Polypeptides having protease activity and polynucleotides encoding the same

Abstract

The present invention relates to isolated polypeptides having protease activity, and animal feed additives comprising the polypeptides which improve the nutritional value of animal feed by adding proteases; the present invention also relates to polynucleotides encoding these polypeptides, nucleic acid constructs, vectors and host cells comprising these polynucleotides, and methods for producing these polypeptides.

Description

[0001] References to sequence lists

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

[0003] This invention relates to polypeptides with protease activity, polynucleotides encoding these polypeptides, nucleic acid constructs containing these polynucleotides, vectors, and host cells, along with methods for producing and using the polypeptides, and to animal feeds or animal feed additives containing polypeptides with protease activity. Background Technology

[0004] When using proteases (in vivo) in animal feed, and / or when using such proteases to process plant proteins (in vitro), it should be noted that protein is an essential nutrient for both animals and humans. Most livestock and many people obtain these essential proteins from plant-based protein sources. Important plant-based protein sources include, for example, oilseed crops, legumes, and cereals.

[0005] When protein sources (such as soybean meal) are included in the feed of monogastric animals (such as pigs and poultry), a significant proportion of the soybean meal is not effectively digested (the apparent ileal nitrogen digestibility in piglets, growing pigs, and poultry such as broilers, laying hens, and roosters is only around 80%). By improving protein digestibility, animals can absorb more protein, thereby improving performance, such as increased body weight gain.

[0006] The gastrointestinal tract of animals consists of a series of segments, each exhibiting a distinct pH environment. In monogastric animals such as pigs and poultry, as well as many types of fish, the stomach is potentially highly acidic with a pH as low as 2–3, while the intestine has a more neutral pH of around 6–7.5. In addition to the stomach and intestine, poultry possess a crop preceding the stomach. The pH of the crop is primarily determined by the digested feed and is therefore typically within the pH range of 4–6. Protein digestion by proteases can occur throughout the digestive tract, provided that the protease is active and survives the conditions of that digestive tract. Therefore, proteases that are highly acid-stable, can survive in the gastric environment, and are simultaneously effectively active across a wide range of physiological pH levels in the target animal's digestive tract are highly desirable.

[0007] One way to determine whether a protease can improve protein absorption is by studying whether ileal nitrogen digestibility is improved when the protease is added to an animal's diet. In vivo assays can confirm the gastric stability of the protease and its effectiveness in degrading proteins. One object of the present invention is to provide a protease that demonstrates improved growth performance, for example, through apparent ileal nitrogen digestibility. Summary of the Invention

[0008] This invention provides isolated or purified polypeptides with protease activity and polynucleotides encoding these polypeptides.

[0009] Therefore, the present invention relates to isolated or purified polypeptides having protease activity, wherein these isolated or purified polypeptides are selected from the group consisting of:

[0010] (a) A polypeptide having at least 90% sequence identity with the mature polypeptide of SEQ ID NO:1, for example, at least 95% sequence identity;

[0011] (b)(a) fragments of polypeptides that have protease activity.

[0012] The present invention also relates to isolated or purified polynucleotides encoding the polypeptides of the present invention; nucleic acid constructs comprising these polynucleotides, recombinant expression vectors, recombinant host cells; and methods for producing these polypeptides.

[0013] The present invention further relates to an animal feed additive comprising one or more polypeptides having protease activity, wherein the polypeptide is an S8 protease, and wherein the S8 protease is selected from the group consisting of:

[0014] (a) A polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:1;

[0015] (b) A polypeptide encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide encoding sequence of SEQ ID NO:1;

[0016] (c) A variant of any one of SEQ ID NO:1, wherein the variant has protease activity and contains one or more substitutions, and / or one or more deletions, and / or one or more insertions or any combination thereof at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50;

[0017] (d) A polypeptide comprising (a), (b) or (c) and a polypeptide with an N-terminal and / or C-terminal His-tag and / or HQ-tag;

[0018] (e) A polypeptide comprising (a), (b), or (c) and a polypeptide extended at the N-terminus and / or C-terminus of up to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and

[0019] (f) A fragment of a polypeptide of (a), (b) or (c) having protease activity and having at least 90% of the length of a mature polypeptide.

[0020] The present invention also relates to methods for improving one or more performance parameters of animals, the method comprising administering an animal feed additive comprising the polypeptide of the present invention to one or more animals; methods for preparing animal feed comprising mixing an animal feed additive or liquid formulation comprising the polypeptide of the present invention with at least one protein or protein source; methods for processing proteins comprising adding an animal feed additive or liquid formulation of the present invention to at least one protein or protein source; methods for increasing the digestibility and / or solubility of proteins comprising mixing an animal feed additive or liquid formulation of the present invention with at least one protein or protein source; and methods for improving the nutritional value of animal feed comprising adding an animal feed additive or liquid formulation of the present invention to feed.

[0021] The present invention also relates to isolated or purified polynucleotides encoding signal peptides comprising or consisting of amino acids 1 to 25 of SEQ ID NO:1, which are operatively linked to genes encoding proteins; nucleic acid constructs comprising these polynucleotides, expression vectors, and recombinant host cells; and methods for producing proteins.

[0022] This invention further relates to animal feed additives, comprising:

[0023] (a) at least one polypeptide according to any one of claims 1-5; and

[0024] (b) at least one fat-soluble vitamin, and / or

[0025] (c) at least one water-soluble vitamin, and / or

[0026] (d) At least one trace mineral.

[0027] One related aspect involves animal feeds that include animal feed additives as defined in this invention.

[0028] Sequence List Overview

[0029] SEQ ID NO:1 is the amino acid sequence of the S8 protease from Bacillus oceanisediminis.

[0030] SEQ ID NO:2 is the DNA sequence encoding the S8 protease from SEQ ID NO:1 of Bacillus subtilis.

[0031] definition

[0032] Based on this detailed description, the following definitions apply. Note that the singular forms “a / an” and “the” include plural indicators unless the context explicitly indicates otherwise.

[0033] This article refers to the value or parameter “about” as including aspects of that value or parameter itself. For example, a description of “about X” includes aspect “X”.

[0034] Unless otherwise defined or explicitly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] Animals: The term "animal feed" refers to all animals other than humans. Examples of animals include non-ruminants and ruminants. Ruminants include, for example, animals such as sheep, goats, cattle (e.g., beef cattle, dairy cattle, and calves), deer, yaks, camels, llamas, and kangaroos. Non-ruminant animals include monogastric animals such as pigs or live pigs (including but not limited to piglets, growing pigs, and sows); poultry such as turkeys, ducks, and chickens (including but not limited to broilers and laying hens); horses (including but not limited to hot-blooded horses, cold-blooded horses, and warm-blooded horses), calves; fish (including but not limited to amberjack, arapaima, flounder, perch, bluefish, bocachico, cyprinids, catfish, cachama, carp, catfish, catfish, flounder, char, cichlids, cod, sunfish, golden snapper, croaker, eel, goby, goldfish, gudgeon, grouper, guapote, halibut, Java fish, and mullet). Fish, including lai, loach, mackerel, milkfish, silver perch, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pomfret, bream, salmon, sampa, Canadian yellowtail, black perch, sea bream, shiner, sleeper shark, blackfish, sea bream, sawfish, flounder, sashimi, sturgeon, ocean sunfish, sweetfish, trevally, tilapia, trout, tuna, turbot, white trout, whitebait, and whitefish; and crustaceans (including but not limited to shrimp and prawns).

[0036] Animal feed: The term "animal feed" refers to any compound, preparation, or mixture suitable for or intended for use by animals. Animal feed for monogastric animals typically contains concentrates along with vitamins, minerals, enzymes, direct fed microbial, amino acids, and / or other feed ingredients (as in premixes), while animal feed for ruminants typically contains forage (including roughage and silage) and may also contain concentrates along with vitamins, minerals, enzymes, direct fed microbial, amino acids, and / or other feed ingredients (as in premixes).

[0037] Apparent ileal nitrogen digestibility: The term "apparent ileal nitrogen digestibility" (or AIDN) is the percentage difference in nitrogen concentration between ileal digestate and feed, taking into account the apparent loss of dry matter in the terminal small intestine (ileum). AIDN is used as an estimate of crude protein digestibility in the small intestine, without considering the release of endogenous protein from the small intestine. This means that true crude protein digestibility is always greater than AIDN. An increase in AIDN is generally associated with increased absorption of amino acids in the small intestine and is a marker of improved animal performance. Apparent ileal nitrogen digestibility (AIDN) is calculated using the following formula:

[0038] AIDN(%)=100-[(CMf / CMe)x(CNe / CNf)]x 100;

[0039] in

[0040] CMf = Concentration labeled in feed; CMe = Concentration labeled in ileal digestate;

[0041] CNf = concentration of nutrients in feed; CNe = concentration of nutrients in ileal digestate.

[0042] The term “improves ileal nitrogen digestibility by at least x% (e.g., 4%) compared to the negative control” means that if the apparent ileal nitrogen digestibility of the negative control (i.e., the same feed, but without the addition of protease to the diet) is y% (e.g., 75%), then the percentage of apparent ileal nitrogen digestibility of the protease group is at least y% + x% (so >79% in this example).

[0043] Body weight gain: The term “body weight gain” refers to the increase in the live weight of an animal during a given period of time, such as the body weight gain from day 1 to day 21.

[0044] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules obtained from eukaryotic or prokaryotic cells. cDNA lacks the intron sequences that can be present in the corresponding genomic DNA. The initial primary RNA transcript is the precursor of mRNA, which undergoes a series of steps (including splicing) to become mature spliced ​​mRNA.

[0045] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are typically defined by a read frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0046] Composition: The term "composition" refers to a composition comprising a carrier and at least one enzyme of the present invention. The compositions described herein may be mixed with animal feed and may be referred to as "powdered feed".

[0047] Concentrates: The term “concentrate” refers to feeds with high protein and energy concentrations, such as fishmeal, molasses, oligosaccharides, sorghum, seeds and grains (e.g., whole or prepared from corn, oats, rye, barley, wheat, or by crushing, milling, etc.), oilseed filter cake (e.g., from cottonseed, safflower, sunflower, soybean, rapeseed / canola, peanut, or ground peanut), palm kernel cake, yeast-derived materials, and distillers' grains (e.g., wet distillers' grains (WDS) and dry distillers' grains with solubles (DDGS)).

[0048] Control Sequence: The term "control sequence" refers to the nucleic acid sequence necessary for the expression of the polynucleotide encoding the polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or heterologous (i.e., from different genes) for the polynucleotide encoding the polypeptide, or native or heterologous relative to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction sites that facilitate the linking of control sequences to the coding region of the polynucleotide encoding the polypeptide.

[0049] European Production Efficiency Factor (EPEF): The term "European Production Efficiency Factor" is a term used to determine production efficiency, taking into account feed conversion ratio, mortality rate, and daily weight gain. EEF is calculated as [(survival rate (%) × body weight gain (kg)) / (study duration in days) × FCR)] × 100.

[0050] Expression: The term “expression” refers to any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0051] Expression vector: The term "expression vector" refers to a straight or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operatively linked to a control sequence that provides for its expression.

[0052] Feed conversion ratio: The term "feed conversion ratio" refers to the amount of feed given to an animal to gain a specified amount of body weight. Improved feed conversion ratio means a lower feed conversion ratio. A "lower feed conversion ratio" or "improved feed conversion ratio" means that, when the feed does not contain the feed additive composition, the use of the feed additive composition in the feed results in a smaller amount of feed being given to the animal to gain a specified amount of body weight compared to the amount of feed required to gain the same amount of body weight.

[0053] Feed efficiency: The term "feed efficiency" refers to the amount of weight gain per unit of feed when an animal is fed arbitrarily or in a specified amount of food over a period of time. "Increased feed efficiency" means that the use of the feed additive composition according to the invention in feed results in an increased weight gain per unit of feed intake compared to animals fed without the presence of the feed additive composition.

[0054] Forage: As defined herein, the term "forage" also includes coarse grains. Forage is fresh plant material, such as hay and silage derived from forage plants (grasses) and other forage plants (seagrass, germinated grains, and legumes) or any combination thereof. Examples of forage plants are alfalfa (lucerne), birdsfoot, brassica (e.g., kale, rapeseed (canola, turnip), radish), clover (e.g., mixed clover, red clover, ground clover, white clover), grasses (e.g., Bermuda grass, bromegrass, false oat grass, fescue, heath grass, Kentucky bluegrass, orchardgrass). Forage includes grass, ryegrass, timothy-grass, corn, millet, barley, oats, rye, sorghum, soybeans, and wheat, as well as vegetables (such as sugar beets). Forage further includes crop residues from grain production (such as corn stalks; straw from wheat, barley, oats, rye, and other grains); residues from vegetables like beettop; residues from oilseed production such as stems and leaves from soybeans, rapeseed, and other legumes; and portions from grain refining for animal or human consumption, or from fuel production or other industries.

[0055] Fragment: The term "fragment" refers to a polypeptide having one or more (e.g., several) amino acids deleted from the amino and / or carboxyl termini of a mature polypeptide or domain; wherein the fragment has protease activity. In some embodiments, the fragment contains at least 320 amino acid residues (e.g., amino acids 26 to 345 of SEQ ID NO:1), at least 340 amino acid residues (e.g., amino acids 26 to 365 of SEQ ID NO:1), or at least 360 amino acid residues (e.g., amino acids 26 to 385 of SEQ ID NO:1).

[0056] Fusion polypeptide: The term "fusion polypeptide" is a polypeptide in which one polypeptide is fused to the N-terminus or C-terminus of the polypeptide of the present invention. Fusion polypeptides are generated by fusing a polynucleotide encoding another polypeptide to the polynucleotide of the present invention. Techniques for generating fusion polypeptides are known in the art and include linking the coding sequences of the polypeptides such that they conform to reading frames, and that the expression of the fusion polypeptide is under the control of one or more identical promoters and terminators. Fusion polypeptides can also be constructed using intronomer technology, wherein the fusion polypeptide is generated post-translational (Cooper et al., 1993, EMBO J. [Journal of the European Society for Molecular Biology] 12:2575-2583; Dawson et al., 1994, Science 266:776-779). Fusion polypeptides may further include a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved, thereby releasing both polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. [Journal of Industrial Microbiology and Biotechnology] 3:568-576; Svetina et al., 2000, J. Biotechnol. [Journal of Biotechnology] 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 63:3488-3493; Ward et al., 199 5. Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0057] Heterogeneous: For host cells, the term "heterogeneous" means that the polypeptide or nucleic acid is not naturally present in the host cell. For polypeptides or nucleic acids, the term "heterogeneous" means that the control sequence (e.g., the promoter or domain of the polypeptide or nucleic acid) is not naturally associated with that polypeptide or nucleic acid; that is, the control sequence is from a gene other than the gene encoding the mature polypeptide of SEQ ID NO:1.

[0058] Host cell: The term "host cell" means any microbial or plant cell in which a nucleic acid construct or expression vector containing the polynucleotides of the present invention has been introduced. Methods of introduction include, but are not limited to, protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. In some embodiments, the host cell is an isolated recombinant host cell that is partially or completely separated from at least one other component (including, but not limited to, proteins, nucleic acids, cells, etc.).

[0059] Hybrid polypeptide: The term "hybrid polypeptide" refers to a polypeptide that contains domains from two or more polypeptides, such as a binding domain from one polypeptide and a catalytic domain from another polypeptide. The domains can be fused at the N-terminus or C-terminus.

[0060] Hybridization: The term "hybridization" refers to the pairing of substantially complementary strands of nucleic acids using a standard DNA blotting procedure. Hybridization can be performed under medium, medium-high, high, or very high stringency conditions. Medium stringency conditions refer to pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours, followed by washing three times at 55°C with 0.2X SSC and 0.2% SDS for 15 minutes each. Medium-high stringency conditions refer to pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours, followed by washing three times at 60°C with 0.2X SSC and 0.2% SDS for 15 minutes each. Highly stringent conditions refer to pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cut and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours, followed by washing three times at 65°C with 0.2X SSC and 0.2% SDS for 15 minutes each. Very high stringent conditions refer to pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cut and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours, followed by washing three times at 70°C with 0.2X SSC and 0.2% SDS for 15 minutes each.

