Polypeptides having serine protease activity and polynucleotides encoding them and their use in animal feed

Abstract

The present invention relates to animal feed or animal feed additive comprising a polypeptide having protease activity and uses thereof. Specifically, the protease is a serine S1 protease from the genera Microspora, Terracoccus and Knoellia, all belonging to the family Microbacteriaceae of the suborder Micrococineae. These proteases have high activity over a broad pH range (pH 3-7) and are thus highly active during the entire passage through the digestive tract. It further relates to methods for producing these proteases as well as using the proteases to improve animal performance and the nutritional value of animal feed.

Description

[0001] References to sequence lists

[0002] This application contains a sequence list in a computer-readable form, which is incorporated herein by reference. Background of the Invention

[0003] Invention Field

[0004] This invention relates to animal feeds or animal feed additives containing polypeptides with protease activity and their uses. It also relates to methods for producing these proteases and for using proteases to improve animal performance and the nutritional value of animal feed. Background of the Invention

[0006] In the use of proteases (in vivo) in animal feed, and / or the use of such proteases for processing plant proteins (in vitro), it is important to note 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.

[0007] When soybean meal, for example, is included in the feed of monogastric animals such as pigs and poultry, a significant proportion of soybean meal is not effectively digested (the apparent ileal protein digestibility in piglets, growing pigs, and poultry such as broilers, laying hens, and roosters is only about 80%).

[0008] 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 1–2, 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 in the pH range of 4–6. Protein digestion by a protease 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 and thus can survive in the gastric environment, while simultaneously being effectively active across a wide range of physiological pH levels in the target animal's digestive tract, are particularly desirable.

[0009] Since animal feed is typically formulated in granular form, where steam is used in the granulation process, it is desirable that the proteases used in animal feed remain active after exposure to the steam treatment.

[0010] In order to produce proteases for industrial use, it is important to produce proteases in high yields so that a sufficient quantity of the product can be obtained to supply the proteases at a favorable price.

[0011] Related technical specifications

[0012] Proteases from the S1 family are known in the art and are used in animal feed. For example, WO01 / 58275 discloses the use of acid-stable proteases from the subtilis protease family in animal feed. WO 01 / 58276 discloses the use of acid-stable proteases (10R proteases) derived from Nocardia spp. NRRL 18262 and proteases derived from Nocardia albopictus DSM 14010 in animal feed. WO 04 / 072221, WO 04 / 111220, WO 04 / 111223, WO 05 / 035747, and WO 05 / 123911 disclose proteases related to 10R proteases and their use in animal feed. WO 04 / 072279 discloses the use of other proteases in animal feed. WO 04 / 034776 discloses the use of Bacillus subtilis protease / keratinase, PWD-1 from Bacillus licheniformis, in poultry feed.

[0013] Soybeans and corn are two highly used protein sources in agriculture, and therefore, it is important that proteases exhibit good activity against these substrates. Numerous studies have disclosed the activity of proteases from various bacterial sources on soybean-corn flour, including species of *Kribbella* (WO 2013 / 026796), *Rhodotorula sacchariformis* (WO 2013 / 110766 and WO 2014 / 122161), *Saccharomyces viride* (WO 2013 / 189972), *Saccharomyces australis* (WO 2013 / 041689), and species of *Dactylogyrus* (WO 2014 / 096259).

[0014] Furthermore, proteases isolated from other bacterial species (e.g., Janibacter sp.) are known in the art. Thrash et al. performed a shotgun genome sequencing of the bacterium HTCC2649, as described in “Genome sequence of the Marine Janibacter Sp. Strain HTCC2649”, 2011, J. Bacteriol. 193:584-585, which has been submitted to EMBL / GenBank under accession number AAMN01000001. From this genome sequencing, the polypeptide with Uniprot number A3TJ83 (SEQ ID NO:2 in this paper) was annotated as a serine protease.

[0015] Yoshida et al. performed a whole-genome shotgun biopsy of the bacterium Austwickia chelonae NBRC 105200, which was submitted to the EMBL / GenBank / DDBJ databases. The peptidase (Uniprot:K6VM97, SEQ ID NO:9) from these databases was annotated with SEQ ID NO:5 (corresponding to the mature polypeptides of SEQ ID NO:2 and 4 in this paper) having 62.1% sequence identity.

[0016] Commercial products containing a protease and sold for use in animal feed include ProAct (DSM NP / Novozymes) (DuPont) (DuPont) (DuPont), Allzyme TM (Alltech) (BioResources, Int., Poultrygrow) TM (Jefo) and (Novus).

[0017] However, there is still a need to find proteases that exhibit improved properties in the field of animal feed. Invention Overview

[0019] This invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, wherein the polypeptide is selected from the group consisting of the following items:

[0020] (a) A polypeptide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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:5;

[0021] (b) A polypeptide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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:14;

[0022] (c) A polypeptide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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:20;

[0023] (d) A polypeptide encoded by a polynucleotide that hybridizes with the following under low-strict, medium-strict, medium-high-strict, high-strict, or very high-strict conditions:

[0024] (i) The mature polypeptide coding sequence of SEQ ID NO:1;

[0025] (ii) The mature polypeptide coding sequence of SEQ ID NO:10;

[0026] (iii) The mature polypeptide coding sequence of SEQ ID NO:16; or

[0027] (iv)(i), (ii) or (iii) full-length complements;

[0028] (e) A polypeptide encoded by a polynucleotide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 coding sequence of SEQ ID NO:1;

[0029] (f) A polypeptide encoded by a polynucleotide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 coding sequence of SEQ ID NO:10;

[0030] (g) A polypeptide encoded by a polynucleotide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 coding sequence of SEQ ID NO:16;

[0031] (h) A variant of SEQ ID NO:5, SEQ ID NO:14, or SEQ ID NO:20, wherein the variant has protease activity and includes 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; and

[0032] (i) A fragment of a polypeptide of (a), (b), (c), (d), (e), (f), (g), or (h) having protease activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids.

