity of beer with and without added protease (EBC 90° and 25°). Forced turbidity was measured according to the EBC TOHA method, and blind values, initial total turbidity, and final total turbidity are shown. Standard deviation (Std.) was determined from two measurements.

[0316]

[0317]

[0318]

[0319] The measurements of initial and final total turbidity clearly show that all whitening enzymes significantly reduced beer turbidity compared to a reference without proteases. Equally clear is that proteases from *Aspergillus brasiliensis*, *Aspergillus transatlansus*, and *Aspergillus niger* (AniPro_2), at high doses, all resulted in a surprisingly significant increase in the reduction of final total turbidity compared to AnPro. This was observed in turbidity measurements taken under 90° scattering (small particles).

[0320] Example 17

[0321] Beer with endopeptide production during fermentation exhibits foam stability throughout its shelf life.

[0322] As described in Example 14, beer was produced in a semi-industrial pilot brewery, and proteases AnPro and AniPro2 were added at the start of the beer fermentation process as described in Example 14, using a dosage of 20.5 mg protease / hL wort. Depending on the substrate specificity of a given protease, proteases can potentially affect various foam-stabilizing proteins during beer production. Therefore, the foam stability of beer produced with proteases AnPro and AniPro2 was compared with that of similar beer without the addition of any enzymes during fermentation. The beer was tested within its shelf life (stored at 12°C–14°C for up to 9 months) and the foam stability was determined using a NIBEM-T meter (using a Haffmans Nibem foam stability tester from Pentair) according to EBC Analytica Method 9.42.1 “Foam Stability of Beer Using NIBEM-T Meter – 2004”. The results are shown in Table 17.

[0323] Clearly, the addition of AniPro_2 improved beer foam stability compared to AnPro, showing similar stability to the control beer without the enzyme. Surprisingly, the foam stability after adding AniPro_2 was significantly improved compared to the control beer without the enzyme, as observed after one month of storage, and even became clearer after nine months of storage.

[0324] Table 17. NIBEM foam stability of beers with various protease additions after 0-9 months (measured at 19°C-22°C).

[0325]

[0326]

[0327] Relative foam stability after 9 months can be calculated for beer without any enzymes based on the NIBEM foam test (30 mm) as follows: (collapse time) NIBEM 30mm具有酶(collapse time) / (collapse time) NIBEM 30mm没有酶 The values ​​were calculated as follows: (x 100%), and the values ​​for AnPro and AniPro_2 are shown in Table 18 below. Compared to AnPro, AniPro_2 was observed to improve foam stability, with a foam stability exceeding 100.0%. Therefore, the addition of AniPro_2 surprisingly increased foam stability.

[0328] Table 18. Relative foam stability (NIBEM-30 mm) calculated for the absence of enzymes and given as a percentage.

[0329]

[0330] Example 18 Cloning of the Aspergillus pseudostem CBS 117616 protease ApsPro1 (CRC21071-WT)

[0331] Aspergillus pseudocaelatus CBS 117616 was selected as a potential source of enzymes for various industrial applications. One of the genes identified in Aspergillus pseudocaelatus CBS 117616 (named ApsPro1 (CRC21071-WT)) encodes a protein homologous to a protease identified from a BLAST search (Altschul et al., J Mol Biol, 215: 403–410, 1990). The protein encoded by the ApsPro1 gene is shown in SEQ ID NO:5 (JGI reference sequence: Asppsec1_294119). At the N-terminus, the protein has a signal peptide of 22 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen et al. (2011) Nature Methods, 8:785-786). The presence of the signal sequence indicates that ApsPro1 is a secreted enzyme.

[0332] Example 19 Cloning of Aspergillus aureus CBS 112787 protease AnePro2 (CRC21072-WT)

[0333] Aspergillus neoauricomus CBS 112787 was selected as a potential source of enzymes for various industrial applications. One of the genes identified in Aspergillus neoauricomus CBS 112787 (named AnePro2 (CRC21072-WT)) encodes a protein homologous to a protease identified from a BLAST search (Altschul et al., J Mol Biol, 215: 403–410, 1990). The protein encoded by the AnePro2 gene is shown in SEQ ID NO:6 (JGI reference sequence: Aspneoa1_131875). At the N-terminus, the protein has a signal peptide of 22 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen et al. (2011) Nature Methods, 8:785-786). The presence of the signal sequence indicates that AnePro2 is a secreted enzyme.