[0061] Isolated: The term "isolated" refers to a polypeptide, nucleic acid, cell, or other specific material or component that is separated from at least one other material or component (including, but not limited to, other proteins, nucleic acids, cells, etc.) found in nature and naturally associated with it. Isolated polypeptides include, but are not limited to, culture media containing secreted polypeptides.

[0062] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form following translation and any post-translational modifications (e.g., N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and removal of the signal peptide, propeptide, and protopeptide). In one aspect, a mature polypeptide is amino acids 26-425 of SEQ ID NO:1. It is known in the art that host cells can produce mixtures of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide. It is also known in the art that different host cells can process polypeptides differently, and therefore a host cell expressing a polynucleotide can produce different mature polypeptides (e.g., with different C-terminal and / or N-terminal amino acids) compared to another host cell expressing the same polynucleotide.

[0063] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to the polynucleotide that encodes a mature polypeptide with protease activity.

[0064] Natural: The term "natural" refers to nucleic acids or polypeptides that are naturally present in host cells.

[0065] Nucleic acid constructs: The term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a way that does not normally exist in nature to contain segments of nucleic acid, or is synthesized and contains one or more control sequences.

[0066] Operable ligation: The term “operable ligation” refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide, such that the control sequence directs the expression of the coding sequence.

[0067] Pellet: The terms “pellet” and / or “pelleting” refer to solid round, spherical and / or cylindrical flakes or pellets, and the processes used to form such solid shapes, particularly feed pellets and solid extruded animal feed. As used herein, the term “extrusion” (or extruding) is a well-known term in the art and refers to the process of passing a composition through an orifice under pressure, as described herein.

[0068] Performance Parameters: The term "performance parameter" refers to one of many items selected from the following list, which consists of: body weight gain, European Production Efficiency Factor (EPEF), European Production Effectiveness Factor (EFF), and Free Flow Rate (FCR). The term "improvement of one or more performance parameters" means an increase in body weight gain, an increase in the European Production Efficiency Factor (EPEF), an increase in the European Production Effectiveness Factor (EFF), and / or a decrease in FCR in one or more animals.

[0069] Protease: The term “protease” is defined herein as an enzyme that hydrolyzes peptide bonds. It includes any enzyme belonging to the EC 3.4 enzyme group (including each of its thirteen subclasses http: / / en.wikipedia.org / wiki / Category:EC_3.4). EC numbers refer to the 1992 Enzyme Nomenclature of NC-IUBMB, Academic Press, San Diego, California, and include supplements 1-5 published in the following: Eur. J. Biochem. 223:1-5 (1994) Eur. J. Biochem. 232:1-6 (1995) Eur. J. Biochem. 237:1-5 (1996) Eur. J. Biochem. 250:1-6 (1997) and Eur. J. Biochem. 264:610-650 (1999). The term "subtilisinase" refers to the serine protease subgroup according to Siezen et al., 1991, Protein Engineering, 4:719-737 and Siezen et al., 1997, Protein Science, 6:501-523. Serine proteases, or serine peptidases, are subgroups of proteases characterized by the presence of a serine residue at their active site that forms a covalent adduct with the substrate. Furthermore, subtilisinase (and serine proteases) is characterized by having two additional active site amino acid residues besides serine: histidine and aspartic acid residues. Subtilisinases can be classified into six subgroups: the subtilisinase family, the thermophilic protease family, the proteinase K family, the lanoxanthionein family, the Kexin family, and the Pyrolysin family.

[0070] Protease activity: The term "protease activity" refers to protein hydrolysis activity (EC 3.4). Peptides or proteases exhibiting protease activity are sometimes also designated as peptidases, prions, peptide hydrolases, or protein hydrolases. Proteases can be exopeptidases that hydrolyze peptides originating from either end or endopeptidases that function within the polypeptide chain. Endopeptidases exhibit activity against N- and C-terminated peptide substrates, the specificity of which depends on the protease in question.

[0071] Several types of protease activity exist, such as trypsin-like proteases that cleave at the carboxyl terminus of Arg and Lys residues, and chymotrypsin-like proteases that cleave at the carboxyl terminus of hydrophobic amino acid residues. The protease of the present invention is a serine endopeptidase (EC 3.4.21) with a slightly alkaline optimal pH (optimal pH 8-9.5).

[0072] Any assay can be used to measure protease activity, employing a substrate that includes peptide bonds specific to the protease in question. Assays of pH and temperature are equally applicable to the protease in question. Examples of pH measurements are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of temperatures are 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 80°C, 90°C, or 95°C. Examples of common protease substrates are casein, bovine serum albumin, and hemoglobin. In the classic Anson and Mirsky method, denatured hemoglobin is used as a substrate, and the amount of trichloroacetic acid-soluble hemoglobin is determined as a measure of protease activity after incubation with the protease in question (Anson and Mirsky, 1932, J. Gen. Physiol. [Journal of General Physiology] 16:59 and Anson, 1938, J. Gen. Physiol. [Journal of General Physiology] 22:79).

[0073] For the purposes of this invention, assays described in "Materials and Methods" are used to determine protease activity, such as the Suc-AAPF-pNA assay and the Protazyme AK assay. For the Protazyme AK assay, when incubated with a protease, the insoluble Protazyme AK (azurin-crosslinked casein) substrate releases a blue color, and this color is determined as a measure of protease activity. For the Suc-AAPF-pNA assay, when incubated with a protease, the colorless Suc-AAPF-pNA substrate releases a yellow p-nitroaniline, and this yellow color is determined as a measure of protease activity.

[0074] The polypeptides of the present invention have at least 20%, for example at least 40%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, and at least 100% of the protease activity of the polypeptide of SEQ ID NO:1.

[0075] The S8 peptidase family contains serine endopeptidases and is the second largest family of serine peptidases in terms of both sequence number and characteristic peptidases. In the S8A subfamily, active site residues frequently occur within the motif. Asp -Thr / Ser-Gly、 His -Gly-Thr-His and Gly-Thr- Ser In Met-Ala-Xaa-Pro, the catalytic residues were identified as Asp-41, His-103, and Ser-278, with the fourth active site amino acid identified as Asn-208. Mutations in any of these catalytic residues will result in altered or lost enzyme activity.

[0076] Purified: The term "purified" refers to nucleic acids or peptides that are substantially free of other components, as determined by analytical techniques well known in the art (e.g., in electrophoresis gels, chromatographic eluates, and / or media after density gradient centrifugation, where purified peptides or nucleic acids form discrete bands). Purified nucleic acids or peptides are at least about 50% pure, and typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or more pure (e.g., by molar weight percentage). In a relevant sense, when there is a significant increase in the concentration of the molecule after the application of purification or enrichment techniques, the composition is enriched for that molecule. The term “enrichment” refers to the presence of compounds, peptides, cells, nucleic acids, amino acids, or other specified materials or components in a composition at a relative or absolute concentration higher than that of the starting composition.

[0077] Recombinant: When used to refer to cells, nucleic acids, proteins, or vectors, the term "recombinant" means that it has been modified from its natural state. Thus, for example, a recombinant cell expresses a gene not found in the natural (non-recombinant) form of the cell, or expresses a natural gene at a different level or under different conditions compared to its natural counterpart. The difference between a recombinant nucleic acid and its natural sequence lies in the operative linking of one or more nucleotides and / or a foreign sequence (e.g., a foreign promoter in an expression vector). The difference between a recombinant protein and its natural sequence may lie in the fusion of one or more amino acids and / or a foreign sequence. A vector containing a nucleic acid encoding a polypeptide is a recombinant vector. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0078] Whole grains: The term "whole grains" refers to dry plant materials with a high level of fiber, such as fiber, bran, and husks from seeds and grains, as well as crop residues (e.g., straw, copra, rice straw, husks, sugar beet waste).

[0079] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0080] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Niedle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 6.6.0 or later) is used to determine the sequence identity between two amino acid sequences as the output of “longest identity”. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 substitution matrix (the EMBOSS version of BLOSUM62). For the Niedle program to report the longest identity, the nobrief option must be specified in the command line. The Niedle-marked “longest identity” output is calculated as follows:

[0081] (identical residues x 100) / (alignment length - total number of vacancies in the alignment)

[0082] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (preferably version 6.6.0 or later) is used to determine the sequence identity between two polynucleotide sequences as the output of "longest identity". The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. For the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of the "longest identity" marked by Needleman is calculated as follows:

[0083] (identical deoxyribonucleotides x 100) / (alignment length - total number of vacancies in the alignment)

[0084] Silage: The term "silage" refers to a fermented, high-moisture stored feed that can be fed to ruminants (cud-chewing animals, such as cattle and sheep) or used as a biofuel feedstock for anaerobic digesters. It is fermented and stored in a process called silage (ensilage, ensiling, or silaging) and is typically made from whole green plants (not just grains) of grasses or cereal crops (such as corn, sorghum, oats, rye, timothy, etc.) or legume crops (such as clover, alfalfa, peas). Silage can be made from many field crops and, depending on the type, may use specific terms (oatlage for oats, haylage for alfalfa). Silage is made by placing cut green vegetation in silage pits, by piling it in large heaps covered with plastic sheets, or by wrapping large bales in plastic film.

[0085] Subsequence: The term “subsequence” refers to a polynucleotide that is missing one or more (e.g., several) nucleotides from the 5' and / or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having protease activity.

[0086] Variants: The term "variant" refers to a polypeptide that has protease activity and contains artificial mutations (i.e., substitutions, insertions, and / or deletions, such as truncations) at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

[0087] Wild-type: The term "wild-type" in relation to amino acid or nucleic acid sequences means that the amino acid or nucleic acid sequence is a naturally occurring or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., protein, amino acid, or nucleic acid sequences). Conversely, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences). Detailed Implementation

[0088] polypeptides with protease activity

[0089] In some embodiments, the present invention relates to isolated polypeptides having at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:1, and these isolated polypeptides having protease activity. In one aspect, these polypeptides differ from the mature polypeptide of SEQ ID NO:1 by up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0090] In a preferred embodiment, the polypeptide of the present invention has protease activity and is an S8 protease.

[0091] The polypeptide preferably comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO:1 or its mature polypeptide; or is a fragment thereof with protease activity.

[0092] In some embodiments, the present invention relates to isolated polypeptides having protease activity encoded by polynucleotides hybridizing with the full-length complementary sequence of their mature polypeptide or their cDNA under medium-strict, medium-high-strict, high-strict, or very high-strict conditions (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York).

[0093] Nucleic acid probes can be designed using polynucleotides or their subsequences, together with the mature polypeptide or fragment of SEQ ID NO:1, to identify and clone DNA encoding protease-active polypeptides from strains of different genera or species, according to methods well known in the art. Such probes can be used to hybridize with the genomic DNA or cDNA of target cells following standard DNA blotting procedures to identify and isolate the corresponding genes therein. Such probes may be significantly shorter than the complete sequence, but should be at least 15 nucleotides long, for example, at least 25, at least 35, or at least 70 nucleotides. Preferably, the nucleic acid probe is at least 100 nucleotides long, for example, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, or at least 900 nucleotides. Both DNA and RNA probes can be used. Probes are typically labeled (e.g., with...). 32 P, 3 H, 35 Probes (such as biotin or avidin) are used to detect corresponding genes. Such probes are covered in this invention.

[0094] DNA from genomic DNA or cDNA libraries prepared from other strains of this type that hybridizes to the probes described above and encodes peptides with protease activity can be screened. Genomic DNA or other DNA from other strains of this type can be separated by agarose or polyacrylamide gel electrophoresis or other separation techniques. DNA from the library or separated DNA can be transferred and immobilized on nitrocellulose or another suitable carrier material. The carrier material is used in DNA blotting to identify clones or DNA that hybridize to polynucleotides or their daughter sequences.

[0095] For the purposes of this invention, hybridization refers to the hybridization of a polynucleotide with a labeled nucleic acid probe under medium to very high stringency conditions. Molecules hybridized under these conditions can be detected using, for example, X-ray film or any other detection method known in the art.

[0096] In some embodiments, the nucleic acid probe is a polynucleotide encoding a mature polypeptide or a fragment thereof of SEQ ID NO:1.

[0097] In some embodiments, the present invention relates to variants of the mature polypeptide of SEQ ID NO:1 comprising substitutions, deletions, and / or insertions at one or more (e.g., several) sites. In one aspect, the number of amino acid substitutions, deletions, and / or insertions introduced into the mature polypeptide of SEQ ID NO:1 is up to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1-30 amino acids; small N-terminal or C-terminal extensions, such as methionine residues at the N-terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering net charge or another function (e.g., polyhistidine fragments, antigenic epitopes, or binding modules).

[0098] Essential amino acids in peptides can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the protease activity of the resulting molecule is tested to identify amino acid residues critical to the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, such as by techniques including nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutation of the amino acid at the putative contact site. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of essential amino acids can also be inferred from comparisons with related peptides.

[0099] Using known mutagenesis, recombination, and / or tampering methods, followed by relevant screening procedures, one or more amino acid substitutions, deletions, and / or insertions can be made and tested. These relevant screening procedures are disclosed, for example, those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US Patent No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0100] Mutagenesis / reorganization methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenesis-encoded peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenesis-encoded DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0101] In some embodiments, the polypeptide contains at least 340 amino acid residues (e.g., amino acids 26 to 365 of SEQ ID NO:1), at least 360 amino acid residues (e.g., amino acids 26 to 385 of SEQ ID NO:1), or at least 380 amino acid residues (e.g., amino acids 26 to 405 of SEQ ID NO:1).

[0102] The polypeptide can be a hybrid polypeptide or a fusion polypeptide.

[0103] When mixed with animal feed, the peptides of the present invention exhibit a certain level of protease activity, thereby increasing the digestibility and / or solubility of the protein and thus improving the nutritional value of the animal feed.

[0104] Sources of peptides with protease activity

[0105] The protease-active polypeptides of the present invention can be obtained from microorganisms of any genus. For the purposes of this invention, the term "obtained from" as used herein in conjunction with a given source should mean that the polypeptide encoded by the polynucleotide is produced by that source or by a strain that has inserted a polynucleotide from that source. In one aspect, polypeptides obtained from a given source are secreted extracellularly.

[0106] On the other hand, the polypeptide is a Bacillus subtilis polypeptide.

[0107] It should be understood that, for the aforementioned species, this invention covers both perfect and imperfect states, and other taxonomic equivalents, such as asexual forms, regardless of their known species names. Those skilled in the art will readily identify the appropriate equivalents.

[0108] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial and Cell Culture Collection (DSMZ), the Centralbureau Voor Schimmelcultures (CBS), and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0109] The probes mentioned above can be used to identify and obtain polypeptides from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.). Techniques for directly isolating microorganisms and DNA from natural habitats are well known in the art. The polynucleotide encoding the polypeptide can then be obtained by similarly screening a library of genomic DNA or cDNA from another microorganism or a mixed DNA sample. Once the polynucleotide encoding the polypeptide has been detected with one or more probes, the polynucleotide can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, ibid.).

[0110] Polynucleotides

[0111] This invention also relates to isolated polynucleotides encoding the polypeptides of this invention, as described herein.

[0112] Techniques for isolating or cloning polynucleotides are known in the art and include isolation from genomic DNA or cDNA or combinations thereof. Cloning of polynucleotides from genomic DNA can be achieved, for example, by using polymerase chain reaction (PCR) or screening with expression library antibodies to detect DNA fragments of clones having common structural features. See, for example, Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures can be used, such as ligase chain reaction (LCR), ligation-activated transcription (LAT), and polynucleotide-based amplification (NASBA). These polynucleotides can be cloned from strains of the genus Bacillus or related organisms, and therefore, for example, can be species variants of the polypeptide coding region of the polynucleotide.

[0113] Modification of the polynucleotide encoding the polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term “substantially similar” to the polypeptide refers to a non-naturally occurring form of the polypeptide. These polypeptides may differ from polypeptides isolated from their natural sources due to some form of engineering, for example, in variations in specific activity, thermal stability, optimal pH, etc. These variants can be constructed as follows: based on a polynucleotide encoding the mature polypeptide of SEQ ID NO:1 or a subsequence thereof, and / or by introducing nucleotide substitutions that do not alter the amino acid sequence of the polypeptide but correspond to the codon usage intended for the host organism to produce the enzyme, or by introducing nucleotide substitutions that may produce a different amino acid sequence. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2:95-107.