[0033] The present invention further relates to the use of the protease of the present invention in animal feed, methods for preparing animal feed, methods for improving the nutritional value of animal feed, methods for processing proteins, methods for improving the digestibility and / or solubility of proteins, methods for improving one or more performance parameters in animals, and methods for producing the polypeptides of the present invention.

[0034] Sequence List Overview

[0035] SEQ ID NO:1 is the DNA sequence of S1 protease 1 from the genus *Hypericum* HTCC2649.

[0036] SEQ ID NO:2 is the amino acid sequence as deduced from SEQ ID NO:1.

[0037] SEQ ID NO:3 is a DNA sequence of the recombinant expressed DNA sequence from SEQ ID NO:1.

[0038] SEQ ID NO:4 is the amino acid sequence as deduced from SEQ ID NO:3.

[0039] SEQ ID NO:5 is the amino acid sequence of mature S1 protease 1 from the genus *Hypericum* HTCC2649.

[0040] SEQ ID NO:6 is a secretion signal from Bacillus clausii.

[0041] SEQ ID NO:7 is the DNA sequence of proteinase 10R (WO 05 / 035747, SEQ ID NO:1).

[0042] SEQ ID NO:8 is the amino acid sequence of proteinase 10R (WO 05 / 035747, SEQ ID NO:2).

[0043] SEQ ID NO:9 is the amino acid sequence of a peptidase from Austwickia chelonae NBRC 105200 (Uniprot:K6VM97).

[0044] SEQ ID NO:10 is the DNA sequence of S1 protease 1 from the genus *Terracoccus*.

[0045] SEQ ID NO:11 is the amino acid sequence as deduced from SEQ ID NO:10.

[0046] SEQ ID NO:12 is a DNA sequence derived from the recombinant expressed DNA sequence of SEQ ID NO:10.

[0047] SEQ ID NO:13 is the amino acid sequence as deduced from SEQ ID NO:12.

[0048] SEQ ID NO:14 is the amino acid sequence of mature S1 protease 1 from the genus *Terracoccus*.

[0049] SEQ ID NO:15 is the conserved motif VCG[E / Q]KVGQP.

[0050] SEQ ID NO:16 is the DNA sequence of S1 protease 1 from Knoellia flava.

[0051] SEQ ID NO:17 is the amino acid sequence as deduced from SEQ ID NO:16 (Uniprot:A0A0A0JF07).

[0052] SEQ ID NO:18 is the DNA sequence of the synthetic gene with codon optimization of SEQ ID NO:16.

[0053] SEQ ID NO:19 is the amino acid sequence as deduced from SEQ ID NO:18.

[0054] SEQ ID NO:20 is the amino acid sequence of mature S1 protease 1 from Norfolk's bacterium. Brief description of the attached diagram

[0056] Figure 1 The pH-activity profiles of S1 protease 1 from the genus *Herba Alternaria* HTCC2649 on the Suc-AAPF-pNA substrate are shown at 25 °C, compared to protease 10R.

[0057] Figure 2 The pH-stability curves (residual activity after 2 hours at 37°C) of S1 protease 1 from Alternaria HTCC2649 are shown compared to protease 10R.

[0058] Figure 3 The temperature activity curves of S1 protease 1 from the genus *Hypertricis* HTCC2649 against protazyme AK at pH 7.0 are shown compared to protease 10R at pH 6.5.

[0059] Figure 4 The activity (OD) of S1 protease 1 from *Haloxylon ammodendron* HTCC2649 on soybean-corn flour was shown compared to that of protease 10R. 340 (x dilution factor). The error bar shows 2 standard deviations.

[0060] Figure 5 The activity (OD) of S1 protease 1 from *Haloxylon ammodendron* HTCC2649, *Haloxylon ammodendron* variants S68N and T71N, and S1 protease 1 from *Terracoccus* sp. on soybean-corn flour compared to 10R protease is shown. 340 (x dilution factor).

[0061] definition

[0062] Peptide activity against soybean-corn flour: The term "peptide activity against soybean-corn flour" refers to the protease activity of an enzyme against a 30:70 mixture of soybean and corn flour, determined using o-phthalaldehyde (OPA) assays as described herein. Examples of pH measurements are pH 3.0, 4.0, 5.0, 6.0, and 7.0. Examples of temperature measurements are 30°C, 35°C, 40°C, 45°C, and 50°C. Examples of time measurements are 2, 3, and 4 hours. Examples of enzyme concentrations are 50, 100, 150, 200, 250, and 300 mg enzyme protein / kg dry matter substrate.

[0063] In a preferred embodiment, the activity of the peptide against soybean-corn flour was determined by the following: Soybean-corn flour (1 g) mixed at a 30:70 ratio was added to a buffer solution containing 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CAPS, 1 mM CaCl2, 150 mM KCl, and 0.01% Triton X-100 (10 mL). This buffer solution had been prepared and adjusted to a pH of 3.0, 4.0, 5.0, 6.0, or 7.0 after the soybean-corn flour substrate was mixed with the assay buffer. Then, an aliquot of the substrate slurry (2 mL) was mixed for 30 min. A solution dissolved in 100 μl of 100 mM sodium acetate buffer (9.565 g / L NaOAc, 1.75 g / L acetic acid, 5 mM...) was added. The protease (200 mg enzyme protein / kg dry matter) was prepared in CaCl2, 0.01% BSA, 0.01% Tween 20, pH 6.0; the sample was incubated at 40°C (500 rpm) for 3 hours; the sample was centrifuged (10 min, 4000 rpm, 0°C); and the supernatant was collected for analysis using o-phthalaldehyde (OPA) assay (referred to herein as the “soybean-corn flour assay”). In another preferred embodiment, as described in Example 4 herein, the activity of the peptide against soybean-corn flour was determined.