[0334] Example 20 Cloning of Aspergillus Alberti protease AalPro2 (CRC21076-WT)

[0335] Aspergillus albertensis has been selected as a potential source of enzymes for various industrial applications. One of the genes identified in Aspergillus albertensis (named AalPro2 (CRC21076-WT)) encodes a protein homologous to a protease identified from a BLAST search (Altschul et al., J Mol Biol, 215: 403–410, 1990). The protein encoded by the ApsPro1 gene is shown in SEQ ID NO:7 (JGI reference sequence: Aspalbe1_152875). At the N-terminus, the protein has a signal peptide of 19 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen et al. (2011) Nature Methods, 8:785-786). The presence of the signal sequence indicates that AalPro2 is a secreted enzyme.

[0336] Example 21 Cloning of Aspergillus core CBS 553.77 protease AcoPro2 (CRC21078-WT)

[0337] Aspergillus coremiiformis CBS 553.77 was selected as a potential source of enzymes for various industrial applications. One of the genes identified in Aspergillus coremiiformis CBS 553.77 (named AcoPro2 (CRC21078-WT)) encodes a protein homologous to a protease identified from a BLAST search (Altschul et al., J Mol Biol, 215: 403–410, 1990). The protein encoded by the AcoPro2 gene is shown in SEQ ID NO:8 (JGI reference sequence: Aspcor1_141732). At the N-terminus, the protein has a signal peptide of 22 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen et al. (2011) Nature Methods, 8:785-786). The presence of the signal sequence indicates that AcoPro2 is a secreted enzyme.

[0338] Example 23 Cloning of Aspergillus winterii protease AwePro2 (CRC21080-WT)

[0339] Aspergillus wentii has been selected as a potential source of enzymes for various industrial applications. One of the genes identified in Aspergillus wentii (named AwePro2 (CRC21080-WT)) encodes a protein homologous to a protease identified from a BLAST search (Altschul et al., J Mol Biol, 215: 403–410, 1990). The protein encoded by the AwePro2 gene is shown in SEQ ID NO:9 (JGI reference sequence: Aspwe1_188244). At the N-terminus, the protein has a signal peptide of 20 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen et al. (2011) Nature Methods, 8:785-786). The presence of the signal sequence indicates that AwePro2 is a secreted enzyme.

[0340] Example 24 Cloning of Aspergillus niger protease AbrPro1 (CRC21202-WT)

[0341] Aspergillus brasiliensis has been selected as a potential source of enzymes for various industrial applications. One of the genes identified in Aspergillus brasiliensis (named AbrPro1 (CRC21202-WT)) encodes a protein homologous to a protease identified from a BLAST search (Altschul et al., J Mol Biol, 215: 403–410, 1990). The protein encoded by the AbrPro1 gene is shown in SEQ ID NO:10 (JGI reference sequence: Aspbr1_41430). At the N-terminus, the protein has a signal peptide of 22 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen et al. (2011) Nature Methods, 8:785-786). The presence of the signal sequence indicates that AbrPro1 is a secreted enzyme.

[0342] Example 25 Cloning of Aspergillus discus CBS115572 protease AscPro5 (CRC21204-WT)

[0343] Aspergillus sclerotioniger CBS115572 was selected as a potential source of enzymes for various industrial applications. One of the genes identified in Aspergillus sclerotioniger CBS115572 (named AscPro5 (CRC21204-WT)) encodes a protein homologous to a protease identified from a BLAST search (Altschul et al., J Mol Biol, 215: 403–410, 1990). The protein encoded by the AscPro5 gene is shown in SEQ ID NO:11 (JGI reference sequence: Aspscl1_500682). At the N-terminus, the protein has a signal peptide of 22 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen et al. (2011) Nature Methods, 8:785-786). The presence of the signal sequence indicates that AscPro5 is a secreted enzyme.