[0114] Nucleic acid constructs

[0115] The present invention also relates to nucleic acid constructs comprising the polynucleotide of the present invention, wherein the polynucleotide is preferably operatively linked to one or more control sequences, which, under conditions compatible with the control sequences, direct the expression of the coding sequence in a suitable host cell.

[0116] The polynucleotide can be manipulated in many ways to provide polypeptide expression. Depending on the expression vector, manipulating the polynucleotide before insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0117] The control sequence may be a promoter, i.e., a polynucleotide recognized by the host cell for expressing a polynucleotide encoding the polypeptide of the present invention. The promoter contains a transcriptional control sequence mediating polypeptide expression. The promoter can be any polynucleotide exhibiting transcriptional activity in the host cell, including mutant, truncated, and heterozygous promoters, and can be a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0118] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of this invention in bacterial host cells are promoters obtained from the following genes: Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis α-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus thermophilus maltose amylase gene (amyM), Bacillus subtilis fructan sucrase gene (sacB), Bacillus subtilis xylA and xylB genes, and Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular). Microbiology [Molecular Microbiology] 13:97-107), Escherichia coli lac operon, Escherichia coli trc promoter (Egon et al., 1988, Gene [Gene] 69:301-315), Streptomyces agar hydrolase gene (dagA) and prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 75:3727-3731), and tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 80:21-25). Other promoters are described in Gilbert et al., 1980, Scientific American 242:74-94, “Useful proteins from recombinant bacteria”; and in Sambrook et al., 1989, ibid. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0119] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of this invention in filamentous fungal host cells are promoters obtained from the following genes: Aspergillus nidulans acetamase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucosylamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amylase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 00 / 56900), and Rhizomucor *Trichoderma reesei* lipase, *Rhizopus reesei* aspartic protease, *Trichoderma reesei* β-glucosidase, *Trichoderma reesei* cellobiose hydrolase I, *Trichoderma reesei* cellobiose hydrolase II, *Trichoderma reesei* endoglucanase I, *Trichoderma reesei* endoglucanase II, *Trichoderma reesei* endoglucanase III, *Trichoderma reesei* endoglucanase V, *Trichoderma reesei* xylanase I, *Trichoderma reesei* xylanase II, *Trichoderma reesei* xylanase III, *Trichoderma reesei* β-xylosidase, and *Trichoderma reesei* translation elongation factor, along with NA2-tpi Promoters (modified promoters from Aspergillus neutral α-amylase genes, wherein an untranslated leader sequence has been replaced with an untranslated leader sequence from an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters from Aspergillus niger neutral α-amylase genes, wherein an untranslated leader sequence has been replaced with an untranslated leader sequence from an Aspergillus nidus or Aspergillus oryzae triose phosphate isomerase gene); and mutant, truncated, and heterozygous promoters thereof. Other promoters are described in U.S. Patent No. 6,011,147.

[0120] In yeast hosts, useful promoters are derived from the following genes: *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), *Saccharomyces cerevisiae* triose phosphate isomerase (TPI), *Saccharomyces cerevisiae* metallothionein (CUP1), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase. Other useful promoters in yeast host cells are described by Romanos et al., 1992, Yeast [Yeast] 8:423-488.

[0121] The control sequence may also be a transcription terminator recognized by the host cell to terminate transcription. This terminator is operatively linked to the 3' end of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell can be used in this invention.

[0122] The preferred terminator for bacterial host cells is derived from the following genes: Bacillus clausti alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0123] Preferred terminators for filamentous fungal host cells are derived from the following genes: Aspergillus nidulans acetamase, Aspergillus nidulans o-aminobenzoic acid synthase, Aspergillus niger glucosylamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiose hydrolase I, Trichoderma reesei cellobiose hydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei β-xylosidase, and Trichoderma reesei translation elongation factor.

[0124] Preferred terminators for yeast host cells are derived from the following genes: *Saccharomyces cerevisiae* enolase, *Saccharomyces cerevisiae* cytochrome C (CYC1), and *Saccharomyces cerevisiae* glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al. (1992, ibid.).

[0125] Control sequences can also be mRNA stabilizer regions downstream of the promoter and upstream of the gene's coding sequence, which increase the expression of the gene.

[0126] Examples of suitable mRNA stable regions were obtained from the following: Bacillus thuringiensis cryIIIA gene (WO94 / 25612) and Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. [Journal of Bacteriology] 177:3465-3471).

[0127] The control sequence can also be a leader sequence, which is the untranslated region of mRNA that is important for translation in the host cell. This leader sequence is operatively linked to the 5' end of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell can be used.

[0128] Preferred leader sequences for use in filamentous fungal host cells were obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0129] Suitable leader sequences for yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0130] The control sequence can also be a polyadenylation sequence, a sequence operatively linked to the 3' end of the polynucleotide and recognized by the host cell during transcription as a signal to add polyadenylation residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell can be used.

[0131] Preferred polyadenylation sequences for use in filamentous fungal host cells were obtained from the genes of the following enzymes: Aspergillus nidulans o-aminobenzoic acid synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0132] Useful polyadenylation sequences in yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. [Molecular Cell Biology] 15: 5983-5990.

[0133] The control sequence can also be a signal peptide coding region encoding a signal peptide that is linked to the N-terminus of the polypeptide and guides the polypeptide into the secretory pathway of the cell. The 5' end of the polynucleotide coding sequence itself may contain a signal peptide coding sequence naturally linked to the coding sequence segment of the polypeptide within the translation reading frame. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is heterologous to that coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, a heterologous signal peptide coding sequence may be required. Alternatively, a heterologous signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance polypeptide secretion. However, any signal peptide coding sequence that guides the secretory pathway of the expressed polypeptide into the host cell can be used.

[0134] Effective signal peptide coding sequences for bacterial host cells are obtained from the genes of Bacillus NCIB11837 producing maltose amylase, Bacillus subtilis protease, Bacillus subtilis β-lactamase, Bacillus thermophilus α-amylase, Bacillus thermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described by Simonen and Palva, 1993, Microbiol. Rev. [Microbial Reviews] 57:109-137.

[0135] Effective signal peptide coding sequences for filamentous fungal host cells are obtained from the genes of the following enzymes: Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, Aspergillus oryzae cellulase, Aspergillus oryzae endoglucanase V, Aspergillus sparseis lipase, and Rhizopus oryzae aspartic protease.

[0136] Useful signal peptides for yeast host cells are derived from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al. (1992, ibid.).

[0137] The control sequence can also be a propeptide-coding sequence encoding the propeptide located at the N-terminus of the polypeptide. The resulting polypeptide is called a proenzyme or propeptide progenitor (or, in some cases, a zymogen). The propeptide progenitor is usually inactive and can be converted into an active polypeptide by catalytic cleavage or autocatalytic cleavage of the propeptide progenitor. Propeptide-coding sequences can be obtained from the following genes: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Thermophilus laccase (WO 95 / 33836), Rhizopus oryzae aspartic protease, and Saccharomyces cerevisiae α-factor.

[0138] When both the signal peptide sequence and the propeptide sequence are present, the propeptide sequence is located immediately adjacent to the N-terminus of the polypeptide, and the signal peptide sequence is located immediately adjacent to the N-terminus of the propeptide sequence.

[0139] It is also desirable to add regulatory sequences that regulate the expression of host cell growth-related peptides. Examples of regulatory sequences are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operon systems. In yeast, the ADH2 or GAL1 system can be used. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKA α-amylase promoter and *Aspergillus oryzae* glucosylamylase promoter, the *Trichoderma reesei* cellobiose hydrolase I promoter, and the *Trichoderma reesei* cellobiose hydrolase II promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the peptide is operatively linked to the regulatory sequence.

[0140] expression carrier

[0141] The present invention also relates to recombinant expression vectors comprising the polynucleotide of the present invention, a promoter, and transcription and translation termination signals. Multiple nucleotides and control sequences may be linked together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located in the vector such that the coding sequence is operatively linked to a suitable control sequence for expression.

[0142] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed circular plasmids.

[0143] The vector can be a self-replicating vector, that is, a vector that exists as an extrachromosomal entity and replicates independently of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that integrates into the genome when introduced into a host cell and replicates along with one or more chromosomes in which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids collectively containing the total DNA of the host cell genome to be introduced, or transposons can be used.

[0144] The vector preferably contains one or more selective markers that allow for convenient selection of cells such as transformed cells, transfected cells, and transduced cells. A selective marker is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, or prototrophic auxotrophic traits, etc.

[0145] Examples of selective bacterial markers include the dal gene in Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance (such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance). Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for use in filamentous fungal host cells include, but are not limited to, adeA (phosphogluconoylaminoimidazolium-succinate synthase), adeB (phosphogluconoylaminoimidazolium synthase), amdS (acetamipase), argB (ornithine carbamoyltransferase), bar (glufosinate acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotate nucleoside-5'-phosphate decarboxylase), sC (adenosyl sulfate transtransferase), and trpC (o-aminobenzoic acid synthase) along with their equivalents. Preferred genes for use in Aspergillus cells include the amdS and pyrG genes from Aspergillus nidulans or Aspergillus oryzae, and the bar gene from Streptomyces hygroscopicus. Preferred genes for use in Trichoderma cells include the adeA, adeB, amdS, hph, and pyrG genes.

[0146] Selective labeling can be a biselective labeling system as described in WO 2010 / 039889. In one aspect, the biselective labeling is the hph-tk biselective labeling system.

[0147] The vector preferably contains one or more elements that allow the vector to integrate into the host cell's genome or to replicate autonomously in the cell independently of the genome.

[0148] For integration into the host cell genome, the vector can rely on a polynucleotide sequence encoding the polypeptide or any other element of the vector for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector can contain additional polynucleotides to guide integration into the host cell genome at a precise location on the chromosome via homologous recombination. To increase the likelihood of integration at a precise location, the integrative element should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that have high sequence identity with the corresponding target sequence to enhance the probability of homologous recombination. The integrative element can be any sequence homologous to the target sequence within the host cell genome. Furthermore, the integrative element can be a non-coding or coding polynucleotide. On the other hand, the vector can integrate into the host cell genome via non-homologous recombination.

[0149] For autonomous replication, the vector may further include an origin of replication, which enables the vector to replicate autonomously within the host cell discussed in this context. The origin of replication can be any plasmid replicon that mediates autonomous replication and functions within the cell. The terms "origin of replication" or "plasmid replicon" refer to the polynucleotide that enables a plasmid or vector to replicate in vivo.

[0150] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in Escherichia coli, and the origins of replication of plasmids pUB110, pE194, pTA1060, and pAMβ1, which allow replication in Bacillus.

[0151] Examples of replication origins used in yeast host cells include the 2-micron replication origin, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6.

[0152] Examples of useful origins of replication in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. 15:9163-9175; WO00 / 24883). The AMA1 gene can be isolated and plasmids or vectors containing the gene can be constructed according to the methods disclosed in WO 00 / 24883.

[0153] More than one copy of the polynucleotide of the present invention can be inserted into a host cell to increase polypeptide production. An increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selective marker gene along with the polynucleotide, wherein cells containing the amplified copy of the selective marker gene and thus additional copies of the polynucleotide can be selected by culturing the cells in the presence of a suitable selective reagent.

[0154] The procedures for connecting the above-described elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, ibid.).

[0155] host cells

[0156] The present invention also relates to recombinant host cells containing polynucleotides of the invention operably linked to one or more control sequences that direct the production of polypeptides of the invention. A construct or vector containing the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrase or as an autonomously replicating extrachromosomal vector, as previously described. The choice of host cell will depend largely on the gene encoding the polypeptide and its origin.

[0157] In some embodiments, the peptide and the recombinant host cell are heterologous.

[0158] In some embodiments, at least one of the control sequences is heterologous to the polynucleotide encoding the polypeptide.

[0159] In some embodiments, the recombinant host cell contains at least two copies, such as three, four, or five, of the polynucleotide of the present invention.

[0160] The host cell can be any microbial or plant cell that can be used to recombinantly generate the polypeptides of the present invention, such as prokaryotic cells or fungal cells.

[0161] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Bacillus macrocephala, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Coliform, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0162] The bacterial host cell can be any Bacillus genus cell, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus croceus, Bacillus coagulans, Bacillus sclerosus, Bacillus splenium, Bacillus stenosis, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus thermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0163] The bacterial host cell can also be any streptococcus cell, including but not limited to Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus.

[0164] The bacterial host cell can also be any Streptomyces cell, including but not limited to: Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces griseus, and Streptomyces lividans cells.

[0165] DNA can be introduced into Bacillus cells by the following methods: protoplast transformation (see, for example, Chang and Cohen, 1979, Mol. Gen. Genet. [Molecular and General Genetics] 168:111-115), competent cell transformation (see, for example, Young and Spizizen, 1961, J. Bacteriol. [Journal of Bacteriology] 81:823-829; or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. [Journal of Molecular Biology] 56:209-221), electroporation (see, for example, Shigekawa and Dower, 1988, Biotechniques [Biotechnology] 6:742-751), or conjugation (see, for example, Koehler and Thorne, 1987, J. Bacteriol. [Journal of Bacteriology] 169:5271-5278). DNA can be introduced into E. coli cells by protoplast transformation (see, for example, Hanahan, 1983, J. Mol. Biol. [Journal of Molecular Biology] 166:557-580) or electroporation (see, for example, Dower et al., 1988, Nucleic Acids Res. [Nucleic Acid Research] 16:6127-6145). DNA can be introduced into Streptomyces cells in the following ways: protoplast transformation, electroporation (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, for example, Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, for example, Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294). DNA can be introduced into Pseudomonas cells by means of electroporation (see, for example, Choi et al., 2006, J. Microbiol. Methods 64:391-397) or conjugation (see, for example, Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:51-57).DNA can be introduced into Streptococcus cells in the following ways: natural competence (see, for example, Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, for example, Catt and Jollick, 1991, Microbios 68:189-207), electroporation (see, for example, Buckley et al., 1999, Appl. Environ. Microbiol. 65:3800-3804), or conjugation (see, for example, Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0166] The host cell can be a fungal cell. As used herein, “fungus” includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Oomycota, and all mitotic fungi (as defined by Hawksworth et al. in Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0167] The host cell for fungi can be a yeast cell. As used herein, “yeast” includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeasts belonging to the Fungi Imperfecti (Blastomycetes). Since yeast classification may change in the future, for the purposes of this invention, yeast should be defined as described in *Biology and Activities of Yeast* (edited by Skinner, Passmore, and Davenport, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0168] Yeast host cells can be cells from the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia*, *Saccharomyces*, *Schizosaccharomyces*, or *Yarrowia*, such as *Kluyveromyces lactis*, *Saccharomyces carlsbergensis*, *Saccharomyces cerevisiae*, *Saccharomyces diastaticus*, *Saccharomyces douglasii*, *Saccharomyces kluyveri*, *Saccharomyces norbensis*, *Saccharomyces oviformis*, or *Yarrowia lipolytica*.

[0169] Fungal host cells can be filamentous fungal cells. "Filamentous fungi" includes all filamentous forms within the phylum Eumycota and subphyla Oomycota (as defined by Hawksworth et al., 1995, ibid.). Filamentous fungi are typically characterized by a hyphal wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal extension, and carbon metabolism is obligate aerobic. In contrast, yeast (such as Saccharomyces cerevisiae) undergoes vegetative growth through budding of single-celled cells, and carbon metabolism can be fermentative.

[0170] The host cells of filamentous fungi can be cells from genera such as *Apertoire*, *Aspergillus*, *Bjerkandera*, *Pseudomonas*, *Aureospora*, *Coprinus*, *Coriolus*, *Cryptococcus*, *Filibasidium*, *Fusarium*, *Pyrophyllus*, *Pleurotus*, *Mucor*, *Pyrophyllus*, *Neurophyllus*, *Penicillium*, *Penicillium*, *Phlebia*, *Pleurotus*, *Schizophyllum*, *Ascomycetes*, *Clostridium*, *Cytotrichum*, *Phytotrichum*, *Pleurotus*, *Schizophyllum*, *Cytotrichum*, *Clostridium*, *Trametes*, or *Trichoderma*.