[0064] In one embodiment, the polypeptide of the present invention, such as SEQ ID NO:5, exhibits at least 30%, for example, at least 40%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% activity against soybean-corn flour at pH 4. In another embodiment, the polypeptide of the present invention, such as SEQ ID NO:5, exhibits at least 40%, for example, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% activity against soybean-corn flour at pH 5.

[0065] Allelic variants: The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variations arise naturally from mutations and can lead to polymorphism within a population. Gene mutations can be silent (without alteration in the encoded polypeptide) or can encode a polypeptide with a modified amino acid sequence. Allelic variants of a polypeptide are polypeptides encoded by allelic variants of a gene.

[0066] 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 cows, and calves), deer, yank, camels, llamas, and kangaroos. Non-ruminant animals include monogastric animals, such as 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, bass, bluefish, bocachico, cyprinids, catfish, cachama, carp, catfish, catfish, flounder, charfish, cichlids, cod, sunfish, golden snapper, croaker, eel, goby, goldfish, gudgeon, grouper, guapote, halibut, Java fish, and scad). Fish, 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, 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).

[0067] Animal feed: The term "animal feed" refers to any compound, preparation, or mixture suitable for, or intended for, to be ingested by animals. Animal feed for monogastric animals typically includes concentrates along with vitamins, minerals, enzymes, direct-feeding microorganisms, amino acids, and / or other feed ingredients (e.g., in premixes), while animal feed for ruminants typically includes forage (including roughage and silage) and may further include concentrates along with vitamins, minerals, enzymes, direct-feeding microorganisms, amino acids, and / or other feed ingredients (e.g., in premixes).

[0068] Weight gain: The term “weight gain” refers to the increase in the live weight of an animal during a given period of time, such as the weight gain from day 1 to day 21.

[0069] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules derived from eukaryotic or prokaryotic cells. cDNA lacks the intron sequences that can be present in the corresponding genomic DNA. Early initial RNA transcripts are precursors to mRNA, undergoing a series of processing steps, including splicing, before becoming mature, spliced ​​mRNA.

[0070] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly identifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are generally determined by an open reading frame, which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0071] Composition: The term "composition" refers to a composition comprising a carrier and at least one enzyme of the present invention. The composition described herein can be mixed with animal feed and can be referred to as "powdered feed".

[0072] 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 peanut kernel), palm kernel cake, yeast-derived materials, and distillers' grains (e.g., wet distillers' grains (WDS) and dry distillers' grains with solubles (DDGS)).

[0073] Control Sequences: The term "control sequence" refers to the nucleic acid sequence necessary for the expression of the polynucleotide encoding the mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from different genes) for the polynucleotide encoding the polypeptide, or native or exogenous relative to each other. Such control sequences include, but are not limited to, precursors, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters, as well as transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction enzyme sites that facilitate the linking of these control sequences to the coding regions of the polynucleotide encoding the polypeptide.

[0074] 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.

[0075] Expression: The term “expression” includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0076] Expression vector: The term "expression vector" refers to a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide and that the polynucleotide is operatively linked to a control sequence provided for its expression.

[0077] Feed conversion ratio: The term "feed conversion ratio" refers to the amount of feed given to an animal to increase its body weight by a specified amount. Improved feed conversion ratio refers to a lower feed conversion ratio. "Lower feed conversion ratio" or "improved feed conversion ratio" means that the use of the feed additive composition in the feed results in a lower amount of feed being given to the animal to increase its body weight by a specified amount, compared to the amount of feed required to increase the animal's body weight by the same amount when the feed does not contain the feed additive composition.

[0078] Feed efficiency: The term "feed efficiency" refers to the weight gain per unit of feed when an animal is fed randomly or in a specified amount of food over a period of time. "Increased feed efficiency" refers to the increased weight gain per unit of feed intake resulting from the use of the feed additive composition according to the invention in feed, compared to animals fed without the present feed additive composition.

[0079] 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, 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, orchard grass, ryegrass, timothy-grass), corn, millet, barley, oats, rye, sorghum, soybeans, and wheat, and vegetables (e.g., sugar beets). Forage further includes crop residues from cereal products (such as corn stalks, straw from wheat, barley, oats, rye and other cereals), residues from vegetables such as beet tops, residues from oilseed products such as stems and leaves from soybeans, rapeseed and other legumes, and portions from cereal refining processes for animal or human consumption or from fuel production or other industries.

[0080] Fragment: The term "fragment" refers to a polypeptide in which one or more (e.g., several) amino acids are missing from the amino and / or carboxyl termini of a mature polypeptide; wherein the fragment has protease activity. In one aspect, the fragment comprises at least 173 amino acid residues (e.g., amino acids 16 to 188 of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5), at least 177 amino acid residues (e.g., amino acids 12 to 188 of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5), at least 181 amino acid residues (e.g., amino acids 10 to 190 of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5), at least 185 amino acid residues (e.g., amino acids 9 to 193 of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5), at least 190 amino acid residues (e.g., amino acids 7 to 196 of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5), or at least 195 amino acid residues (e.g., amino acids 5 to 199 of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5).

[0081] In another embodiment, the fragment comprises at least 174 amino acid residues (e.g., amino acids 16 to 189 of SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:14), at least 178 amino acid residues (e.g., amino acids 12 to 189 of SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:14), at least 182 amino acid residues (e.g., amino acids 10 to 191 of SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:14), at least 186 amino acid residues (e.g., amino acids 9 to 194 of SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:14), at least 191 amino acid residues (e.g., amino acids 7 to 197 of SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:14), or at least 196 amino acid residues (e.g., amino acids 5 to 200 of SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:14).

[0082] In another aspect, the fragment comprises at least 174 amino acid residues (e.g., amino acids 16 to 189 of SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:20), at least 178 amino acid residues (e.g., amino acids 12 to 189 of SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:20), at least 182 amino acid residues (e.g., amino acids 10 to 191 of SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:20), at least 186 amino acid residues (e.g., amino acids 9 to 194 of SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:20), at least 191 amino acid residues (e.g., amino acids 7 to 197 of SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:20), or at least 196 amino acid residues (e.g., amino acids 5 to 200 of SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:20).