[0344] Example 26: Expression, fermentation, and purification of ApsPro1, AnePro2, AalPro2, AcoPro2, AwePro2, AbrPro1, and AscPro5

[0345] DNA sequences encoding ApsPro1, AnePro2, AalPro2, AcoPro2, AwePro2, AbrPro1, or AscPro5 were chemically synthesized and inserted into the Trichoderma reesei expression vector pGXT (identical to the pTTTpyr2 vector described in published PCT application WO 2015 / 017256, which is incorporated herein by reference) from Shanghai Jierui Biotechnology Co., Ltd. (Shanghai, China). The resulting plasmids were labeled as pGXT-ApsPro1, pGXT-AnePro2, pGXT-AalPro2, pGXT-AcoPro2, pGXT-AwePro2, pGXT-AbrPro1, or pGXT-AscPro5, respectively.

[0346] Each individual expression plasmid was then transformed into a suitable *Trichoderma reesei* strain using protoplast transformation (Te'o et al. (2002) J. Microbiol. Methods [Microbial Methods Journal] 51:393-99) (described in published PCT application WO 05 / 001036). Transformants were selected on a medium containing acetamide (as the sole nitrogen source). After 5 days of growth on acetamide plates, the transformants were collected and fermented via DASGIP (Eppendorf, Jülich, Germany).

[0347] To initiate fermentation of ApsPro1, AnePro2, AalPro2, AcoPro2, AwePro2, AbrPro1, or AscPro5, seed cultures were grown in 1 L shake flasks, each containing 100 mL of defined medium (pH 5.5 before sterilization). This medium consisted of 50 g / L glucose monohydrate, 6 g / L glycine, 5 g / L (NH4)2SO4, 4.5 g / L KH2PO4, 1 g / L CaCl2·2H2O, 1 g / L MgSO4·7H2O, 2 g / L Mazu 6000K, and 2.5 mL of 400× Trichoderma reesei trace metal stock solution (approximately pH 1) containing 175 g / L C6H8O7·H2O, 200 g / L FeSO4·7H2O, 16 g / L ZnSO4·7H2O, and 3.2 g / L... The incubation solution consisted of CuSO4·5H2O, 1.4 g / L MnSO4·H2O, and 0.8 g / L H3BO3. The seed culture was incubated at 250 rpm and 30°C for 48 hours with shaking. After incubation, 200 mL of the seed culture was transferred to a 2 L bioreactor (DASGIP).

[0348] The fermentation medium in the 2 L bioreactor (DASGIP) contained 60 g / L dextrose, 6 g / L glycine, 1 g / L CaCl2·2H2O, 4.5 g / L KH2PO4, 4 g / L (NH4)2SO4, 1 g / L MgSO4·7H2O, 1.2 g / L Mazu 6000K, and 2.5 ml of 400× Trichoderma reesei trace metals. An induction solution containing 250 g glucose / sophorose per kg was prepared and sterilized.

[0349] After inoculation, batch fermentation was initiated with a working volume of 1 L, maintained at pH 3.5 and 34°C. Dissolved oxygen levels were maintained above 35% throughout the fermentation process by adjusting airflow rate, oxygen supply, and agitation. After 22 hours of fermentation, the glucose in the fermentation broth was depleted, at which point a feed of 250 g (glucose / sophorose) / kg solution was introduced. Gradual feed rates of 4 mL / h and 6 mL / h were applied at intervals of 22–46 hours and 46–72 hours, respectively. With the start of the fed-batch phase, the pH was linearly adjusted to 4.0, and the temperature was adjusted to 28°C. Fermentation was completed after 72 hours of operation. The fermentation broth was harvested by centrifugation, filtered, and subsequently concentrated.

[0350] To purify ApsPro1, AnePro2, AalPro2, AcoPro2, AwePro2, AbrPro1, and AscPro5, the crude products from a 1 L Dasgip fermenter were concentrated, and ammonium sulfate was added to a final concentration of 1 M. The solution was then loaded into HiPrep pre-equilibrated with 20 mM NaAc (pH 5.0). TM A Phenyl FF 16 / 10 column was used, supplemented with an additional 1 M ammonium sulfate. The target protein was eluted from the column with 0.5 M ammonium sulfate. The resulting active protein fractions were then pooled, concentrated, and buffer-exchanged to 20 mM NaAc (pH 5.0) and 150 mM NaCl via a 10K Amicon Ultra device, and stored at -20°C in 40% glycerol until use.