[0171] For example, the host cells of filamentous fungi can be *Aspergillus bubomori*, *Aspergillus sulphureus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus oryzae*, *Bjerkandera adusta*, *Ceriporiopsis saneirina*, *Ceriporiopsis caregiea*, *Ceriporiopsis gilvescens*, *Ceriporiopsis pannocinta*, *Ceriporiopsis rivulosa*, *Ceriporiopsis subrufa*, *Ceriporiopsis subvermispora*, *Chrysosporium inops*, *Chrysosporium keratinophilum*, *Chrysosporium lucknowense*, and *Chrysosporium foetida*. *Chrysosporium pannicola*, *Chrysosporium queenslandicum*, *Chrysosporium tropicum*, *Chrysosporium zonatum*, *Coprinus cinereus*, *Coriolushirsutus*, *Fusarium moniliforme*, *Fusarium graminearum*, *Fusarium graminearum*, *Fusarium graminearum*, *Fusarium graminearum*, *Fusarium graminearum*, *Fusarium moniliforme ... Chrysosporium), Phlebiaradiata, Pleurotus eryngii, Emerson basket fungus, Clostridium terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma corningensis, Trichoderma longibranchii, Trichoderma reesei, or Trichoderma viridans cells.

[0172] Fungal cells can be transformed in a manner known per se through methods involving protoplast formation, protoplast transformation, and cell wall regeneration. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in the following references: EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 81:1470-1474 and Christensen et al., 1988, Bio / Technology [Biology / Technology] 6:1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene [Gene] 78:147-156 and WO 96 / 00787. Yeast can be transformed using procedures described in the following literature: Becker and Guarente, in Abelson, JN and Simon, MI (eds.), Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, pp. 182-187, Academic Press, Inc. (New York); Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:1920.

[0173] Generation method

[0174] The present invention also relates to methods for producing the polypeptides of the invention, the methods comprising (a) culturing cells under conditions favorable to the production of the polypeptide, the cells producing the polypeptide in their wild-type form; and optionally (b) recovering the polypeptide. In one aspect, the cells are Bacillus cells. In another aspect, the cells are Bacillus subtilis cells.

[0175] The present invention also relates to methods for producing the polypeptides of the invention, the methods comprising (a) culturing the recombinant host cells of the invention under conditions conducive to the production of the polypeptide; and optionally (b) recovering the polypeptide.

[0176] The host cells are cultured in a nutrient medium suitable for producing peptides using methods known in the art. For example, cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in a laboratory or industrial fermenter, in a suitable medium and under conditions that allow for peptide expression and / or isolation. Using procedures known in the art, the culture occurs in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the American Type Culture Collection). If the peptide is secreted into the nutrient medium, it can be recovered directly from the medium. If the peptide is not secreted, it can be recovered from cell lysates.

[0177] Peptides can be detected using methods known in the art that are specific to peptides. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, enzyme assays can be used to determine the activity of a peptide.

[0178] Peptides can be recovered using methods known in the art. For example, peptides can be recovered from fermentation media by conventional methods, including but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the entire fermentation broth containing peptides can be recovered.

[0179] The polypeptide can be purified by a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, focusing chromatography, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric focusing electrophoresis), differential dissolution (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, edited by Janson and Ryden, VCH Publishers, New York, 1989) to obtain a substantially pure polypeptide.

[0180] Removal or reduction of protease activity

[0181] The present invention also relates to methods for generating mutant parental cells, methods comprising disrupting or deleting polynucleotides or portions thereof encoding polypeptides of the present invention, which results in the mutant cells producing fewer polypeptides compared to parental cells when cultured under the same conditions.

[0182] Mutant cells can be constructed by reducing or eliminating the expression of the polynucleotide using methods well known in the art, such as insertion, disruption, substitution, or deletion. In a preferred aspect, the polynucleotide is inactivated. For example, the polynucleotide to be modified or inactivated can be a coding region or a portion thereof essential for activity, or a regulatory element required for the expression of the coding region. Examples of such regulatory or control sequences can be promoter sequences or functional portions thereof, i.e., portions sufficient to affect the expression of the polynucleotide. Other control sequences that can be modified include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, signal peptide sequences, transcription terminators, and transcription activators.

[0183] The modification or inactivation of this polynucleotide can be performed by subjecting parental cells to mutagenesis and selecting mutant cells in which the expression of the polynucleotide is reduced or eliminated. This mutagenesis can be specific or random, for example, by using suitable physical or chemical mutagens, by using suitable oligonucleotides, or by mutagenesis generated by PCR of the DNA sequence. Furthermore, mutagenesis can be performed by any combination of these mutagens.

[0184] Examples of physical or chemical mutagens suitable for the purposes of this invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), o-methylhydroxylamine, nitrous acid, ethyl methanesulfonic acid (EMS), sodium bisulfite, formic acid, and nucleotide analogs.

[0185] When using these reagents, mutagenesis is generally performed under suitable conditions in the presence of the selected mutagen by incubating the parental cells to be mutated and screening and / or selecting mutant cells that exhibit reduced or absent gene expression.

[0186] The modification or inactivation of this polynucleotide can be accomplished by inserting, substituting, or deleting one or more nucleotides in the gene or in regulatory elements required for its transcription or translation. For example, the insertion or removal of nucleotides can result in the introduction of a stop codon, the removal of a start codon, or a change in the reading frame. Such modification or inactivation can be accomplished by site-directed mutagenesis or PCR-induced mutagenesis according to methods known in the art. Although, in principle, modification can be performed in vivo, i.e., directly on cells expressing the polynucleotide to be modified, it is preferred to perform the modification in vitro as illustrated in the example below.

[0187] Examples of convenient methods for eliminating or reducing the expression of polynucleotides are based on gene substitution, gene deletion, or gene disruption techniques. For example, in gene disruption methods, a nucleic acid sequence corresponding to an endogenous polynucleotide is mutagenized in vitro to produce a defective nucleic acid sequence, which is then transformed into parental cells to produce a defective gene. Through homologous recombination, the defective nucleic acid sequence replaces the endogenous polynucleotide. Desiredly, the defective polynucleotide also encodes a marker that can be used to select transformants in which the polynucleotide has been modified or disrupted. In one aspect, the polynucleotide is disrupted using selective markers such as those described herein.

[0188] The present invention further relates to mutant cells containing a disrupted or missing parental cell in the polynucleotide encoding the polypeptide or its control sequence or in the silent gene encoding the polypeptide, which results in the mutant cells producing less or no polypeptide compared to the parental cells.

[0189] Peptide-deficient mutant cells are useful as host cells for the expression of natural and heterologous peptides. Therefore, the present invention further relates to methods for producing natural or heterologous peptides, methods comprising: (a) culturing the mutant cells under conditions conducive to the production of the peptide; and (b) recovering the peptide. The term "heterologous peptide" means a peptide that is not natural to the host cell, such as a variant of a natural protein. The host cell may contain more than one copy of a polynucleotide encoding the natural or heterologous peptide.

[0190] The methods for culturing and purifying the target product can be carried out by methods known in the art.

[0191] The method of the present invention for producing products that are substantially free of proteases is of interest in the production of polypeptides (e.g., fungal proteins, such as enzymes). Protease-deficient cells can also be used to express heterologous proteins of pharmaceutical interest, such as hormones, growth factors, receptors, etc.

[0192] In another aspect, the present invention relates to protein products that are substantially free of protease activity, produced by the method of the present invention.

[0193] protease particles

[0194] The present invention also relates to enzyme particles / granules comprising the protease of the present invention. In one embodiment, the particle comprises a core and optionally one or more coatings (outer layers) surrounding the core.

[0195] The diameter of the core (measured as equivalent sphere diameter (average particle size based on volume)) is 20-2000 μm, particularly 50-1500 μm, 100-1500 μm or 250-1200 μm.

[0196] In one embodiment, the core comprises one or more polypeptides having the protease activity of the present invention.

[0197] The core may include additional materials such as fillers, fibrous materials (cellulose or synthetic fibers), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants, and fragrances.

[0198] The core may contain binders such as synthetic polymers, waxes, fats, or carbohydrates.

[0199] The core, typically a homogeneous blend, may contain salts of polyvalent cations, reducing agents, antioxidants, peroxide decomposition catalysts, and / or acidic buffer components.

[0200] The core may contain inert particles into which enzymes are adsorbed or applied (e.g., by fluidized bed coating) to the surface of the inert particles.

[0201] The diameter of the core can be 20-2000μm, specifically 50-1500μm, 100-1500μm, or 250-1200μm.

[0202] The core may be coated with at least one coating, for example, to improve storage stability, to reduce dust formation during processing, or to color the particles. The one or more optional coatings may include salt coatings or other suitable coating materials such as polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA).

[0203] The coating may be applied in an amount of at least 0.1% (e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%) based on the weight of the core. This amount may be at most 100%, 70%, 50%, 40%, or 30%.

[0204] The coating is preferably at least 0.1 μm thick, particularly at least 0.5 μm, at least 1 μm, or at least 5 μm thick. In some embodiments, the coating thickness is less than 100 μm, for example less than 60 μm or less than 40 μm.

[0205] The coating should seal the core unit by forming a substantially continuous layer. A substantially continuous layer should be understood as a coating with very few or no pores, such that the sealed / enclosed core unit has very few or no uncoated areas. The layer or coating should in particular be uniform in thickness.

[0206] The coating may further contain other materials known in the art, such as fillers, anti-sticking agents, pigments, dyes, plasticizers and / or adhesives, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0207] Salt coatings may contain at least 60% salt by weight, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% salt by weight.

[0208] To provide acceptable protection, the salt coating is preferably at least 0.1 μm thick, for example at least 0.5 μm, at least 1 μm, at least 2 μm, at least 4 μm, at least 5 μm, or at least 8 μm. In a particular embodiment, the salt coating thickness is less than 100 μm, for example less than 60 μm or less than 40 μm.

[0209] The salt can be added from a salt solution (where the salt is completely dissolved) or from a salt suspension (where the fine particles are less than 50 μm, for example less than 10 μm or less than 5 μm).

[0210] The salt coating may contain a single salt or a mixture of two or more salts. The salt may be water-soluble, particularly having a solubility of at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g / 100 g of water, for example at least 1 g / 100 g of water, or at least 5 g / 100 g of water.

[0211] The salt can be an inorganic salt, such as a sulfate, sulfite, phosphate, phosphonate, nitrate, chloride, or carbonate, or a salt of a simple organic acid (less than 10 carbon atoms, such as 6 or fewer carbon atoms), such as a citrate, malonate, or acetate. Examples of cations in these salts are alkali or alkaline earth metal ions, ammonium ions, or first transition metal ions, such as sodium, potassium, magnesium, calcium, zinc, or aluminum. Examples of anions include chloride, bromine, iodine, sulfate, sulfite, bisulfite, thiosulfate, phosphate, dihydrogen phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, silicate, citrate, malate, maleate, malonic acid, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate, or gluconate. In particular, alkali or alkaline earth metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate, or salts of simple organic acids such as citrate, malonate or acetate can be used.

[0212] The salt in the coating may have a constant humidity of more than 60% at 20°C, particularly more than 70%, more than 80% or more than 85%, or it may be another hydrated form of the salt (e.g., anhydrous). The salt coating may be as described in WO 00 / 01793 or WO2006 / 034710.

[0213] A specific example of a suitable salt is NaCl (CH3)20℃ =76%), Na2CO3(CH 20℃ =92%), NaNO3(CH 20℃ =73%), Na2HPO4(CH 20℃ =95%), Na3PO4(CH 25℃ =92%), NH4Cl(CH 20℃ =79.5%), (NH4)2HPO4(CH 20℃ =93.0%), NH4H2PO4(CH 20℃ =93.1%), (NH4)2SO4(CH 20℃ =81.1%), KCl(CH 20℃ =85%), K2HPO4(CH 20℃ =92%), KH2PO4(CH 20℃ =96.5%), KNO3(CH 20℃ =93.5%), Na2SO4(CH 20℃ =93%), K2SO4(CH 20℃ =98%), KHSO4(CH 20℃ =86%), MgSO4(CH 20℃ =90%), ZnSO4(CH 20℃ =90%) and sodium citrate (CH 25℃ =86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, and magnesium acetate.

[0214] The salt can be in anhydrous form, or it can be a hydrated salt, i.e., a crystalline salt hydrate with one or more bound water crystals, as described, for example, in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na₂SO₄), anhydrous magnesium sulfate (MgSO₄), and magnesium sulfate heptahydrate (MgSO₄). . 7H2O), zinc sulfate heptahydrate (ZnSO4) . 7H2O), disodium hydrogen phosphate heptahydrate (Na2HPO4) . 7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate, and magnesium acetate tetrahydrate.

[0215] Preferably, the salt is used as a salt solution, for example, in a fluidized bed.

[0216] These coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenol having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols, wherein the alcohol contains 12 to 20 carbon atoms and therein 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for use in fluidized bed technology applications are given in GB 1483591.

[0217] The particles may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA). Examples of enzyme particles with multiple coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0218] The core can be prepared by blends of granulated components, for example by methods including granulation techniques such as crystallization, precipitation, pan-coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, granulation, spheronization, particle size reduction, drum granulation, and / or high-shear granulation.

[0219] Methods for preparing the core can be found in the Handbook of Powder Technology; CECapes, Particle Size Enlargement, Volume 1, 1980; Elsevier. Preparation methods include known feed and pellet formulation techniques, such as:

[0220] a) Spray-dried products, wherein a liquid enzyme-containing solution is atomized in a spray drying tower to form small droplets, which are dried as they descend along the drying tower to form enzyme-containing particulate material; very small particles can be produced in this way (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0221] b) Layered products, wherein the enzyme is coated in layers around pre-formed inert core particles, wherein the enzyme-containing solution is typically atomized in a fluidized bed apparatus, in which the pre-formed core particles are fluidized and the enzyme-containing solution adheres to the core particles and is dried until a dry enzyme layer remains on the surface of the core particles. If useful core particles of the desired size can be found, particles of the desired size can be obtained in this manner. This type of product is described, for example, in WO 97 / 23606.

[0222] c) Absorbed core particles, wherein the enzyme is absorbed not by coating the core in layers, but by absorbing the enzyme on and / or within the surface of the core. Such a method is described in WO 97 / 39116.

[0223] d) Extruded or pelletized products, in which an enzyme-containing paste is pressed into pellets or extruded under pressure through small openings and cut into particles, which are then dried. These particles typically have a fairly large size because the material with the extrusion openings (usually a flat plate with perforations) limits the pressure drop allowed through the openings. Furthermore, when using small openings, the very high extrusion pressure increases the heat generated in the enzyme paste, which is detrimental to the enzyme (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Decker).

[0224] e) A granulated product in which enzyme-containing powder is suspended in molten wax and the suspension is sprayed, for example, through a rotary sprayer into a cooling chamber, where the droplets rapidly solidify (Michael S. Showell (ed.); Powdered Detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Decker). The resulting product is one in which the enzyme is uniformly distributed throughout the inert material rather than concentrated on its surface. This technique is described in U.S. Patent Nos. 4,016,040 and 4,713,245.

[0225] f) A mixer-granulated product in which an enzyme-containing liquid is added to a dry powder composition of commonly used granulation components. The liquid and powder are mixed in a suitable ratio, and because the water content of the liquid is absorbed by the dry powder, the dry powder components begin to adhere and aggregate, and the particles accumulate to form enzyme-containing granules. Such a method is described in U.S. Patent No. 4,106,991 and related documents EP 170360, EP 304332, EP 304331, WO 90 / 09440, and WO 90 / 09428. In specific products of this method, various high-shear mixers can be used as granulators. Granules consisting of enzymes, fillers, and binders are mixed with cellulose fibers to reinforce the granules, resulting in so-called T-granulates. The reinforced granules are more robust and release less enzyme dust.

[0226] g) Particle size reduction, in which core particles are generated by grinding or crushing larger enzyme-containing particles, pellets, flat sheets, briquettes, etc. The desired core particle fraction is obtained by sieving the grinding or crushing product. Oversized and undersized particles can be recovered. Particle size reduction is described in Martin Rhodes (ed.); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0227] h) Fluidized bed granulation. Fluidized bed granulation involves suspending microparticles in an airflow and spraying liquid through a nozzle onto the fluidized particles. The particles hit by the sprayed droplets become wetted and sticky. The sticky particles collide with and adhere to other particles to form granules.