[0083] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using a nucleic acid construct or expression vector containing the polynucleotides of the present invention. The term "host cell" also encompasses any offspring of the parent cell that is not identical to its parent cell due to mutations that occur during replication.

[0084] Separate: The term “separate” means a substance in a form or environment that does not exist in nature. Non-limiting examples of separated substances include (1) any substance that is not naturally occurring, (2) any substance including but not limited to any enzyme, variant, nucleic acid, protein, peptide or cofactor that is at least partially removed from one or more of the naturally occurring components associated with it; (3) any substance that is artificially modified relative to a naturally found substance; or (4) any substance modified by increasing the amount of the substance relative to other components naturally associated with it (e.g., recombinant production in a host cell; multiple copies of the gene encoding the substance; and the use of a promoter stronger than the promoter naturally associated with the gene encoding the substance).

[0085] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, based on the SignalP program (Nielsen et al., 1997, Protein Engineering 10:1-6), the mature polypeptide is amino acids 1 to 203 of SEQ ID NO:2, and amino acids -200 to -171 of SEQ ID NO:2 are the signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 203 of SEQ ID NO:4 (defined herein as SEQ ID NO:5) based on EDMAN N-terminal sequencing data and complete MS data, and amino acids -197 to -171 of SEQ ID NO:4 are the signal peptide.

[0086] In another instance, based on the SignalP procedure (Nielsen et al., 1997, Protein Engineering 10:1-6), the mature polypeptide is amino acids 1 to 204 of SEQ ID NO:11, and amino acids -196 to -171 of SEQ ID NO:11 are the signal peptide. In yet another instance, the mature polypeptide is amino acids 1 to 204 of SEQ ID NO:13 (defined herein as SEQ ID NO:14) based on EDMAN N-terminal sequencing data and complete MS data, and amino acids -197 to -171 of SEQ ID NO:13 are the signal peptide.

[0087] In another instance, based on the SignalP procedure (Nielsen et al., 1997, Protein Engineering 10:1-6), the mature polypeptide is amino acids 1 to 204 of SEQ ID NO:17, and amino acids -198 to -170 of SEQ ID NO:17 are the signal peptide. In yet another instance, the mature polypeptide is amino acids 1 to 204 of SEQ ID NO:19 (defined herein as SEQ ID NO:20) based on EDMAN N-terminal sequencing data and complete MS data, and amino acids -198 to -170 of SEQ ID NO:19 are the signal peptide.

[0088] 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 from the same polynucleotide. It is also known in the art that different host cells 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.

[0089] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide encoding a mature polypeptide with protease activity. In one aspect, based on the SignalP program (Nielsen et al., 1997, see above), the mature polypeptide coding sequence is nucleotides 601 to 1209 of SEQ ID NO:1, and nucleotides 1 to 90 of SEQ ID NO:1 encode a signal peptide. In another aspect, based on EDMAN N-terminal sequencing data and complete MS data of the polypeptide of SEQ ID NO:4, the mature polypeptide coding sequence is nucleotides 592 to 1200 of SEQ ID NO:3, and nucleotides 1 to 81 of SEQ ID NO:3 encode a signal peptide.

[0090] In another instance, based on the SignalP procedure (Nielsen et al., 1997, see above), the mature polypeptide encoding sequence is nucleotides 589 to 1200 of SEQ ID NO:10, and nucleotides 1 to 78 of SEQ ID NO:10 encode a signal peptide. In yet another instance, based on EDMAN N-terminal sequencing data and complete MS data of the polypeptide of SEQ ID NO:13, the mature polypeptide encoding sequence is nucleotides 592 to 1203 of SEQ ID NO:12, and nucleotides 1 to 81 of SEQ ID NO:12 encode a signal peptide.

[0091] In another instance, based on the SignalP procedure (Nielsen et al., 1997, see above), the mature polypeptide encoding sequence is nucleotides 595 to 1206 of SEQ ID NO:16, and nucleotides 1 to 87 of SEQ ID NO:16 encode the signal peptide. In yet another instance, based on EDMAN N-terminal sequencing data and complete MS data of the polypeptide, the mature polypeptide encoding sequence is nucleotides 589 to 1200 of SEQ ID NO:18, and nucleotides 1 to 81 of SEQ ID NO:18 encode the signal peptide.

[0092] 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 includes one or more control sequences.

[0093] Obtained from or available from: The term "obtained from or available from" means that the polypeptide can be found in organisms from a specific taxonomic class. In one embodiment, the polypeptide is obtained from or available from the order Micrococcales (formerly known as Micrococcineae), where the term "order" is a taxonomic class. In another preferred embodiment, the polypeptide is obtained from or available from the family Intrasporangiaceae, where the term "family" is a taxonomic class.

[0094] If the taxonomic class of a polypeptide is unknown, it can be readily determined by a person skilled in the art by performing a BLASTP search of the polypeptide (using, for example, the National Center for Biotechnology Information (NCIB) website http: / / www.ncbi.nlm.nih.gov / ) and comparing it with the closest homologue. Unknown polypeptides that are fragments of known polypeptides are considered to belong to the same taxonomic species. Unknown native polypeptides or artificial variants, including substitutions, deletions, and / or insertions at up to 10 positions, are considered to originate from the same taxonomic species as known polypeptides.

[0095] Operable ligation: The term “operable ligation” refers to a construction in which a control sequence is positioned relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0096] Granulation: The terms “granulation” and / or “granulation” refer to solid round, spherical and / or cylindrical flakes or granules, and the processes used to form such solid shapes, particularly feed granules 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 forcing a composition through an orifice under pressure, as described herein.

[0097] Performance Parameters: The term “performance parameter” refers to one of a number of items selected from the following list, which consists of: 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 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.