[0351] Example 27

[0352] The turbidity reduction performance of ApsPro1, AnePro2, AalPro2, AcoPro2, AwePro2, AbrPro1 and AscPro5

[0353] The turbidity reduction performance of ApsPro1, AnePro2, AalPro2, AcoPro2, AwePro2, AbrPro1, and AscPro5 was calculated using a gliadin-catechin assay. Prior to the reaction, the enzymes were diluted with water to specific concentrations. Gliadin substrate (Sigma, catalog number G3375) was dissolved in 20 mM acetate / phosphate buffer (pH 4.5), supplemented with an additional 0.2% ethanol to a final concentration of 2 mg / mL. Catechin substrate (Sigma, catalog number C1251) was dissolved in 20 mM citrate / phosphate buffer (pH 4.5), supplemented with an additional 0.2% ethanol to a final concentration of 2 mg / mL. To begin the assay, mix 100 μL of gliadin solution with 5 μL of appropriately diluted ApsPro1, AnePro2, AalPro2, AcoPro2, AwePro2, AbrPro1, and AscPro5 (or purified AnPro (Brewer's Clarex)) in 96-MTP. TM (Using 96-MTP as a reference) was mixed; after incubation at 45°C for 90 min in a thermostat, the resulting 96-MTP was placed on ice for 5 min, followed by the addition of 100 μl of catechin solution. Turbidity formed at room temperature for 30 min. The absorbance (A) of the turbidity formed at 600 nm was measured using a SpectraMax 190. 600 Then, graphs were plotted for different enzyme concentrations. Each value is the average of three replicate measurements. For example... Figure 15 As shown, when compared with the baseline (AnPro), ApsPro1, AnePro2, AalPro2, AcoPro2 and AwePro2 are all more effective in reducing gliadin-catechin turbidity.

[0354] Example 28

[0355] Proteolytic activity of AhoPro3, ApsPro1, AnePro2, AalPro2, AcoPro2, AwePro2, AbrPro1, AscPro5 and AnPro

[0356] Using Ala-Ala-Ala-Pro-p-nitroaniline (AAAP-pNA) (synthesized by GL Biochem, Shanghai, China) as a substrate, the proteolytic activities of purified AhoPro3, ApsPro1, AnePro2, AalPro2, AcoPro2, AwePro2, AbrPro1, AscPro5, and AnPro were measured in 25 mM citrate / phosphate buffer (pH 5). Prior to the reaction, the enzymes were diluted with water to specific concentrations. The AAAP-pNA substrate was dissolved in 100% dimethyl sulfoxide (DMSO) to a final concentration of 10 mM. To initiate the reaction, 5 μL of substrate was mixed with 85 μL of citrate / phosphate buffer in a non-bound 96-well microtiter plate (96-MTP) (Corning Life Sciences, #3641) and pre-incubated at 37°C for 5 min in an Eppendorf mixer. Then, 10 μL of appropriately diluted purified enzyme (or water as a blank) was added. After sealing the 96-MTP, the reaction was allowed to proceed for 10 min in an Eppendorf mixer at 37°C and 650 rpm, and the absorbance (A) of the resulting solution was measured at 405 nm using a SpectraMax 190. 405 ). By from enzyme A 405 Subtract blank control A from the middle 405 To calculate net A 405 And then plotted a graph relative to different protein concentrations. Each value is the average of two repeated measurements. Proteolytic activity is shown as net A. 405 .like Figure 16 As shown, compared with the baseline (AnPro), ApsPro1, AhoPro3, AnePro2, AalPro2, AcoPro2, AbrPro1, and AwePro2 are all proteases that are more effective for the proline-specific substrate (AAAP-pNA). Proteolysis assays using AAAP-pNA as a substrate indicate that ApsPro1, AhoPro3, AnePro2, AalPro2, AcoPro2, AbrPro1, and AwePro2 are all highly active proteases.

[0357] Example 29

[0358] MorPro1 clone

[0359] The fungal strain (Magnaporthe oryzae 70-15) was selected as a potential source of enzymes for various industrial applications. BLAST search (Altschul et al., J Mol Biol [Journal of Molecular Biology], 215: 403–410, 1990) led to the identification of genes encoding proteins homologous to the fungal protease (MorPro1 from Magnaporthe oryzae 70-15).