[0228] i) These cores can be dried, for example, in a fluidized bed dryer. Those skilled in the art can use other known methods for drying granules in the feed or enzyme industry. Drying is preferably carried out at a product temperature from 25°C to 90°C. For some enzymes, it is important that the enzyme-containing core contains a small amount of water before salt coating. If water-sensitive enzymes are salt-coated before excess water removal, moisture will be trapped in the core and may negatively affect enzyme activity. After drying, these cores preferably contain 0.1-10% w / w of water.

[0229] Non-dust particles, such as those disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452, can be optionally coated by methods known in the art.

[0230] The particle can then be further packed with one or more additional enzymes. Each enzyme will then be present in more particles, ensuring a more uniform distribution of the enzymes and also reducing the physical separation of different enzymes due to the varying particle sizes. The method for generating multi-enzyme co-particles is disclosed in IP.com Publication IPCOM000200739D.

[0231] Another example of enzyme formulation using co-particles is disclosed in WO 2013 / 188331.

[0232] The present invention also relates to protected enzymes prepared according to the method disclosed in EP 238,216. Another aspect relates to particles comprising:

[0233] (a) a core containing a polypeptide according to any one of claims 1-6, and optionally (b) a coating consisting of one or more layers surrounding the core.

[0234] In one embodiment, the particles further comprise one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. These one or more additional enzymes are preferably selected from the group consisting of: acetylxylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranylase, cellobiase, cellulase, ferulic acid esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, amylopectinase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof.

[0235] Liquid preparations

[0236] The present invention also relates to liquid compositions comprising the protease of the present invention. The composition may comprise an enzyme stabilizer (examples of enzyme stabilizers include polyols (such as propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or boric acid derivatives (e.g., aromatic borate esters, or phenyl boric acid derivatives (such as 4-formylphenylboronic acid)).

[0237] In some embodiments, one or more fillers or one or more carrier materials are included to increase the volume of such compositions. Suitable fillers or carrier materials include, but are not limited to, various salts of sulfates, carbonates, and silicates, as well as talc, clay, etc. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols (including polyols and diols). Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In some embodiments, the composition contains about 5% to about 90% of such materials.

[0238] In one aspect, the present invention relates to a liquid preparation comprising:

[0239] (A) 0.001% to 25% w / w of one or more polypeptides having the protease activity of the present invention; and

[0240] (B) Water.

[0241] In another embodiment, the liquid formulation comprises 20% to 80% w / w of a polyol. In one embodiment, the liquid formulation comprises 0.001% to 2.0% w / w of a preservative.

[0242] In another embodiment, the present invention relates to a liquid preparation comprising:

[0243] (A) 0.001% to 25% w / w of one or more polypeptides having the protease activity of the present invention;

[0244] (B) 20% to 80% w / w polyols;

[0245] (C) Optionally, 0.001% to 2.0% w / w of a preservative; and

[0246] (D) Water.

[0247] In another embodiment, the present invention relates to a liquid preparation comprising:

[0248] (A) 0.001% to 25% w / w of one or more polypeptides having the protease activity of the present invention;

[0249] (B) 0.001% to 2.0% w / w of preservative;

[0250] (C) optionally 20% to 80% w / w of polyols; and

[0251] (D) Water.

[0252] In another embodiment, the liquid formulation comprises one or more formulations, such as formulations selected from the group consisting of: polyols, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate, and phosphate, preferably selected from the group consisting of: sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin, and calcium carbonate. In one embodiment, the polyol is selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propanediol or 1,3-propanediol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600, and polypropylene glycol (PPG) with an average molecular weight of less than about 600, more preferably selected from the group consisting of: glycerol, sorbitol, and propylene glycol (MPG), or any combination thereof.

[0253] In another embodiment, the liquid formulation comprises 20%-80% of a polyol (i.e., the total amount of polyols), for example, 25%-75% polyols, 30%-70% polyols, 35%-65% polyols, or 40%-60% polyols. In one embodiment, the liquid formulation comprises 20%-80% of a polyol, for example, 25%-75% polyols, 30%-70% polyols, 35%-65% polyols, or 40%-60% polyols, wherein the polyol is selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propanediol or 1,3-propanediol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600, and polypropylene glycol (PPG) with an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20%-80% of a polyol (i.e., the total amount of polyols), for example, 25%-75% polyols, 30%-70% polyols, 35%-65% polyols, or 40%-60% polyols, wherein the polyols are selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0254] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof. In one embodiment, the liquid formulation contains 0.02% to 1.5% w / w of a preservative, for example, 0.05% to 1.0% w / w, or 0.1% to 0.5% w / w. In one embodiment, the liquid formulation contains 0.001% to 2.0% w / w of a preservative (i.e., the total amount of preservative), for example, 0.02% to 1.5% w / w, 0.05% to 1.0% w / w, or 0.1% to 0.5% w / w, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof.

[0255] In another embodiment, the liquid formulation further comprises one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. These one or more additional enzymes are preferably selected from the group consisting of: acetylxylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranylase, cellobiase, cellulase, ferulic acid esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, amylopectinase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof.

[0256] Composition

[0257] The present invention also relates to compositions comprising the protease of the present invention. Preferably, these compositions are enriched with such a protease. The term “enrichment” means that the protease activity of the composition has increased, for example, by an enrichment factor of at least 1.1, such as at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2.0, at least 3.0, at least 4.0, at least 5.0, or at least 10.

[0258] These compositions may contain the polypeptide of the present invention as the main enzyme component, for example, a single-component composition. Such compositions may further contain formulations as described below. Alternatively, these compositions may contain more than one polypeptide of the present invention (e.g., a single-activity composition) and / or multiple enzyme activities, such as one or more (e.g., several) enzymes selected from the group consisting of: phytase, xylanase, galactanase, α-galactosidase, additional proteases, phospholipase A1, phospholipase A2, lysophospholipase, phospholipase C, phospholipase D, amylase, lysozyme, arabinofuranase, β-xylosidase, acetylxylan esterase, ferulic acid esterase, cellulase, cellobiase, β-glucosidase, amylopectinase, and β-glucanase or any combination thereof.

[0259] In one embodiment, the composition comprises a polypeptide of the third aspect of the invention and optionally a formulation. In one embodiment, the composition comprises a polypeptide of the fourth aspect of the invention and optionally a formulation. In one embodiment, the composition comprises a polypeptide of the fifth aspect of the invention and optionally a formulation. In one embodiment, the composition comprises a polypeptide of the sixth aspect of the invention and optionally a formulation.

[0260] Preparations

[0261] The enzymes of the present invention can be formulated as liquids or solids. For liquid formulations, the formulation may comprise polyols (such as, for example, glycerol, ethylene glycol, or propylene glycol), salts (such as, for example, sodium chloride, sodium benzoate, potassium sorbate), or sugars or sugar derivatives (such as, for example, dextrin, glucose, sucrose, and sorbitol). Thus, in one embodiment, the composition is a liquid composition comprising the polypeptides of the present invention and one or more formulations selected from the following list, which consists of: glycerol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, sodium chloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose, and sorbitol.

[0262] For solid formulations, the formulation can be, for example, in the form of granules, spray-dried powders, or agglomerates. The formulation may contain salts (organic or inorganic zinc, sodium, potassium, or calcium salts, such as, for example, calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch, or sugars or sugar derivatives (such as, for example, sucrose, dextrin, glucose, lactose, sorbitol).

[0263] In one embodiment, the solid composition is in particulate form. The particles may have a matrix structure in which the components are homogeneously mixed. However, the particles typically comprise a core particle and one or more coatings, typically salt and / or waxy coatings. The core particle may be a protease of the invention, optionally combined with one or more other enzymes, and optionally a homogeneous blend of one or more salts, or an inert particle (containing the protease of the invention, optionally combined with one or more other enzymes applied thereto).

[0264] In one embodiment, the material of the core particle is selected from the group consisting of: inorganic salts (such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or sugar or sugar derivatives (such as, for example, sucrose, dextrin, glucose, lactose, sorbitol), sugar or sugar derivatives (such as, for example, sucrose, dextrin, glucose, lactose, sorbitol), small organic molecules, starch, flour, cellulose, and minerals.

[0265] Salt coatings are typically at least 1 μm thick and can be a single salt or a mixture of salts, such as Na₂SO₄, K₂SO₄, MgSO₄, and / or sodium citrate. Other examples are those described, for example, in WO 2008 / 017659, WO 2006 / 034710, WO 1997 / 05245, WO 1998 / 54980, WO 1998 / 55599, WO 2000 / 70034, or polymer coatings, for example, as described in WO 2001 / 00042.

[0266] In another embodiment, the composition is a solid composition comprising the protease of the present invention and one or more formulations selected from the following list, which consists of: sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, and cellulose. In a preferred embodiment, the formulation is selected from one or more compounds selected from: sodium sulfate, dextrin, cellulose, sodium thiosulfate, and calcium carbonate. In a preferred embodiment, the solid composition is in granular form. In one embodiment, the solid composition is in granular form and comprises a core particle, an enzyme layer comprising the protease of the present invention, and a salt coating.

[0267] In another embodiment, the formulation is selected from one or more of the following compounds: glycerol, ethylene glycol, 1,2-propanediol or 1,3-propanediol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, and cellulose. In a preferred embodiment, the formulation is selected from one or more of the following compounds: 1,2-propanediol, 1,3-propanediol, sodium sulfate, dextrin, cellulose, sodium thiosulfate, and calcium carbonate.

[0268] Animal feed and feed additives

[0269] This invention also relates to animal feed compositions and animal feed additives. The animal feed compositions or diets have a relatively high protein content. Poultry and pig diets can be characterized as shown in columns 2-3 of Table B of WO 01 / 58275. Fish diets can be characterized as shown in column 4 of Table B. Furthermore, such fish diets typically have a crude fat content of 200-310 g / kg.

[0270] The animal feed composition according to the invention has a crude protein content of 50-800 g / kg, and further contains at least one protease as described herein or more than one protease as described herein.

[0271] In addition, or in alternatives to (the above-mentioned crude protein content), the animal feed composition of the present invention has a metabolizable energy of 10-30 MJ / kg; and / or a calcium content of 0.1-200 g / kg; and / or an available phosphorus content of 0.1-200 g / kg; and / or a methionine content of 0.1-100 g / kg; and / or a methionine plus cysteine ​​content of 0.1-150 g / kg; and / or a lysine content of 0.5-50 g / kg.

[0272] In certain embodiments, the contents of metabolizable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine ​​and / or lysine fall within any one of ranges 2, 3, 4 or 5 (R.2-5) in Table B of WO 01 / 58275.

[0273] Crude protein was calculated by multiplying nitrogen (N) by a factor of 6.25, i.e., crude protein (g / kg) = N (g / kg) x 6.25. Nitrogen content was determined by the Kjeldahl method (AOAC, 1984, Official Methods of Analysis, 14th edition, Association of Official Analytical Chemists, Washington, D.C.).

[0274] Metabolizable energy can be calculated from the following sources: NRC publication *Nutrient Requirements in Swine*, Ninth Reprint 1988, Subcommittee on Swine Nutrition, Committee on Animal Nutrition, Board of Agriculture, National Research Council. National Academy Press, Washington, D.C., pp. 2-6; and the European Table of Energy Values ​​for Poultry Feed-stuffs, Spelderholt Centre for Poultry Research and Extension, 7361DA Beckbergen, Netherlands, Grafisch bedrijf Ponsen & looijen bv, Wageningen. ISBN 90-71463-12-5.

[0275] The dietary content of calcium, available phosphorus, and amino acids in the whole animal diet was calculated based on feed tables such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6,8219pk Lelystad. ISBN 90-72839-13-7.

[0276] In one particular embodiment, the animal feed composition of the present invention comprises at least one plant protein as defined above.

[0277] The animal feed compositions of the present invention may also contain animal protein, such as meat and bone meal, feather meal and / or fish meal, typically in an amount of 0-25%. The animal feed compositions of the present invention may also contain dried distillers grains with solubles (DDGS), typically in an amount of 0-30%.

[0278] In yet another specific embodiment, the animal feed composition of the present invention contains 0-80% corn; and / or 0-80% sorghum; and / or 0-70% wheat; and / or 0-70% barley; and / or 0-30% oats; and / or 0-40% soybean meal; and / or 0-25% fish meal; and / or 0-25% meat and bone meal; and / or 0-20% whey.

[0279] Animal feed may contain plant-based protein. In specific embodiments, the plant-based protein content is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (w / w). The plant-based protein may be derived from plant protein sources such as legumes and cereals, for example, materials derived from plants of the Fabaceae, Brassicaceae, Chenopodiaceae, and Poaceae families, such as soybean meal, lupin meal, rapeseed meal, and combinations thereof.

[0280] In one specific embodiment, the plant protein source is material from one or more plants of the Fabaceae family (e.g., soybean, lupin, pea, or common bean). In another specific embodiment, the plant protein source is material from one or more plants of the Chenopodiaceae family (e.g., beet, sugar beet, spinach, or quinoa). Other examples of plant protein sources are rapeseed and cabbage. In yet another specific embodiment, soybean is a preferred plant protein source. Other examples of plant protein sources are cereals such as barley, wheat, rye, oats, corn, rice, and sorghum.

[0281] Animal diets can be formulated, for example, into powdered (non-pelletized) or pelleted feed. Typically, the milled feed is mixed and supplemented with adequate amounts of essential vitamins and minerals according to the instructions for the type discussed. Enzymes are added as solid or liquid enzyme formulations. For example, for powdered feeds, solid or liquid enzyme formulations can be added before or during the ingredient mixing step. For pelleted feeds, the (solid or liquid) protease / enzyme preparation can also be added before or during the feed formulation step. Typically, the liquid protease / enzyme preparation contains the protease of the present invention, optionally accompanied by a polyol (e.g., glycerol, ethylene glycol, or propylene glycol), and is added after the pelleting step, for example, by spraying the liquid formulation onto pellets. The enzyme can also be incorporated into feed additives or premixes.

[0282] Alternatively, proteases can be prepared by freezing a mixture of liquid enzyme solution and a leavening agent (e.g., ground soybean flour) and then freeze-drying the mixture.

[0283] In one embodiment, the composition comprises one or more additional enzymes. In one embodiment, the composition comprises one or more microorganisms. In one embodiment, the composition comprises one or more vitamins. In one embodiment, the composition comprises one or more minerals. In one embodiment, the composition comprises one or more amino acids. In one embodiment, the composition comprises one or more other feed ingredients.

[0284] In another embodiment, the composition comprises one or more polypeptides of the present invention, one or more formulations, and one or more additional enzymes. In one embodiment, the composition comprises one or more polypeptides of the present invention, one or more formulations, and one or more microorganisms. In one embodiment, the composition comprises one or more polypeptides of the present invention, one or more formulations, and one or more vitamins. In one embodiment, the composition comprises one or more polypeptides of the present invention and one or more minerals. In one embodiment, the composition comprises one or more polypeptides of the present invention, one or more formulations, and one or more amino acids. In one embodiment, the composition comprises one or more polypeptides of the present invention, one or more formulations, and one or more other feed ingredients.

[0285] In another embodiment, the composition comprises one or more polypeptides of the present invention, one or more formulations, and one or more components selected from the list of the following: one or more additional enzymes; one or more microorganisms; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.

[0286] The final protease concentration in the diet is in the range of 0.01-200 mg protease protein / kg diet, preferably between 0.5-100 mg / kg diet, more preferably 2-50 mg, and even more preferably 5-25 mg protease protein / kg animal diet.

[0287] The protease is currently expected to be administered in one or more of the following amounts (dosage ranges): 0.01-200; 0.01-100; 0.5-100; 1-50; 5-100; 5-50; 10-100; 0.05-50; 5-25; or 0.10-10 - all of these ranges are in mg protease protein / kg feed (ppm).

[0288] To determine the number of mg of protease protein per kg of feed, the protease was purified from the feed composition, and the specific activity of the purified protease was determined using relevant tests (see Protease Activity). The protease activity of the feed composition was determined using the same tests, and the dosage, in mg of protease protein per kg of feed, was calculated based on these two determinations.