[0098] Protease: The term “protease” is defined herein as an enzyme that hydrolyzes peptide bonds. The definition of protease also applies to the protease portion of the terms “parental protease” and “protease variant” as used herein. The term “protease” includes enzymes belonging to the EC 3.4.21 enzyme group (serine proteases). EC numbers are referenced to the 1992 Enzyme Nomenclature of NC-IUBMB Academic Press, San Diego, California, including supplements 1-5 published in *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 naming conventions are regularly supplemented and updated; see, for example, the World Wide Web (WWW) at http: / / www.chem.qmw.ac.uk / iubmb / enzyme / index.html.

[0099] The proteases of the present invention and the proteases used according to the invention are selected from serine proteases of the peptidase family S1, as described in Biochem.J. 290:205-218 in 1993 and in the MEROPS protease database, issue 9.9 (August 23, 2013) (www.merops.ac.uk). This database is described in Rawlings, ND, Barrett, AJ, and Bateman, A., 2010, “MEROPS: the peptidase database,” Nucleic Acids Res. 38:D227-D233.

[0100] To determine whether a given protease is a serine protease or an S1 family protease, refer to the aforementioned manual and the principles described therein. This determination can be performed on all types of proteases, regardless of whether they are naturally occurring or wild-type, or genetically engineered or synthetic.

[0101] Protease activity: The term "protease activity" refers to proteolytic activity (EC 3.4). Peptides or proteases exhibiting protease activity are sometimes also designated as peptidases, prions, peptide hydrolases, or proteolytic enzymes. Proteases can be exopeptidases that hydrolyze peptides originating from either end or endopeptidases that function within the polypeptide chain. Endopeptidases exhibit activity against peptide substrates with N- and C-terminal closures, the specificity of which depends on the protease in question.

[0102] 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).

[0103] Any assay can be used to measure protease activity, employing a substrate that includes peptide bonds specific to the protease in question. pH and temperature assays are equally applicable to the protease in question. Examples of pH assays are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of temperature assays 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, ML and Mirsky, AE, 1932, J. Gen. Physiol. 16:59 and Anson, ML, 1938, J. Gen. Physiol. 22:79).

[0104] 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 the 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 the protease, the colorless Suc-AAPF-pNA substrate releases a yellow p-nitroaniline, and this yellow color is determined as a measure of protease activity.

[0105] 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% protease activity of the polypeptide of SEQ ID NO:5. In another embodiment, 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% protease activity of the polypeptide of SEQ ID NO:14. In another embodiment, 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% protease activity of the polypeptide of SEQ ID NO:20.

[0106] 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 (such as straw, copra, rice straw, husks, beet waste).

[0107] Sequence consistency: The correlation between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence consistency".

[0108] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: European Open Software Suite for Molecular Biology, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 3.0.0 or later) is used to determine the degree of sequence identity between two amino acid sequences. Version 6.1.0 is used. Optional parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the "longest consistency" annotated by Needle (obtained using the -non-simplification option) is used as the percentage consistency and calculated as follows:

[0109] (identical residues x 100) / (length of alignment - total number of vacancies in alignment)

[0110] For the purposes of this invention, the Niedleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) implemented in the Niedle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (preferably version 3.0.0 or later) is used to determine the degree of sequence identity between two deoxyribonucleotide sequences. Version 6.1.0 is used. Optional 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. The Niedle-annotated "longest consistency" output (obtained using the -non-simplified option) is used as the percentage of consistency and calculated as follows:

[0111] (Consistent deoxyribonucleotides x 100) / (Alignment length - total number of vacancies in the alignment)

[0112] 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, trefoils, 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.

[0113] Strict conditions: Different strict conditions are defined as follows.

[0114] The term "very low stringency conditions" means that for probes of at least 100 nucleotides in length, a standard DNA blotting procedure is followed, involving pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Vector material is finally washed three times at 65°C for 15 minutes each time with 1.2X SSC and 0.2% SDS.

[0115] The term "low stringency conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following standard DNA blotting procedures at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Vector materials are finally washed three times at 70°C for 15 minutes each time with 1.2X SSC and 0.2% SDS.

[0116] The term "medium-strict conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following standard DNA blotting procedures 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. Vector material is finally washed three times at 70°C for 15 minutes each time with 0.6X SSC and 0.2% SDS.

[0117] The term "medium-high stringent conditions" refers to pre-hybridization and hybridization for probes at least 100 nucleotides in length, following a standard DNA blotting procedure 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. Vector material is finally washed three times at 75°C for 15 minutes each time with 0.6X SSC and 0.2% SDS.

[0118] The term "highly stringent conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following standard DNA blotting procedures at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Vector materials are finally washed three times at 75°C for 15 minutes each time with 0.3X SSC and 0.2% SDS.

[0119] The term "very high stringency conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following standard DNA blotting procedures at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Vector materials are finally washed three times at 75°C for 15 minutes each time with 0.15X SSC and 0.2% SDS.

[0120] Subsequence: The term "subsequence" refers to a polynucleotide in which one or more (e.g., several) nucleotides are deleted from the 5' and / or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having protease activity. In one aspect, the subsequence comprises at least 519 nucleotides (e.g., nucleotides 646 to 1164 of SEQ ID NO:1), at least 531 nucleotides (e.g., nucleotides 634 to 1164 of SEQ ID NO:1), at least 543 nucleotides (e.g., nucleotides 628 to 1170 of SEQ ID NO:1), at least 555 nucleotides (e.g., nucleotides 625 to 1179 of SEQ ID NO:1), at least 570 nucleotides (e.g., nucleotides 619 to 1188 of SEQ ID NO:1), or at least 585 nucleotides (e.g., nucleotides 613 to 1197 of SEQ ID NO:1).