[0360] The full-length nucleic acid sequence of the MorPro1 gene, identified from the NCBI database (NCBI reference sequence: NC_017851.1 from 2214046 to 2215835; complement), is provided in SEQ ID NO: 12. The corresponding full-length protein encoded by the MorPro1 gene is shown in SEQ ID NO: 13 (NCBI reference sequence: XP_003716615.1). As predicted by SignalP version 4.0 (Nordahl Petersen et al., (2011) Nature Methods [Natural Methods] 8:785-786), MorPro1 has an N-terminal signal peptide, indicating that it is a secretase. The corresponding, predicted, mature enzyme sequence of MorPro1 is provided in SEQ ID NO: 14.

[0361] Example 30

[0362] MorPro1 expression

[0363] A chemically synthesized DNA sequence encoding the full-length MorPro1 (SEQ ID NO: 12) was inserted into the *Trichoderma reesei* expression vector pTrex3gM (described in U.S. Publication No. 2011 / 0136197A1) from Shanghai Jierui Biotechnology Co., Ltd. (Shanghai, China). The synthetic nucleotide sequence of the full-length MorPro1 is shown in SEQ ID NO: 15. The pTrex3gM expression vector contains a *Trichoderma reesei* cbhI-derived promoter (cbhI) and a cbhI terminator region, which allows for strong inducible expression of the target gene. The *Aspergillus nidulans* amdS selection marker caused the transformants to grow with acetamide as the sole nitrogen source. The resulting plasmid was labeled pGX256 (Trex3gM-MorPro1), and then the expression plasmid was transformed into a tetradeleted *Trichoderma reesei* strain (described in WO 05 / 001036) using the biological projectile method (Te'o VS et al., *J Microbiol Methods*, 51:393-9, 2002). Transformants were selected on a medium containing acetamide as the sole nitrogen source (acetamide 0.6 g / L; cesium chloride 1.68 g / L; glucose 20 g / L; potassium dihydrogen phosphate 15 g / L; magnesium sulfate heptahydrate 0.6 g / L; calcium chloride dihydrate 0.6 g / L; ferric(II) sulfate 5 mg / L; zinc sulfate 1.4 mg / L; cobalt(II) chloride 1 mg / L; manganese(II) sulfate 1.6 mg / L; agar 20 g / L; pH 4.25). Transformed colonies (approximately 50-100) appeared within about one week. After growth on acetamide plates, transformants were picked and transferred individually to acetamide agar plates. After 5 days of growth on acetamide plates, transformants exhibiting stable morphology were inoculated into 200 μL of glucose / sophorose determinant medium in 96-well microtiter plates. The microtiter plates were incubated at 28°C for 5 days in an oxygen growth chamber. The supernatant from these cultures was used to confirm protein expression by SDS-PAGE analysis. Stable strains with the highest protein expression were selected and fermented in 250 mL shake flasks with glucose / sophorose determinant medium.

[0364] Example 31

[0365] Peptide and protease-specific analyses of beer containing endopeptidase production during fermentation

[0366] As described in Example 14, beer was produced in a semi-industrial pilot brewery where proteases AnPro, AniPro2, AhoPro, and MorPro1 were added at the start of the beer fermentation process as described in Example 14, using the dosages shown in Table 7. The 2 hL beer brewed at the pilot brewery was filtered (without silica or PVPP), but the unstabilized all-malt Pilsner had approximately 66% RDF and an alcohol content of 4.7% (v / v). The beer was filtered using a diatomaceous earth filter as described in Example 13. 15 ml of the final beer was centrifuged in a 10 kDa rotary filter (Vivaspin 20, Sartorius), and 100 µl of the filtrate was added to a desalting column (Oasis HLB 1cc (10 mg) extraction cartridge, material number 186000383, Waters, USA). The column was activated with 0.5 mL MeOH and conditioned with 0.5 mL 0.1% TFA before loading the desalting column. Add 0.5 mL of 0.1% TFA and 100 µl of sample to the sample (slowly load for about 1 min), and wash twice with 0.5 mL of 0.1% TFA. Elute with 0.5 mL of 50% ACN and 0.1% TFA, collect the eluent fraction and freeze-dry overnight. Send the dried sample for MS analysis as described below.