[0289] In a particular embodiment, the animal feed additive of the present invention is intended to be contained (or specified to be required to be contained) in animal diets or feeds at a level of 0.01% to 10.0%, more particularly 0.05% to 5.0% or 0.2% to 1.0% (% means g additive / 100g feed). The same applies to premixes.

[0290] The same principle is used to determine the mg of protease protein in feed additives. Of course, if a sample of the protease used to prepare the feed additive or feed is available, the specific activity can be determined from that sample (without needing to purify the protease from the feed composition or additive).

[0291] Other enzymes

[0292] In another embodiment, the compositions described herein optionally comprise one or more enzymes. Enzymes can be classified according to the handbook Enzyme Nomenclature (from NC-IUBMB, 1992), and also see the ENZYME website on the Internet: http: / / www.expasy.ch / enzyme / . ENZYME is an information repository relative to enzyme nomenclature. It is primarily based on the recommendations of the International Union of Biochemistry and Molecular Biology Nomenclature Committee (IUB-MB), Academic Press, Inc., 1992, and describes each type of enzyme characterized, providing the enzyme with an EC (Enzyme Committee) number (Bairoch. 2000, The ENZYME database, Nucleic Acids Res. 28:304-305). This IUB-MB enzyme nomenclature is based on their substrate specificity, and sometimes on their molecular mechanisms; this classification does not reflect the structural characteristics of these enzymes.

[0293] Henrissat et al. described another classification of certain glycosidases (such as endoglucanases, galactanases, mannanases, glucanases, lysozymes, and galactosidases) in “The carbohydrate-active enzymes database (CAZy) in 2013”, Nucl. Acids Res. (January 1, 2014) 42(D1):D490-D495; see also www.cazy.org.

[0294] Therefore, the compositions of the present invention may further comprise at least one other enzyme selected from the group consisting of: acetylxylan esterase (EC 3.1.1.23), acylglycerol lipase (EC 3.1.1.72), α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), arabinofuranylase (EC 3.2.1.55), cellobiose hydrolase (EC 3.2.1.91), cellulase (EC 3.2.1.4), ferulic acid esterase (EC 3.1.1.73), galactanase (EC 3.2.1.89), α-galactosidase (EC 3.2.1.22), β-galactosidase (EC 3.2.1.23), β-glucanase (EC 3.2.1.6), β-glucosidase (EC 3.2.1.21), triacylglycerol lipase (EC 3.2.1.6), β-glucosidase (EC 3.2.1.21), and triacylglycerol lipase (EC 3.2.1.23). 3.1.1.3), lysophospholipase (EC 3.1.1.5), lysozyme (EC 3.2.1.17), α-mannosidase (EC 3.2.1.24), β-mannosidase (mannanase) (EC 3.2.1.25), phytase (EC 3.1.3.8, EC 3.1.3.26, EC 3.1.3.72), phospholipase A1 (EC 3.1.1.32), phospholipase A2 (EC 3.1.1.4), phospholipase D (EC 3.1.4.4), protease (EC 3.4), amylopectinase (EC 3.2.1.41), pectin esterase (EC 3.1.1.11), xylanase (EC 3.2.1.8, EC 3.2.1.136), β-xylosidase (EC 3.1.1.3), lysophospholipase (EC 3.1.1.5), lysozyme (EC 3.2.1.17), α-mannosidase (EC 3.2.1.24), β-mannosidase (EC 3.2.1.25), phytase (EC 3.1.3.8, EC 3.1.3.26, EC 3.1.3.72), phospholipase A1 (EC 3.1.1.32), phospholipase A2 (EC 3.1.1.4), phospholipase D (EC 3.1.4.4), protease (EC 3.4), amylopectinase (EC 3.2.1.41), pectin esterase (EC 3.1.1.11), xylanase (EC 3.2.1.8, EC 3.2.1. 3.2.1.37) or any combination thereof.

[0295] In one embodiment, the composition of the present invention comprises galactanase (EC 3.2.1.89) and β-galactosidase (EC 3.2.1.23).

[0296] In one embodiment, the composition of the present invention comprises phytase (EC 3.1.3.8 or 3.1.3.26). Examples of commercially available phytases include Bio-Feed. TM Phytase (Novozymes) P, NP and HiPhos (DSM Nutritional Products), Natuphos TM (BASF) and Natuphos TM E (BASF) and Blue (AB Enzymes), (Huvepharma) Phytase (Aveve Biochem) XP (Verenium / DuPont) and PHY (DuPont). Other preferred phytases include those described, for example, WO 98 / 28408, WO 00 / 43503, and WO 03 / 066847.

[0297] In one embodiment, the composition of the present invention comprises xylanase (EC 3.2.1.8). Examples of commercially available xylanases include... WX (Dissmann Nutrition Products Company) XT and Barley (ABVista Corporation (AB Vista)), (Verenium) X (Haowei Pharmaceutical Company) XB (xylanase / β-glucanase, DuPont) and XAP (xylanase / amylase / protease, DuPont) XG 10 (xylanase / glucanase) and 02CS (xylanase / glucanase / pectinase, Aveve Biochem), Naturgrain (BASF).

[0298] In one embodiment, the composition of the present invention comprises a protease (EC 3.4). Examples of commercially available proteases include... ProAct (DISM Nutrition Products), Winzyme Pro (Suntaq International Limited) and DP100 (Novus International).

[0299] In one embodiment, the composition of the present invention comprises α-amylase (EC 3.2.1.1). Examples of commercially available α-amylases include A and RumiStar TM (DISM Nutrition Products Company)

[0300] In one embodiment, the composition of the present invention comprises a multi-component enzyme product, such as... Octazyme (Framelco) G2 VP and MultiGrain (DISM Nutrition Products) Excel or Advance (Adisseo) DC (endo-1,3(4)-β-glucanase and endo-1,4-β-xylanase, Andres Pintaluba SA) or (Endo-1,3(4)-β-glucanase and endo-1,4-β-xylanase and α-amylase, Andres Pintaluba SA).

[0301] Health products

[0302] Eubiotics are compounds intended to provide a healthy balance of gut microbiota. Eubiotics include many different feed additives, such as probiotics, prebiotics, phytonutrients (essential oils), and organic acids, which are described in more detail below.

[0303] Probiotics

[0304] In one embodiment, the animal feed composition further comprises one or more additional probiotics. In one embodiment, the animal feed composition further comprises bacteria from one or more genera including: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnivora, Propionibacterium, Bifidobacterium, Clostridium, and Megacoccus, or any combination thereof.

[0305] In one embodiment, the animal feed composition further comprises bacteria from one or more of the following strains: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circumflex, Enterococcus faecalis, Enterococcus species and Pediococcus species, Lactobacillus species, Bifidobacterium species, Lactobacillus acidophilus, Pediococcus lactis, Lactococcus lactis, Bifidobacterium bifidum, Propionibacterium tergenti, Lactobacillus sausageii, Lactobacillus rhamnosus, Clostridium butyricum, Bifidobacterium animalis subsp. animalis, Lactobacillus reuteri, Lactobacillus salivarius subsp. salivarius, Giant cocci, and Propionibacterium species.

[0306] In another embodiment, the composition, animal feed additive, or animal feed further comprises bacteria from one or more of the following Bacillus subtilis strains: 3A-P4 (PTA-6506), 15A-P4 (PTA-6507), 22C-P1 (PTA-6508), 2084 (NRRL B-500130), LSSA01 (NRRL-B-50104), BS27 (NRRL B-50105), BS 18 (NRRLB-50633), BS 278 (NRRL B-50634), DSM 29870, DSM 29871, DSM 32315, NRRL B-50136, NRRLB-50605, NRRL B-50606, NRRL B-50622, and PTA-7547.

[0307] In another embodiment, the composition, animal feed additive, or animal feed further comprises bacteria from one or more of the following Bacillus pumilus strains: NRRL B-50016, ATCC 700385, NRRL B-50885, or NRRL B-50886.

[0308] In another embodiment, the composition, animal feed additive, or animal feed further comprises bacteria from one or more of the following Bacillus licheniformis strains: NRRL B 50015, NRRL B-50621, or NRRL B-50623.

[0309] In another embodiment, the composition, animal feed additive, or animal feed further comprises bacteria from one or more of the following Bacillus amyloliquefaciens strains: DSM 29869, DSM 29869, NRRL B 50607, PTA-7543, PTA-7549, NRRL B-50349, NRRL B-50606, NRRL B-50013, NRRL B-50151, NRRL B-50141, NRRL B-50147, or NRRL B-50888.

[0310] The bacterial count of each bacterial strain in this animal feed composition is in the range of 1 x 10^6. 4 and 1x 10 14 The CFU / kg dry matter content is preferably between 1 x 10⁻⁶. 6 and 1x 10 12 Between CFU / kg of dry matter, and more preferably between 1 x 10⁻⁶. 7 Up to 1x10 11 The CFU / kg dry matter content is between [value missing]. In another embodiment, the bacterial count of each bacterial strain in the animal feed composition is between 1 x 10 [value missing]. 8 and 1x 10 10 Between CFU / kg of dry matter.

[0311] The bacterial count of each bacterial strain in this animal feed composition is in the range of 1 x 10^6. 5 and 1x 10 15 Between CFU / animal / day, preferably in 1x10 7 and 1x 10 13 Between CFU / animal / day, and more preferably in 1 x 10 8 and 1x 10 12 Between CFU / animal / day. In another embodiment, the bacterial count for each bacterial strain in the animal feed composition is between 1 x 10⁻⁶. 9 and 1x 10 11 Between CFU / animal / day. In one embodiment, the amount of probiotics is 0.001% to 10% by weight of the composition.

[0312] In another embodiment, the one or more bacterial strains exist in a stable spore form.

[0313] Examples of commercial products are (DISM Nutrition Products), Alterion (Adisseo), Enviva PRO (DuPont Animal Nutrition) (Mixed enzymes + probiotics, DuPont Animal Nutrition) and (Norel / Evonik) and PY1 (Evonik).

[0314] prebiotics

[0315] Prebiotics are substances that induce the growth or activity of microorganisms (e.g., bacteria and fungi) that contribute to the health of the host. Prebiotics are typically indigestible fibrous compounds that pass undigested through the upper gastrointestinal tract and stimulate the growth or activity of beneficial bacteria that colonize the large intestine by acting as their substrate. Generally, prebiotics increase the number or activity of Bifidobacteria and Lactobacilli in the gastrointestinal (GI) tract.

[0316] Yeast derivatives (inactivated whole yeast cells or yeast cell walls) can also be considered prebiotics. They typically contain mannan oligosaccharides, yeast beta-glucan, or protein contents, and are usually derived from the cell walls of yeast (Saccharomyces cerevisiae).

[0317] In one embodiment, the amount of prebiotic is 0.001% to 10% by weight of the composition. An example of a yeast product is... And Agrimos (Lallemand Animal Nutrition).

[0318] Phytobiotics

[0319] Phytokines are a group of natural or non-antibiotic growth promoters derived from herbs, spices, or other plants and used as feed additives. Phytokines can be single substances prepared from essential oils / extracts, essential oils / extracts, single plants, and plant mixtures (herbal products) or mixtures of essential oils / extracts / plants (specialty products).

[0320] Examples of phytochemicals include rosemary, sage, oregano, thyme, clove, and lemongrass. Examples of essential oils include thymol, eugenol, m-cresol, vanillin, salicylates, resorcinol, guajacol, gingerol, lavender oil, ionone, irisone, eucalyptol, menthol, peppermint oil, α-pinene, limonene, anethole, linalool, methyl dihydrojasmonate, carvacrol, propionic acid / propionic acid ester, acetate / acetic acid ester, butyric acid / butyric acid ester, rosemary oil, clove oil, geraniol, terpineol, citronellol, amyl salicylate and / or benzyl salicylate, cinnamaldehyde, plant polyphenols (tannins), turmeric, and turmeric extract.

[0321] In one embodiment, the amount of phytochemical is 0.001% to 10% by weight of the composition. Examples of commercial products are... (DISM Nutrition Products), Cinergy TM Biacid TM , ProHacidTM Classic and ProHacidTM Advance TM (all Promivi Corporation / Cargill) and Envivo EO (DuPont Animal Nutrition).

[0322] organic acids

[0323] Organic acids (C1-C7) are widely distributed in nature as normal components of plant or animal tissues. They are also formed through microbial fermentation of carbohydrates, primarily in the large intestine. They are commonly used in swine and poultry production as alternatives to antibiotic growth promoters because they have a preventative effect against intestinal problems such as necrotizing enterocolitis in chickens and E. coli infection in piglets. Organic acids can be sold as single components or, typically, as mixtures of two or three different organic acids. Examples of organic acids include short-chain fatty acids (e.g., formic acid, acetic acid, propionic acid, butyric acid), medium-chain fatty acids (e.g., hexanoic acid, octanoic acid, capric acid, lauric acid), di / tricarboxylic acids (e.g., fumaric acid), hydroxy acids (e.g., lactic acid), aromatic acids (e.g., benzoic acid), citric acid, sorbic acid, malic acid, tartaric acid, or their salts (usually sodium or potassium salts, such as potassium diformate or sodium butyrate).

[0324] In one embodiment, the amount of organic acid is 0.001% to 10% by weight of the composition. An example of a commercial product is... (DISM Nutrition Products Company) (BASF), AF (OXEA) and Adimix Precision (Nutriad).

[0325] premix

[0326] In one embodiment, the animal feed may include a premix, which may contain, for example, vitamins, minerals, enzymes, amino acids, preservatives, antibiotics, other feed ingredients, or any combination thereof mixed into the animal feed.

[0327] amino acids

[0328] The compositions of the present invention may further comprise one or more amino acids. Examples of amino acids for use in animal feed are lysine, alanine, β-alanine, threonine, methionine, and tryptophan. In one embodiment, the amount of amino acids is from 0.001% to 10% by weight of the composition.

[0329] Vitamins and minerals

[0330] In another embodiment, the animal feed may contain one or more vitamins, such as one or more fat-soluble vitamins and / or one or more water-soluble vitamins. In another embodiment, the animal feed may optionally contain one or more minerals, such as one or more trace minerals and / or one or more macro minerals.

[0331] Typically, fat-soluble vitamins and water-soluble vitamins, along with trace minerals, form part of what is known as a premix intended to be added to feed, while macro-minerals are usually added to feed separately.

[0332] Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, such as vitamin K3.

[0333] Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and pantothenates, such as Ca-D-pantothenate.

[0334] Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, and zinc.

[0335] Non-limiting examples of major minerals include calcium, magnesium, potassium, and sodium.

[0336] The nutritional requirements for these components (taking poultry and piglets / pigs as examples) are listed in Table A of WO 01 / 58275. Nutritional requirements refer to the specified concentrations of these components that should be provided in the diet.

[0337] In an alternative, the animal feed additive of the present invention comprises at least one of the individual components specified in Table A of WO 01 / 58275. At least one means any one or more of one, two, three, four, etc., up to all thirteen or all fifteen individual components. More specifically, this at least one individual component is included in the additive of the present invention in an amount providing an in-feed concentration within the range described in column four, five, or six of Table A.

[0338] In another embodiment, the animal feed additive of the present invention contains at least one of the following vitamins, preferably at a concentration in feed that falls within the ranges defined in Table 1 below (for piglet diets and broiler diets, respectively).

[0339] Table 1: Recommended Typical Vitamins

[0340]

[0341]

[0342] Other feed ingredients

[0343] The compositions of the present invention may further comprise colorants, stabilizers, growth-improving additives and aromatic compounds / flavorings, polyunsaturated fatty acids (PUFAs); reactive oxygen species, antimicrobial peptides, and antifungal peptides.

[0344] Examples of colorants are carotenoids, such as beta-carotene, astaxanthin, and lutein.

[0345] Examples of aromatic compounds / flavorings are methoxycresol, anethole, decadecyl, undecyl and / or dodecyl lactone, ionone, irisone, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin, and tannins.

[0346] Examples of antimicrobial peptides (AMPs) are CAP18, leucocin A, tritrpticin, protegrin-1, thanatin, avidin, lactoferrin, lactoferrin peptide, and oxavirin such as Noviprin (Robert Lehrer, 2000), phenocrine, and statins (including compounds and peptides disclosed in WO 03 / 044049 and WO 03 / 048148) as well as variants or fragments of the above that retain antimicrobial activity.