[0121] In another aspect, the subsequence comprises at least 522 nucleotides (e.g., nucleotides 634 to 1155 of SEQ ID NO:10), at least 534 nucleotides (e.g., nucleotides 622 to 1155 of SEQ ID NO:10), at least 546 nucleotides (e.g., nucleotides 614 to 1161 of SEQ ID NO:10), at least 558 nucleotides (e.g., nucleotides 611 to 1170 of SEQ ID NO:10), at least 573 nucleotides (e.g., nucleotides 605 to 1179 of SEQ ID NO:10), or at least 588 nucleotides (e.g., nucleotides 599 to 1188 of SEQ ID NO:10).

[0122] In another embodiment, the subsequence comprises at least 522 nucleotides (e.g., nucleotides 640 to 1161 of SEQ ID NO:16), at least 534 nucleotides (e.g., nucleotides 628 to 1161 of SEQ ID NO:16), at least 546 nucleotides (e.g., nucleotides 620 to 1167 of SEQ ID NO:16), at least 558 nucleotides (e.g., nucleotides 617 to 1176 of SEQ ID NO:16), at least 573 nucleotides (e.g., nucleotides 611 to 1185 of SEQ ID NO:16), or at least 588 nucleotides (e.g., nucleotides 605 to 1194 of SEQ ID NO:16).

[0123] Substantially pure polypeptide: The term "substantially pure polypeptide" means a formulation containing, by weight, at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, and at most 0.5% of other polypeptide material naturally or recombinantly associated with it. Preferably, the polypeptide is at least 92% pure by weight of the total polypeptide material present in the formulation, for example, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, and 100% pure. The polypeptides of the present invention are preferably in a substantially pure form. This can be accomplished, for example, by preparing the polypeptide using well-known recombinant methods or classical purification methods.

[0124] Variants: The term "variant" means a polypeptide with protease activity that contains alterations (i.e., substitution, insertion, and / or deletion of one or more amino acid residues) at one or more 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 1-3 amino acids adjacent to an amino acid occupying a position. These variants of the present invention have at least 20%, such as at least 40%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the protease activity of SEQ ID NO:5 and / or SEQ ID NO:14 and / or SEQ ID NO:20.

[0125] Nomenclature

[0126] For the purposes of this invention, the nomenclature [E / Q] indicates that the amino acid at that position can be glutamic acid (Glu, E) or glutamine (Gln, Q). Similarly, the nomenclature [V / G / A / I] indicates that the amino acid at that position can be valine (Val, V), glycine (Gly, G), alanine (Ala, A), or isoleucine (Ile, I), and so on, as other combinations described herein. Unless further limited otherwise, amino acid X is defined such that it can be any of the 20 naturally occurring amino acids. Invention Details

[0128] Animal feed or animal feed additives containing peptides with protease activity

[0129] Soybeans and corn are two highly used protein sources in agriculture, and therefore it is important that proteases exhibit good activity toward these substrates. Furthermore, proteases exhibiting high activity across the entire gastrointestinal pH range (i.e., between approximately pH 3 and pH 7.5) would be of great significance, as they can be expected to degrade substrates more effectively shortly after ingestion and throughout most of the gastrointestinal tract.

[0130] Some proteases have been found to have surprisingly good pH-activity profiles for commercially relevant substrates (soybean-corn) and are significantly more active in a lower pH range (4-5) than protease 10R (SEQ ID NO:8), while maintaining comparable activity at neutral pH.

[0131] Therefore, in a first aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, wherein:

[0132] (a) The polypeptide is a serine protease of the S1 family of peptidases;

[0133] (b) The polypeptide exhibits at pH 4 at an activity at least twice that of the soybean-corn flour protein, for example at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3 times, at least 3.25 times, at least 3.5 times, at least 3.75 times, or at least 4 times greater than that of the protein 10R (SEQ ID NO:8) at the same pH.

[0134] (c) The polypeptide exhibits at least twice the activity of protease 10R (SEQ ID NO:8) at the same pH at pH 5, for example, at least 2.25 times, at least 2.5 times, at least 2.75 times, or at least 3 times the activity of protease 10R (SEQ ID NO:8) at the same pH; and

[0135] (d) Compared with protease 10R (SEQ ID NO:8) at the same pH, this polypeptide has at least 50% activity against soybean-corn flour at pH 7, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., at least the same), or at least 105% activity.

[0136] In a preferred embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15).

[0137] In a preferred embodiment, the activity of the peptide against soybean-corn flour is determined using the soybean-corn flour assay as described in the definition section herein.

[0138] In one embodiment, the polypeptide exhibits less than 20 times, for example, less than 15 times, less than 10 times, less than 9 times, or less than 8 times, the activity of protease 10R (SEQ ID NO:8) against soybean-corn flour at pH 4, and less than 20 times, for example, less than 15 times, less than 10 times, less than 9 times, or less than 8 times, the activity of protease 10R (SEQ ID NO:8) against soybean-corn flour at pH 5. In one embodiment, compared to protease 10R (SEQ ID NO:8) at the same pH, the polypeptide exhibits less than 200% activity against soybean-corn flour at pH 7, for example, less than 180%, less than 170%, less than 160%, less than 150%, less than 140%, less than 130%, or less than 125%.

[0139] In one embodiment, the polypeptide exhibits at least twice the activity against soybean-corn flour at pH 4, at least twice the activity against soybean-corn flour at pH 5, and at least 75% activity of protease 10R (SEQ ID NO:8) at pH 7. In another embodiment, the polypeptide exhibits at least twice the activity against soybean-corn flour at pH 4, at least twice the activity against soybean-corn flour at pH 5, and at least 90% activity of protease 10R (SEQ ID NO:8) at pH 7. In yet another embodiment, the polypeptide exhibits at least 2.5 times the activity against soybean-corn flour at pH 4, at least 2.25 times the activity against soybean-corn flour at pH 5, and at least 75% activity of protease 10R (SEQ ID NO:8) at pH 7. In one embodiment, the polypeptide exhibits at least 2.5 times greater activity against soybean-corn flour at pH 4, at least 2.25 times greater activity against soybean-corn flour at pH 5, and at least 90% activity of protease 10R (SEQ ID NO:8) at pH 7.