[0367] LC-MS data acquisition

[0368] Nano LC-MS / MS analysis was performed using an UltiMate™ 3000 RSLCnano system (Thermo Fisher Scientific, Massachusetts, USA) coupled with a Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ mass spectrometer (Thermo Fisher Scientific, Massachusetts, USA). Samples were dissolved in 0.1% TFA and loaded onto a 20 mm nanoViper Trap column (Acclaim™ PepMap™ 100 C18, 3 µm particle size, 0.075 mm inner diameter) coupled to a 250 mm analytical column (PepSep, ReproSil 1.9 µm C18 beads, 120 Å pore size, 0.075 mm inner diameter). Separation was performed using a 20-min gradient of 2%–41% solvent B (100% ACN, 0.1% FA) into a NanosprayFlex ion source (Thermo Scientific) at a flow rate of 300 nL / min. HCD fragments were used in a data-dependent MS / MS operation on a Q Exactive HF instrument. Peptide mass was measured via Orbitrap (MS scans at 60,000 resolution obtained at m / z 200). The top 7 strongest ions were selected and fragmented. Ions were separated using a 2.0 Da quadrupole with a separation window. Fragment spectra recorded in Orbitrap were at 60,000 resolution. Dynamic exclusion was enabled for 10 s, with an exclusion mass tolerance width of ±10 ppm relative to the mass on the list.

[0369] Peptide data analysis

[0370] LC-MS / MS data were processed using Proteome Discoverer (version 2.4, Thermo Scientific) (smoothing, background subtraction, and centroiding). The processed LC-MS / MS data were submitted to a database, and the SwissProt database was searched using the internal Mascot server with GreenPlants as the taxonomy. Non-specific enzymes were selected. Methionine oxidation was set as a variable modification. MS / MS results were searched using a peptide ion mass tolerance of ±10 ppm and a fragment ion mass tolerance of ±0.2 Da. A filter (Percolator) (Kall, Canterbury et al. 2007) was used to calculate the free diametrical density (FDR). Only peptides identified as order 1 peptides with a confidence value of 1% (q < 0.01) were considered for further analysis. Relative quantification of peptides was performed using the built-in quantification module. Abundance sums were scaled to the same level and used for calculations.

[0371] Peptides in beer were analyzed, and the relative amino acid content corresponding to the P3'-P3 peptide terminal positions was determined by protease cleavage. The relative amino acid content of peptides from MorPro1, AnPro, and AniPro_2 at the corresponding P1 positions is shown in Figure 21. The right side shows the relative amino acid content at the peptide positions corresponding to P3-P3', and the graph shows the cumulative content of each amino acid at a given position, thus revealing a detailed protease-specific preference. The relative amino acid content of peptides from beer containing MorPro1, AnPro, and AniPro_2, located to P3'-P3 by protease cleavage, is shown in Tables 19 to 21. Peptide corresponding to the P1 position. It is clear that the relative content of both proline at the P1 position is very high, supporting the action of proline-specific proteases. AnPro was found to have the highest proline (P) specificity (52%), followed by AniPro_2 (47%) and MorPro1 (36%). Surprisingly, relatively high glutamine (Q) specificity was also observed in P1 for AniPro_2 (12%) and AnPro (11%), while MorPro1 showed only 3%. A similarly high preference for proline (P) and glutamine (Q) was observed by the relative sum of contents at the P3'-P3 positions, with AniPro_2 exhibiting 141% Q and 108% P, AnPro 134% Q and 101% P, and MorPro1 53% Q and 70% P. This clearly indicates that AniPro_2 is the most specific glutamine and proline-specific endopeptide, followed by AnPro and MorPro1. This also supports and explains the superior turbidity-reducing performance of AniPro_2. Increased glutamine and proline specificity allows for more efficient degradation of turbidity-sensitive proteins in malt or wheat beers known to have high proline and glutamine content. The average peptide length (number of amino acid residues) of peptides identified in beers with protease production was calculated from over 300 unique peptides identified, as shown in the results. Figure 22 As shown. The average peptide lengths observed in beers produced with AniPro2 (12.1 residues), AnPro (12.2 residues), MorPro1 (12.7 residues), and the control (14.2 residues) were calculated. This clearly supports more efficient degradation of isolated proteins in beer, resulting in shorter peptides in beers produced with proteases that have increased glutamine and proline specificity. The shortest average peptides were found in beers produced with AniPro2, followed by beers produced with AnPro and MorPro1, and the control with the longest average peptide length. Therefore, proteases with increased glutamine and proline specificity produce shorter peptides in beer, thus more efficiently degrading turbidity-sensitive proteins in malt beers.