[0347] Examples of antifungal peptides (AFPs) are peptides from Aspergillus megaterium and Aspergillus niger, along with variants and fragments that retain their antifungal activity, as disclosed in WO 94 / 01459 and WO 02 / 090384.

[0348] Examples of polyunsaturated fatty acids are C18, C20, and C22 polyunsaturated fatty acids, such as arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid, and γ-linolenic acid.

[0349] Examples of reactive oxygen species (ROS) generating substances include chemicals such as perborates, persulfates, or percarbonates; and enzymes such as oxidases, oxygenases, or synthases.

[0350] The compositions of the present invention may further comprise at least one amino acid. Examples of amino acids for use in animal feed are lysine, alanine, β-alanine, threonine, methionine, and tryptophan.

[0351] use

[0352] The present invention also relates to methods for using polypeptides or combinations thereof having protease activity (for example, for animal feed).

[0353] Uses in animal feed

[0354] The proteases of the present invention can also be used in animal feed. In one embodiment, the present invention provides a method for preparing an animal feed composition, the method comprising adding one or more proteases of the present invention to one or more animal feed ingredients.

[0355] According to WO 00 / 21381 and WO 04 / 026334, one or more proteases of the present invention can also be used in animal feed as feed-enhancing enzymes to improve feed digestibility and increase its utilization efficiency.

[0356] In another embodiment, the protease of the present invention can be used in animal feed or as a feed additive, wherein it can have a positive effect on the animal's digestive tract and thereby improve animal production performance, or improve animal health, such as reducing mortality, based on weight gain, feed conversion ratio (FCR), European Production Efficiency Factor (EPEF), European Production Effectiveness Factor (EFF). FCR is calculated as feed intake per animal relative to weight gain per animal.

[0357] In the application according to the invention, the protease can be fed to the animal before, after, or simultaneously with a diet. The latter is preferred.

[0358] In one particular embodiment, the form of the protease when added to or included in feed additives is explicitly defined. "Explicitly defined" means that the protease formulation is at least 50% pure, as determined by size exclusion chromatography (see Example 12 of WO 01 / 58275). In other particular embodiments, the protease formulation is at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, or at least 95% pure, as determined by this method.

[0359] Specific protease formulations are advantageous. For example, it is easier to determine the correct dosage in feed for proteases that are substantially unaffected or uncontaminated by other proteases. The term "dose correctly" specifically refers to the goal of obtaining consistent and stable results, and the ability to optimize dosage based on the desired effect.

[0360] However, for use in animal feed, proteases do not have to be pure; they can include other enzymes, in which case they can be called protease preparations.

[0361] This protease preparation can be (a) added directly to feed, or (b) used in the production of one or more intermediate compositions, such as feed additives or premixes, which are subsequently added to feed (or used in the processing). Regardless of whether it is used according to (a) or (b) above, the purity mentioned above refers to the purity of the original protease preparation.

[0362] In particular, protease preparations with the purity of the aforementioned orders of magnitude can be obtained using recombinant production methods. However, when producing proteases using conventional fermentation methods, obtaining these protease preparations is not easy, and there are significant batch-to-batch variations.

[0363] These types of protease preparations can certainly be mixed with other enzymes.

[0364] The protein can be an animal protein, such as meat and bone meal, feather meal, and / or fish meal; or it can be a plant protein.

[0365] As used herein, the term plant-based protein means any compound, composition, formulation, or mixture comprising at least one protein derived from or derived from a plant, including modified proteins and protein derivatives. In specific embodiments, the plant-based protein contains at least 10%, 20%, 30%, 40%, 50%, or 60% (w / w).

[0366] Plant-based proteins can be derived from plant protein sources such as legumes and cereals, for example, materials derived from plants of the Fabaceae, Brassicaceae, Chenopodiaceae, and Poaceae families, such as soybean flour, lupin flour, and rapeseed flour.

[0367] In one particular embodiment, the plant-based protein source is material derived from one or more plants of the Fabaceae family (e.g., soybean, lupin, pea, or common bean).

[0368] In another specific embodiment, the plant-based protein source is material from one or more plants of the Chenopodiaceae family (e.g., beets, sugar beets, spinach, or quinoa).

[0369] Other examples of plant-based protein sources include rapeseed, sunflower seeds, cottonseed, and cabbage.

[0370] Soybeans are a preferred source of plant-based protein.

[0371] Other examples of plant-based protein sources include cereals such as barley, wheat, rye, oats, corn, rice, black wheat, and sorghum.

[0372] In a particular embodiment of the treatment process, the protease or proteases discussed affect (or exert effects on) these proteins, such as plant proteins or protein sources, or impose hydrolytic or degradative effects. To achieve this, the protein or protein source is generally suspended in a solvent, such as an aqueous solvent like water, and the pH and temperature values ​​are adjusted with due attention to the characteristics of the enzyme in question. For example, treatment may be carried out at pH values ​​that allow the actual protease activity to be at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90%. Similarly, treatment may be carried out, for example, at temperatures that allow the actual protease activity to be at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90%. The above activity percentages are relative to maximum activity. The enzymatic reaction is continued until the desired result is obtained, and the reaction can then be terminated by, for example, inactivating the enzyme through a heat treatment step, or the reaction may not be terminated.

[0373] In another specific embodiment of the processing of the present invention, the action of the protease is maintained, which means that, for example, the protease is added to these proteins, but its hydrolytic effect is not activated until later, when there is a need, once appropriate hydrolytic conditions are established, or once any enzyme inhibitor is inactivated, or whatever other way the action of the enzyme is delayed, its hydrolytic effect will be activated.

[0374] In one embodiment, the treatment is pre-treatment of animal feed or proteins used in animal feed, i.e., these proteins are hydrolyzed before ingestion.

[0375] The term "improving the nutritional value of animal feed" refers to enhancing the availability of nutrients in the feed. In this invention, improving nutritional value specifically refers to improving the availability of the protein portion of the feed, resulting in increased protein extraction, higher protein yield, and / or improved protein utilization. When the nutritional value of the feed increases, the digestibility of protein and / or amino acids increases, and the animal's growth rate and / or weight gain and / or feed conversion (i.e., feed intake relative to weight gain) can be improved.

[0376] Proteases can be added to feed in any form, such as when they are relatively pure proteases, or as a mixture with other components to be added to animal feed, i.e., in the form of animal feed additives, such as so-called animal feed premixes.

[0377] Another aspect relates to the use of the polypeptides of the present invention.

[0378] (a) In animal feed;

[0379] (b) In animal feed additives;

[0380] (c) In the preparation of compositions for use in animal feed;

[0381] (d) Used to improve the nutritional value of animal feed;

[0382] (e) Used to increase the digestible and / or soluble protein in animal feed;

[0383] (f) Used to increase the degree of protein hydrolysis in animal diets; and / or

[0384] (g) is used to process proteins.

[0385] Another aspect of the invention relates to a method for improving the nutritional value of animal feed.

[0386] Improving the nutritional value of animal feed, increasing the digestible and / or soluble protein in animal feed, and increasing the degree of protein hydrolysis in animal diets can save farmers feed costs.

[0387] Another aspect of the invention relates to the use of the polypeptides of the invention in supporting a healthy digestive system in pigs, swine, or poultry.

[0388] Another aspect of the invention relates to the use of the peptides of the invention to reduce the release of phosphorus and nitrogen into the environment.

[0389] Preparation method

[0390] In one embodiment, the invention also relates to a method for preparing animal feed or feed additives, the method comprising preparing animal feed or feed additives comprising animal feed and a protease of the first aspect of the invention.

[0391] In one embodiment, the invention also relates to a method for preparing animal feed or feed additives, the method comprising preparing animal feed or feed additives containing animal feed and the protease of the second aspect of the invention.

[0392] In one embodiment, the invention also relates to a method for preparing animal feed or feed additives, the method comprising preparing animal feed or feed additives containing animal feed and the protease of the third aspect of the invention.

[0393] In one embodiment, the invention also relates to a method for preparing animal feed or feed additives, the method comprising preparing animal feed or feed additives containing animal feed and the protease of the fourth aspect of the invention.

[0394] In one embodiment, the invention also relates to a method for preparing animal feed or feed additives, the method comprising preparing animal feed or feed additives containing animal feed and the protease of the fifth aspect of the invention.

[0395] In one embodiment, the invention also relates to a method for preparing animal feed or feed additives, the method comprising preparing animal feed or feed additives containing animal feed and the protease of the sixth aspect of the invention.

[0396] signal peptide

[0397] The present invention also relates to isolated polynucleotides encoding a signal peptide comprising or consisting of amino acids 1 to 25 of SEQ ID NO:1. The polynucleotide may further comprise a gene encoding a protein operatively linked to the signal peptide and / or propeptide. Preferably, the protein is heterologous to the signal peptide and / or propeptide.

[0398] The present invention also relates to nucleic acid constructs, expression vectors and recombinant host cells containing such polynucleotides.

[0399] The present invention also relates to a method for producing a protein, the method comprising: (a) culturing a recombinant host cell containing such a polynucleotide; and optionally (b) recovering the protein.

[0400] The protein can be native or heterologous to the host cell. The term "protein" is not intended to refer to a coding product of a specific length, and therefore encompasses peptides, oligopeptides, and polypeptides. The term "protein" also encompasses two or more polypeptides that combine to form a coding product. These proteins also include hybrid polypeptides and fusion polypeptides.

[0401] Preferably, the protein is a hormone, enzyme, receptor or a portion thereof, antibody or a portion thereof, or reporter gene. For example, the protein can be a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, such as α-galactosidase, α-glucosidase, aminopeptidase, amylase, β-galactosidase, β-glucosidase, β-xylosidase, glycosylase, carboxypeptidase, catalase, cellobiase, cellulase, chitinase, keratinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucosylamylase, invertase, laccase, lipase, mannosidase, polysaccharidase, oxidase, pectinase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

[0402] This gene can be obtained from any prokaryotic, eukaryotic, or other source.

[0403] Example

[0404] 1. An isolated or purified polypeptide having protease activity, wherein the isolated or purified polypeptide is selected from the group consisting of:

[0405] (a) A polypeptide having at least 95% sequence identity with the mature polypeptide of SEQ ID NO:1;

[0406] (b)(a) fragments of polypeptides that have protease activity.

[0407] 2. The polypeptide according to Example 1, wherein the polypeptide has at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:1.

[0408] 3. The polypeptide according to Example 1 or 2, wherein the polypeptide is a variant of the mature polypeptide containing substitution, deletion, and / or insertion of SEQ ID NO:1 at one or more positions.

[0409] 4. The polypeptide according to any one of Examples 1-3, wherein the polypeptide comprises, is substantially composed of or consists of SEQ ID NO:1 or its mature polypeptide.

[0410] 5. The polypeptide according to any one of Examples 1-4, wherein the polypeptide is a fragment of SEQ ID NO:1 or its mature polypeptide, wherein the fragment has protease activity.

[0411] 6. The polypeptide according to any one of Examples 1-5, wherein the polypeptide is obtained from or can be obtained from Bacillus subtilis polypeptide.

[0412] 7. A fusion polypeptide comprising the polypeptide and the second polypeptide according to any one of Examples 1-6.

[0413] 8. A particle comprising:

[0414] (a) Containing a core of a polypeptide according to any one of Examples 1-6, and optionally

[0415] (b) A coating consisting of one or more layers surrounding the core.

[0416] 9. A composition comprising a polypeptide according to any one of Examples 1-6.

[0417] 10. Use of at least one polypeptide according to any one of Examples 1-6:

[0418] In animal feed;

[0419] In animal feed additives;

[0420] In the preparation of compositions for use in animal feed;

[0421] Used to improve the nutritional value of animal feed;

[0422] Used to increase the digestible and / or soluble protein in animal feed;

[0423] Used to increase the degree of protein hydrolysis in animal diets; and / or

[0424] Used for processing proteins.

[0425] 11. A method for improving the nutritional value of animal feed, the method comprising adding to the feed at least one polypeptide according to any one of Examples 1-6.

[0426] 12. An animal feed additive comprising:

[0427] At least one polypeptide according to any one of Examples 1-6; and

[0428] At least one fat-soluble vitamin, and / or

[0429] At least one water-soluble vitamin, and / or

[0430] At least one trace mineral.

[0431] 13. The animal feed additive according to Example 11 further comprises one or more amylases; phytases; xylanases; galactanases; α-galactosidases; proteases, phospholipases, β-glucanases, or any mixture thereof.

[0432] 14. An animal feed comprising the animal feed additives according to Example 11 or 12.

[0433] 15. The animal feed according to Example 13 has a crude protein content of 50 to 800 g / kg.

[0434] 16. A method for processing a protein, the method comprising the step of adding at least one polypeptide according to any one of Examples 1-6 to at least one protein or protein source.

[0435] 17. The method according to Example 16, wherein soybeans are contained in the at least one protein source.

[0436] 18. Use of at least one polypeptide according to any one of Examples 1-6 in animal feed.

[0437] 19. An animal feed composition comprising at least one polypeptide according to any one of Examples 1-6.

[0438] 20. The animal feed composition according to Example 19, wherein the composition comprises one or more additional enzymes.

[0439] 21. The animal feed composition according to Example 20, wherein these additional enzymes are selected from the group consisting of: amylase, catalase, cellulase, keratinase, endoglucanase, halogenated peroxidase, lipase, mannanase, pectinase, pectin lyase, peroxidase, protease, xanthan gumase and xyloglucanase, or any mixture thereof.

[0440] 22. An isolated or purified polynucleotide encoding a polypeptide according to any one of Examples 1-6.

[0441] 23. A nucleic acid construct or expression vector comprising a polynucleotide according to Example 22, wherein the polynucleotide is preferably operatively linked to one or more control sequences that direct the production of the polypeptide in an expression host.

[0442] 24. A recombinant host cell comprising the polynucleotide according to Example 22, the polynucleotide being operatively linked to one or more control sequences that direct the production of the polypeptide.

[0443] 25. The recombinant host cell according to Example 24, wherein the polypeptide is heterologous to the recombinant host cell.

[0444] 26. The recombinant host cell according to Example 24 or 25, wherein at least one of the one or more control sequences is heterologous to the polynucleotide encoding the polypeptide.

[0445] 27. The recombinant host cell according to any one of Examples 24-26, comprising at least two copies, such as three, four or five, of the polynucleotide described in Example 22.

[0446] 28. The recombinant host cell according to any one of claims 24-27, wherein the recombinant host cell is a yeast recombinant host cell, for example, cells of the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia*, *Saccharomyces*, *Saccharomyces*, or *Yersinia*, such as *Kluyveromyces lactis*, *Kalvatia*, *Saccharomyces cerevisiae*, *Saccharomyces sacchariformis*, *Saccharomyces davidiana*, *Douglas*, *Kluyveromyces kluyvernsis*, *Nordiya*, *Ovoyces*, or *Yersinia lipolytica* cells.

[0447] 29. The recombinant host cell according to any one of Examples 24-27, wherein the recombinant host cell is a filamentous fungal recombinant host cell, such as *Cladosporium*, *Aspergillus*, *Briefomus*, *Cirsium*, *Pseudomonas*, *Aureospora*, *Coprinus*, *Cladosporium*, *Cryptococcus*, *Ustilago*, *Fusarium*, *Pyrophyllus*, *Pyrophyllus*, *Mucor*, *Pyrophyllus*, *Nematophytes*, *Penicillium*, *Penicillium*. Cells of the genera *Penicillium*, *Pleurotus*, *Radix*, *Ruminocytophyte*, *Pleurotus*, *Schizophyllum*, *Basilaria*, *Thermophilic Ascomycetes*, *Fusporium*, *Cyclophorus*, *Pterocaryon*, or *Trichoderma*, especially *Aspergillus buergerianus*, *Aspergillus sulphureus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus oryzae*, *Aspergillus niger*, *Aspergillus oryzae*, *Pterocaryon*, *Carnegiea*, *Pterocaryon*, *Pterocaryon*, *Pterocaryon*, *Pterocaryon*, and *Pterocaryon*. Wax fungi, Insect-like wax fungi, Narrow-sided golden spores, Keratopteric golden spores, Lukenowens golden spores, Coprophytic golden spores, Rent-type golden spores, Queen's golden spores, Tropical golden spores, Brown-thin golden spores, Grey-capped coprinus, Trichoderma, Fusarium solani, Cereal-like Fusarium, Cuvier's Fusarium, Large-scaled Fusarium, Gramineae Fusarium, Gramineae Fusarium, Heterosporium Fusarium, Albizia Fusarium, Fusarium oxysporum, Fusarium multibranchii, Pink Fusarium, Bone-setting Fusarium oxysporum, Fusarium scabiosaefolium, Fusarium pseudobranchii, Fusarium sulfideum, Fusarium rotundum, Fusarium pseudofilariae, Fusarium variegatum, specific humic mold, loose cottony humic mold, Rhizopus spp., thermophilic filamentous mold, Structurally scabiosaefolium, Penicillium purpureoides, Pleurotus ostreatus, Pleurotus eryngii, Pleurotus ostreatus, Clostridium perfringens, Trichoderma harzianum, Trichoderma cornigrin, Trichoderma longibranchii, Trichoderma reesei, or green Trichoderma cells.