[0140] In one embodiment, the polypeptide exhibits at least 3-fold increased activity against soybean-corn flour at pH 4, at least 2.5-fold increased activity against soybean-corn flour at pH 5, and at least 75% activity of protease 10R (SEQ ID NO:8) at pH 7. In another embodiment, the polypeptide exhibits at least 3-fold increased activity against soybean-corn flour at pH 4, at least 2.5-fold increased activity against soybean-corn flour at pH 5, and at least 90% activity of protease 10R (SEQ ID NO:8) at pH 7. In yet another embodiment, the polypeptide exhibits at least 3.5-fold increased activity against soybean-corn flour at pH 4, at least 2.75-fold increased activity against soybean-corn flour at pH 5, and at least 75% activity of protease 10R (SEQ ID NO:8) at pH 7. In one embodiment, the polypeptide exhibits at least 3.5 times greater activity against soybean-corn flour at pH 4, at least 2.75 times greater activity against soybean-corn flour at pH 5, and at least 90% activity of protease 10R (SEQ ID NO:8) at pH 7.

[0141] In one embodiment, the polypeptide exhibits at least 4-fold increased activity against soybean-corn flour at pH 4, at least 3-fold increased activity against soybean-corn flour at pH 5, and at least 75% activity of protease 10R (SEQ ID NO:8) at pH 7. In another embodiment, the polypeptide exhibits at least 4-fold increased activity against soybean-corn flour at pH 4, at least 3-fold increased activity against soybean-corn flour at pH 5, and at least 90% activity of protease 10R (SEQ ID NO:8) at pH 7. In yet another embodiment, the polypeptide exhibits at least 4.5-fold increased activity against soybean-corn flour at pH 4, at least 3-fold increased activity against soybean-corn flour at pH 5, and at least 75% activity of protease 10R (SEQ ID NO:8) at pH 7. In one embodiment, the polypeptide exhibits at least 4.5 times greater activity against soybean-corn flour at pH 4, at least 3 times greater activity against soybean-corn flour at pH 5, and at least 90% activity of protease 10R (SEQ ID NO:8) at pH 7.

[0142] In a preferred embodiment, the polypeptide is obtained from or can be obtained from the order Micrococciles. In an even more preferred embodiment, the polypeptide is obtained from or can be obtained from the family Mesocystaceae.

[0143] In a second aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, wherein:

[0144] (a) The polypeptide is a serine protease of the S1 family of peptidases;

[0145] (b) Compared with its activity at pH 7, the peptide has at least 25% activity against soybean-corn flour at pH 4, for example at least 30%, at least 35%, or at least 40% activity;

[0146] (c) Compared to its activity at pH 7, the peptide exhibits at least 45% activity against soybean-corn flour at pH 5, for example, at least 50%, at least 55%, at least 60%, or at least 65% activity; and

[0147] (d) Compared with protease 10R (SEQ ID NO:8) at the same pH, this polypeptide has at least 50% activity against soybean-corn flour at pH 7, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., at least the same), or at least 105% activity.

[0148] In a preferred embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15).

[0149] In a preferred embodiment, the activity of the peptide against soybean-corn flour is determined using the soybean-corn flour assay as described in the definition section herein.

[0150] In one embodiment, the polypeptide exhibits less than 100% activity against soybean-corn flour at pH 4, for example, less than 90%, less than 80%, less than 75%, or less than 70%, compared to its activity at pH 7, and less than 95% activity against soybean-corn flour at pH 5, for example, less than 90%, less than 85%, or less than 80%, compared to its activity at pH 7. In one embodiment, compared to protease 10R (SEQ ID NO: 8) at the same pH, the polypeptide exhibits less than 200% activity against soybean-corn flour at pH 7, for example, less than 180%, less than 170%, less than 160%, less than 150%, less than 140%, less than 130%, or less than 125% activity.

[0151] In one embodiment, the polypeptide exhibits at least 25% activity at pH 4 compared to its activity at pH 7; at least 45% activity at pH 5 compared to its activity at pH 7; and at least 75% activity of protease 10R (SEQ ID NO:8) at pH 7. In another embodiment, the polypeptide exhibits at least 25% activity at pH 4 compared to its activity at pH 7; at least 45% activity at pH 5 compared to its activity at pH 7; and at least 90% activity of protease 10R (SEQ ID NO:8) at pH 7.

[0152] In one embodiment, the polypeptide exhibits at least 30% activity at pH 4 compared to its activity at pH 7; at least 55% activity at pH 5 compared to its activity at pH 7; and at least 75% activity of protease 10R (SEQ ID NO:8) at pH 7. In another embodiment, the polypeptide exhibits at least 30% activity at pH 4 compared to its activity at pH 7; at least 55% activity at pH 5 compared to its activity at pH 7; and at least 90% activity of protease 10R (SEQ ID NO:8) at pH 7.

[0153] In one embodiment, the polypeptide exhibits at least 35% activity at pH 4 compared to its activity at pH 7; at least 60% activity at pH 5 compared to its activity at pH 7; and at least 75% activity of protease 10R (SEQ ID NO:8) at pH 7. In another embodiment, the polypeptide exhibits at least 35% activity at pH 4 compared to its activity at pH 7; at least 60% activity at pH 5 compared to its activity at pH 7; and at least 90% activity of protease 10R (SEQ ID NO:8) at pH 7.

[0154] In one embodiment, the polypeptide exhibits at least 40% activity at pH 4 compared to its activity at pH 7; at least 65% activity at pH 5 compared to its activity at pH 7; and at least 75% activity of protease 10R (SEQ ID NO:8) at pH 7. In another embodiment, the polypeptide exhibits at least 40% activity at pH 4 compared to its activity at pH 7; at least 65% activity at pH 5 compared to its activity at pH 7; and at least 90% activity of protease 10R (SEQ ID NO:8) at pH 7.