[0372] Table 19. Peptide analysis of malt beer produced on a 1 hL scale with endopeptide, and the relative amino acid (AA) content corresponding to the P3'-P3 peptide terminal positions obtained by protease cleavage. The relative amino acid content of peptides at the P3'-P3 position in beer made with MorPro1 is shown by protease cleavage. The sum of the relative amino acid content of each amino acid from P3' to P3 is given in SUM form.

[0373]

[0374] Table 20. Peptide analysis of malt beer produced on a 1 hL scale with endopeptide, and the relative amino acid (AA) content corresponding to the P3'-P3 peptide terminal positions obtained by protease cleavage. The relative amino acid content of peptides at the P3'-P3 position in beer made with AnPro is shown by protease cleavage. The sum of the relative amino acid content of each amino acid from P3' to P3 is given in SUM form.

[0375]

[0376] Table 21. Peptide analysis of malt beer produced on a 1 hL scale with endopeptide, and the relative amino acid (AA) content corresponding to the P3'-P3 peptide terminal positions obtained by protease cleavage. The relative amino acid content of peptides at the P3'-P3 position in beer made with AniPro2 is shown by protease cleavage. The sum of the relative amino acid content of each amino acid from P3' to P3 is given in SUM form.

[0377]

Claims

1. A method for reducing or preventing turbidity in a beverage, the method comprising adding an endopeptide to the beverage, wherein the endopeptide is an enzyme with an amino acid sequence as shown in SEQ ID NO:

16.

2. The method of claim 1, wherein the beverage contains protein.

3. The method according to any one of claims 1 to 2, wherein the beverage contains polyphenols.

4. The method of any one of claims 1 to 3, wherein the beverage is beer.

5. The method of any one of claims 1 to 3, wherein the beverage is wine.

6. The method of any one of claims 1 to 3, wherein the beverage is fruit juice.

7. The method of claim 4, wherein the endopeptide is added to the wort.

8. The method of claim 4, wherein the endopeptide is added to the beer after turbidity has formed.

9. The method of claim 4, wherein the endopeptide is added to the beer prior to turbidity formation.

10. The method of any one of claims 4 or 7 to 9, wherein the beer has increased relative foam stability and increased relative turbidity reduction.

11. The method of claim 10, wherein the increased relative foam stability is greater than 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 110%.

12. The method of claim 10 or 11, wherein, as measured by 90° scattering, the increase in relative turbidity is reduced by more than 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%.

13. The method of claim 7, wherein the amount of the endopeptide in the wort is 4 to 300 mg protease / hL wort, 10 to 250 mg protease / hL wort, 20 to 200 mg protease / hL wort, 30 to 150 mg protease / hL wort, or 40 to 100 mg protease / hL wort.

14. The method as described in any of the preceding claims, the method further comprising adding one or more of ALDC enzyme, glucosylamylase, maltose α-amylase, amylopectinase, catalase or transglucosidase.

15. The method of claim 14, wherein the ALDC enzyme is acetolactate decarboxylase as described in EC 4.1.1.

5.

16. The method of claim 14, wherein the glucosidase is a 1,4-α-glucosidase as described in EC 3.2.1.

3.

17. The method of claim 14, wherein the maltose α-amylase is a dextran 1,4-α-maltose hydrolase as described in EC 3.3.1.

133.

18. The method of claim 14, wherein the amylopectin is an α-dextrin endoglucosidase, a restriction dextrinase, amylopectin 6-glucan hydrolase, or a debranching enzyme as described in EC 3.2.1.

41.

19. The method of claim 15, wherein the transglucosidase is a 1,4-α-glucan-branched enzyme or an oligoglucan-branched glucosyltransferase as described in EC 2.4.1.