[0448] 30. The recombinant host cell according to any one of Examples 24-27, wherein the recombinant host cell is a prokaryotic recombinant host cell, for example, Gram-positive cells selected from the group consisting of: Bacillus spp., Clostridium spp., Enterococcus spp., Bacillus terrestris spp., Lactobacillus spp., Lactococcus spp., Bacillus spp., Staphylococcus spp., Streptococcus spp., or Streptomyces spp. cells; or Gram-negative bacteria selected from the group consisting of: Campylobacter spp., Escherichia coli, Flavobacterium spp., Fusobacterium spp., Helicobacter spp., Staphylococcus spp., Neisseria spp., Pseudomonas spp. Cells of the genera *Salmonella* and *Ureaplasma*, such as *Bacillus alkalophilus*, *Bacillus amyloliquefaciens*, *Bacillus brevis*, *Bacillus circulans*, *Bacillus croceae*, *Bacillus coagulans*, *Bacillus sclerotiorum*, *Bacillus splenicus*, *Bacillus stenosis*, *Bacillus licheniformis*, *Bacillus megaterium*, *Bacillus pumilus*, *Bacillus thermophilus*, *Bacillus subtilis*, *Bacillus thuringiensis*, *Streptococcus equi*, *Streptococcus pyogenes*, *Streptococcus lactis* and *Streptococcus equi* subsp. *porphyria*, *Streptococcus nonchromogenic*, *Streptococcus pyogenes*, *Streptococcus auricula*, *Streptococcus grayi*, and *Streptococcus purpureus* cells.

[0449] 31. A method for producing a polypeptide according to any one of Examples 1-6, the method comprising culturing cells under conditions favorable for producing the polypeptide, the cells producing the polypeptide in their wild-type form.

[0450] 32. The method according to Example 30, further comprising recovering the polypeptide.

[0451] 33. A method for producing a polypeptide with protease activity, the method comprising culturing a recombinant host cell according to any one of Examples 24-30 under conditions favorable for the production of the polypeptide.

[0452] 34. The method according to Example 33, further comprising recovering the polypeptide.

[0453] 35. A method for generating a mutant of a parent cell, the method comprising inactivating a polynucleotide encoding a polypeptide according to any one of Examples 1-6, which results in the mutant producing less polypeptide compared to the parent cell.

[0454] 36. A mutant cell generated by the method according to Example 35.

[0455] 37. The mutant cell according to Example 36 further comprises a gene encoding a native or heterologous protein.

[0456] 38. A method for producing a protein, the method comprising culturing mutant cells as described in Example 36 or 37 under conditions conducive to the production of the protein.

[0457] 39. The method of claim 37, further comprising recovering the protein.

[0458] 40. An isolated or purified polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 25 of SEQ ID NO:1, the polynucleotide being operatively linked to a polynucleotide encoding a polypeptide heterologous to the signal peptide.

[0459] 41. A nucleic acid construct or expression vector comprising the polynucleotide described in Example 40.

[0460] 42. A recombinant host cell comprising the nucleic acid construct or expression vector according to Example 41.

[0461] 43. A method for producing a protein, the method comprising culturing a recombinant host cell as described in Example 42 under conditions conducive to the production of the protein.

[0462] 44. The method according to Example 43, the method further comprising recovering the protein.

[0463] 45. A whole culture medium formulation or cell culture composition comprising a polypeptide according to any one of Examples 1-6.

[0464] 46. ​​An animal feed additive comprising one or more polypeptides having protease activity, wherein the polypeptide is an S8 protease, and wherein the S8 protease is selected from the group consisting of:

[0465] (a) A polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:1;

[0466] (b) A polypeptide encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide encoding sequence of SEQ ID NO:1;

[0467] (c) A variant of any one of SEQ ID NO:1, wherein the variant has protease activity and contains one or more substitutions, and / or one or more deletions, and / or one or more insertions or any combination thereof at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50;

[0468] (d) A polypeptide comprising (a), (b) or (c) and a polypeptide with an N-terminal and / or C-terminal His-tag and / or HQ-tag;

[0469] (e) A polypeptide comprising (a), (b), or (c) and a polypeptide extended at the N-terminus and / or C-terminus of up to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; and

[0470] (f) A fragment of a polypeptide of (a), (b) or (c) having protease activity and having at least 90% of the length of a mature polypeptide.

[0471] The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0472] Example

[0473] Example 1: Expression of S8 protease from Bacillus subtilis

[0474] The gene encoding the S8 protease (SEQ ID NO:1) from *Bacillus subtilis* was codon-optimized and synthesized using integrated DNA technology (Interleuvenlaan 12A, B-3001 Leuven, Belgium). An expression construct was prepared that expresses the gene as a secretase. In this construct, the natural signal peptide was replaced with the *Bacillus clausti* signal peptide (having the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA (signal peptide from SEQ ID NO:1)).

[0475] The construct was prepared as a linearly integrated construct, wherein the synthesized gene was PCR-fused with a chloramphenicol resistance marker via a strong promoter between two Bacillus subtilis homologous chromosomal regions. Fusion was performed via SOE PCR (Horton et al., 1989, Gene 77:61-68). The SOE PCR method is also described in patent application WO 2003 / 095658. The gene was expressed under the control of a triple promoter system (as described in WO 99 / 43835), consisting of a Bacillus licheniformis α-amylase gene promoter containing a stable sequence, a Bacillus amyloliquefaciens α-amylase gene promoter (amyQ), and a Bacillus thuringiensis cryIIIA promoter. The linear PCR construct was transformed into Bacillus subtilis. Transformants were selected on LB plates supplemented with 6 μg chloramphenicol per ml. The recombinant Bacillus subtilis clone was grown in liquid culture medium. The recombinase accumulated in the supernatant after natural cell lysis. Collect the supernatant containing the enzymes and purify these enzymes as described in Example 2.

[0476] Example 2: Purification of S8 protease from Bacillus subtilis

[0477] Centrifuge the culture medium (20000 x g, 20 min) and carefully decant the supernatant from the precipitate. Filter the supernatant through a Nalgene 0.2 μm filter to remove any remaining Bacillus host cells. Add solid (NH4)2SO4 to the 0.2 μm filtrate to achieve a final concentration of 1.4 M (NH4)2SO4, and apply the S8 protease solution to the phenyl-... FF column (from GE Healthcare). After thoroughly washing the column with equilibration buffer, the S8 protease was eluted with a mixture of 75% (20 mM HEPES, 1 mM CaCl2 (pH 8.0)) and 25% 2-propanol. G25 was used. The column (from GE Healthcare) was used to transfer the eluted S8 protease peak to 10 mM CH3COOH / NaOH, 1 mM CaCl2 (pH 5.0). The G25 column was then used for further processing. The transferred S8 protease was applied to a source equilibrated in 10 mM CH3COOH / NaOH and 1 mM CaCl2 (pH 5.0). TM 30S column (from GE Healthcare). After thorough washing of the column with equilibration buffer, the protease was eluted with five column volumes using a linear gradient between equilibration buffer and 10 mM CH3COOH / NaOH, 1 mM CaCl2, and 500 mM NaCl (pH 5.0). The protease activity of the fractions from this column was analyzed (Suc-AAPF-pNA activity assay at pH 9), and the activity fractions were further analyzed by SDS-PAGE. The fractions (a major band of 38 kDa was visible on a Coomassie blue-stained SDS-PAGE gel) were pooled. The pooled product was a purified formulation used for further characterization.

[0478] Example 3: Apparent jejunal nitrogen digestibility of novel protease homologs in broilers compared to baselines (Cibenza and XAP proteases).

[0479] Animals and feeding

[0480] One-day-old chickens (Cobb 500) obtained from a commercial hatchery (Accouvoir multiplicateur Grelier, La Bohardière, France) were used. The chickens were housed in cable-stayed cages (0.75 m² / cage, 6 chickens / cage). Free access to feed and water was provided until day 7. On day 7, the chickens were weighed and assigned to one of six treatments based on body weight. A similar diet was continued until day 16. The experimental period spanned from day 16 to day 21 of the chickens' lives. During the experiment, the chickens were fed either a negative control diet (NC) or NC + test enzymes (Table 5). The enzymes were provided in liquid form and applied to the treatments via spraying using an ultra-low pressure system coupled to a Forberg F60 mixer. All diets contained TiO2 as a digestibility marker.

[0481] Table 5: Composition and Chemical Analysis of the Diet

[0482]

[0483]

[0484] Data and sample collection

[0485] Between days 16 and 21, mean body weight and feed consumption per cage and per treatment were obtained. On day 21, all chickens were euthanized by cervical dislocation. Chickens were dissected and jejunal contents were collected. The jejunum was defined as the segment of the small intestine that begins at the end of the pancreatic loop (duodenum) and terminates distally 1 cm proximal to the Merkel diverticulum. Jejunal digests were consolidated in one cage, lyophilized, and ground for chemical analysis. Crude protein and TiO2 concentrations were determined in the digests and feed samples for subsequent estimation of the apparent jejunal nitrogen digestibility (AJDN) (%) given in Table 6.

[0486] AJDN(%)=100-[(CMf / CMe)x(CNe / CNf)]x 100

[0487] CMf = Concentration labeled in feed; CMe = Concentration labeled in jejunal digestate;

[0488] CNf = concentration of nutrients in feed; CNe = concentration of nutrients in jejunal digestate.

[0489] According to the Dumas method (Dumas, JBA, Procedes de l'Analyse Organique [Organic Analysis Methods], Ann. Chim. Phys. [Annals of Chemistry and Physics] 247: 198–213 (1831)), through the LECO apparatus FP-528 ( The company determined the nitrogen content. A factor of 6.25 was used to convert the nitrogen content into crude protein.

[0490] According to DIN EN ISO 11885:1997 (DIN EN ISO 1998), after the samples were mineralized with H2SO4, the concentration of titanium dioxide in feed and digests was determined by an inductively coupled plasma (ICP) apparatus ICP-OES 5100 (Agilent Technologies).

[0491] Table 6: Results of in vivo experiments

[0492]

[0493] Compared to both NC and positive benchmarks (Cibenza and XAP), the protease increased apparent jejunal nitrogen digestibility. Cibenza is... DP100 (Novus) is a formulation of Bacillus licheniformis (ATCC 53757), and its protease (EC 3.4.21.19) is an S1 peptidase (trypsin family). It is administered at the recommended dose (0.25-0.5 kg / metric ton) with a minimum activity of 6,000,000 U / g. XAP (DuPont) is a combination of xylanase (X), amylase (A), and protease (P), providing 2,000 U of X, 200 U of A, and 4,000 U of P per kg of diet.

[0494] The invention described and claimed herein is not limited to the specific aspects disclosed herein, as these aspects are intended to illustrate several aspects of the invention. Any equivalent aspects are intended to be within the scope of the invention. In fact, various modifications to the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the disclosure including the definition shall prevail.

Claims

1. An isolated or purified polypeptide having protease activity, the isolated or purified polypeptide being selected from the group consisting of: (a) a polypeptide having at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 1, which is from Bacillus oceanisediminis; wherein the mature polypeptide is amino acids 26-425 of SEQ ID NO: 1; (b) a polypeptide comprising the polypeptide of (a) and an N-terminal and / or C-terminal His-tag and / or HQ-tag; and (c) the polypeptide is SEQ ID NO: 1 or its mature polypeptide, the mature polypeptide is Amino acids 26-425 of SEQ ID NO:

1.

2. The polypeptide according to claim 1, which has 100% sequence identity with the mature polypeptide of SEQ ID NO:

1.

3. Use of at least one polypeptide according to any one of claims 1 to 2: (a) in animal feed; (b) in animal feed additives; (c) in the preparation of a composition for use in animal feed; (d) for improving the nutritional value of animal feed; (e) for increasing digestible and / or soluble protein in animal feed; (f) for increasing the extent of protein hydrolysis in an animal's diet; and / or (g) For processing proteins.

4. A method for improving the nutritional value of animal feed, comprising adding to the feed at least one polypeptide according to any one of claims 1-2.

5. An animal feed additive comprising: (a) at least one polypeptide according to any one of claims 1-2; and (b) at least one fat-soluble vitamin, and / or (c) at least one water-soluble vitamin, and / or (d) at least one trace mineral.

6. The animal feed additive according to claim 5, further comprising one or more of amylase; phytase; xylanase; galactanase; α-galactosidase; protein enzyme, phospholipase, beta-glucanase or any mixture thereof.

7. An animal feed comprising the animal feed additive according to claim 5 or 6.

8. An isolated or purified polynucleotide encoding the polypeptide according to any one of claims 1-2.

9. A nucleic acid construct or expression vector comprising the polynucleotide according to claim 8, wherein the polynucleotide is operably linked to to one or more control sequences that direct the production of the polypeptide in an expression host.

10. A recombinant host cell comprising the polynucleotide of claim 8 operably linked to one or more control sequences that direct the production of the polypeptide.

11. The recombinant host cell according to claim 10, which is a yeast A parent recombinant host cell; or The recombinant host cell is a filamentous fungal recombinant host cell; or The recombinant host cell is a prokaryotic recombinant host cell.

12. The recombinant host cell according to claim 11, wherein the recombinant host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Schizosaccharomyces or a Yarrowia cell; or The recombinant host cell is a member of the genus Acremonium, Aspergillus, Acidis, Nicotiana, Ceroplastes, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Ustilago, Fusarium, Humicola, Pyrospora, Mucor, Myceliophthora, Neotrichia, Alternaria Pseudomonas, Penicillium, Pseudomonas, Radius, Ruminochytrium, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Toxoplasma, Trametes or Trichoderma cells; or The recombinant host cell is a Gram-positive cell selected from the group consisting of: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus Bacillus, Staphylococcus, Streptococcus or Streptomyces cells, or a Gram-negative bacterial cell selected from the group consisting of Negative bacteria, this group consists of: Campylobacter spp., Enterobacter, Flavobacterium, Fusobacterium, Helicobacter, Gluebacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells.

13. The recombinant host cell according to claim 12, wherein the recombinant host cell is a lactobacillus Kluyveromyces acidophilus, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cells; or The recombinant host cell is Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans Aspergillus, black Aspergillus, oryzae, black thorn tobacco pipe mold, dry wax fungus, Carnegie wax fungus, light yellow wax fungus, Pannoshita wax fungus, ring-band wax fungus, slightly red wax fungus, insect wax fungus, narrow-edged golden spore, horny golden spore, Lukenowensi golden spore, fecal golden spore or The recombinant host cell is alkalophilic Bacillus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus radiata, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans cells. The recombinant host cell according to claim 11 , which is a Saccharomyces cerevisiae or Bacillus spp.

15. An animal feed additive comprising one or more polypeptides having protease activity, wherein the polypeptide is an S8 protease, and wherein the S8 protease is selected from the group consisting of: (a) a polypeptide having at least 99%, or 100% sequence identity to SEQ ID NO: 1, which is from Bacillus oceanisediminis; wherein the mature polypeptide is amino acids 26-425 of SEQ ID NO: 1; (b) a polypeptide encoded by a polynucleotide corresponding to SEQ ID NO: 1 has 100% sequence identity with the mature polypeptide coding sequence; (c) the polypeptide is SEQ ID NO: 1 or its mature polypeptide, the mature polypeptide is amino acids 26-425 of SEQ ID NO: 1; and (d) a polypeptide comprising (a), (b) or (c) and an N-terminus and / or C-terminus His-tagged and / or HQ-tagged polypeptides.

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