24.

20. A method for increasing relative foam stability in beer, the method comprising adding an endopeptide to the beverage, wherein the endopeptide is an enzyme with an amino acid sequence as shown in SEQ ID NO:

16.

21. The method of claim 20, wherein the increased relative foam stability is greater than 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 110%.

22. The method of claim 21, wherein the endopeptide is added to the wort.

23. The method of claim 22, wherein the amount of the endopeptide in the wort is 4 to 300 mg protease / hL wort, 10 to 250 mg protease / hL wort, 20 to 200 mg protease / hL wort, 30 to 150 mg protease / hL wort, or 40 to 100 mg protease / hL wort.

24. The method of any one of claims 20 to 21, wherein the endopeptide is added to the beer after turbidity has formed.

25. The method of any one of claims 20 to 21, wherein the endopeptide is added to the beer prior to turbidity formation.

26. The method of any one of claims 20 to 25, wherein the beer has increased relative foam stability and increased relative turbidity reduction.

27. The method of claim 26, wherein, as measured by 90° scattering, the increase in relative turbidity is reduced by more than 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%.

28. The method according to any one of claims 20 to 27, the method further comprising adding one or more of ALDC enzyme, glucosylamylase, maltose α-amylase, amylopectinase, catalase or transglucosidase.

29. The method of claim 28, wherein the ALDC enzyme is acetolactate decarboxylase as described in EC 4.1.1.

5.

30. The method of claim 28, wherein the glucosidase is a 1,4-α-glucosidase as described in EC 3.2.1.

3.

31. The method of claim 28, wherein the maltose α-amylase is a dextran 1,4-α-maltose hydrolase as described in EC 3.3.1.

133.

32. The method of claim 28, wherein the amylopectin is an α-dextrin endoglucosidase, a restriction dextrinase, amylopectin 6-glucan hydrolase, or a debranching enzyme as described in EC 3.2.1.

41.

33. The method of claim 28, wherein the transglucosidase is a 1,4-α-glucan-branched enzyme or an oligoglucan-branched glucosyltransferase as described in EC 2.4.1.

24.

34. An isolated polypeptide composed of an endopeptide, wherein the endopeptide is an enzyme with an amino acid sequence as shown in SEQ ID NO:

16.

35. An isolated polynucleotide comprising a nucleic acid sequence encoding the polypeptide of claim 34.

36. A nucleic acid construct comprising the polynucleotide of claim 35, said polynucleotide being operatively linked to one or more control sequences that direct the production of the polypeptide in a suitable expression host.

37. A recombinant expression vector comprising the nucleic acid construct as described in claim 36.

38. A recombinant host cell comprising the nucleic acid construct of claim 36 or the vector of claim 37.

39. A method for producing a polypeptide as claimed in claim 34, the method comprising culturing a recombinant host cell as claimed in claim 38 to produce a supernatant and / or cells containing the polypeptide; and recovering the polypeptide.

40. Use of the filtrate obtained from the fermentation broth obtained by the method of claim 39 in preventing or reducing turbidity in beverages.

41. A composition comprising a polypeptide, isolated polynucleotide, nucleic acid construct, recombinant expression vector or recombinant host cell as described in any one of claims 34 to 38.

42. Use of any polypeptide, isolated polynucleotide, nucleic acid construct, recombinant expression vector or recombinant host cell as described in any one of claims 34 to 38 for increasing the relative foam stability in a beverage.

43. Use of any polypeptide, isolated polynucleotide, nucleic acid construct, recombinant expression vector or recombinant host cell as described in any one of claims 34 to 38 for increasing turbidity reduction in beverages.

44. A method for reducing turbidity in a beverage, the method comprising adding an endogenous glutamine protease to the beverage having an amino acid sequence as shown in SEQ ID NO: 16, wherein the beverage contains a protein or peptide having glutamine residues, the glutamine residues being cleaved by the protease to reduce turbidity.

45. The method of claim 44, wherein the glutamine endopeptidase further cleaves proline residues.

46. ​​The method of claim 44 or 45, wherein the beverage is fruit juice, wine, or beer.

47. The method of claim 46, wherein the beverage is beer.

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