[0155] In a preferred embodiment, the polypeptide is obtained from or can be obtained from the order Micrococciles. In an even more preferred embodiment, the polypeptide is obtained from or can be obtained from the family Mesocystaceae.

[0156] In a third aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, wherein:

[0157] (a) The polypeptide is a serine protease of the S1 family of peptidases; and

[0158] (b) The polypeptide is obtained from or can be obtained from the family Mesocystaceae.

[0159] In one embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15).

[0160] In one embodiment, the polypeptide exhibits at least twice the activity of the protein 10R (SEQ ID NO:8) at the same pH at pH 4, for example, at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3 times, at least 3.25 times, at least 3.5 times, at least 3.75 times, or at least 4 times higher activity than that of the protein 10R (SEQ ID NO:8) at the same pH.

[0161] In one embodiment, the polypeptide exhibits at least twice the activity of the soybean-corn flour protein at pH 5 compared to the activity of protease 10R (SEQ ID NO:8) at the same pH, for example, at least 2.25 times, at least 2.5 times, at least 2.75 times, or at least 3 times higher activity.

[0162] In one embodiment, the polypeptide has at least 50% activity against soybean-corn flour at pH 7 compared to protease 10R (SEQ ID NO:8) at the same pH, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., at least the same), or at least 105% activity.

[0163] In a preferred embodiment, the polypeptide:

[0164] (a) At pH 4, it has at least 2 times greater activity on soybean-corn flour than the activity of protease 10R (SEQ ID NO:8) at the same pH, for example at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3 times, at least 3.25 times, at least 3.5 times, at least 3.75 times, or at least 4 times greater activity;

[0165] (b) At pH 5, it exhibits at least twice the activity of protease 10R (SEQ ID NO:8) at the same pH, for example, at least 2.25 times, at least 2.5 times, at least 2.75 times, or at least 3 times the activity of protease 10R (SEQ ID NO:8) at the same pH; and

[0166] (c) Having at least 50% activity against soybean-corn flour at pH 7 compared to protease 10R (SEQ ID NO:8) at the same pH, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., at least the same), or at least 105% activity.

[0167] In a preferred embodiment, the polypeptide:

[0168] (a) The polypeptide includes one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15);

[0169] (b) At pH 4, it has at least 2 times greater activity on soybean-corn flour than the activity of protease 10R (SEQ ID NO:8) at the same pH, for example at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3 times, at least 3.25 times, at least 3.5 times, at least 3.75 times, or at least 4 times greater activity.

[0170] (c) At pH 5, it exhibits at least twice the activity of protease 10R (SEQ ID NO:8) at the same pH, for example, at least 2.25 times, at least 2.5 times, at least 2.75 times, or at least 3 times the activity of protease 10R (SEQ ID NO:8) at the same pH; and

[0171] (d) Compared with protease 10R (SEQ ID NO:8) at the same pH, it has at least 50% activity against soybean-corn flour at pH 7, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., at least the same), or at least 105% activity.

[0172] In a fourth aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, wherein the polypeptide has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 of SEQ ID NO:2. In one embodiment, these peptides differ from the mature peptide of SEQ ID NO:2 by up to 50 amino acids, for example, between 1 and 50 amino acids, such as 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5 amino acids, or 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 amino acids. In one embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15).

[0173] In one embodiment, the polypeptide has been isolated. The animal feed or animal feed additive preferably comprises or consists of the amino acid sequence of SEQ ID NO:2 or its allelic variants; is a fragment having protease activity with a deletion of, for example, 30, 26, 22, 18, 13, 11, 8, or 5 amino acids from its N-terminus or C-terminus, or is a protease-active fragment comprising at least 173 amino acids, such as at least 177 amino acids, at least 181 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the animal feed or animal feed additive comprises or consists of the mature polypeptide of SEQ ID NO:2. In another embodiment, the animal feed or animal feed additive comprises or consists of amino acids 1 to 203 of SEQ ID NO:2.

[0174] In a continuation of the fourth aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, wherein the polypeptide has at least 65% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 70% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 75% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 80% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 81% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 82% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 83% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 84% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 85% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 86% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 87% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 88% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 89% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 90% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 91% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 92% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 93% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 94% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 95% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 96% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 97% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 98% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide has at least 99% sequence identity with SEQ ID NO:5. In one embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15).

[0175] In the continuation of the fourth aspect, these polypeptides differ from SEQ ID NO:5 by up to 50 amino acids, for example, between 1 and 50 amino acids, such as 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5 amino acids, or 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 amino acids. In one embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15).

[0176] In one embodiment, the polypeptide has been isolated. The animal feed or animal feed additive preferably comprises or consists of the amino acid sequence of SEQ ID NO:5 or its allele variants; is a fragment having protease activity with a deletion of, for example, 30, 26, 22, 18, 13, 11, 8, or 5 amino acids from its N-terminus or C-terminus, or is a protease-active fragment comprising at least 173 amino acids, such as at least 177 amino acids, at least 181 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the animal feed or animal feed additive comprises or consists of amino acids 1 to 203 of SEQ ID NO:5.

[0177] In a continuation of the fourth aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, these polypeptides being encoded by polynucleotides that hybridize under low-strict, medium-strict, medium-high-strict, high-strict, or very-high-strict conditions with: (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the mature polypeptide coding sequence of SEQ ID NO:3, or (iii) the full-length complement of (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor, New York). In one embodiment, the polypeptide has been isolated.

[0178] In a continuation of the fourth aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, these polypeptides being encoded by a polynucleotide having at least 65%, for example, at least 70%, at least 75%, at least 80%, at least 85%, 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 coding sequence of SEQ ID NO:1. In another embodiment, the polypeptide has been isolated.

[0179] In a continuation of the fourth aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, these polypeptides being encoded by a polynucleotide having at least 65%, for example, at least 70%, at least 75%, at least 80%, at least 85%, 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 coding sequence of SEQ ID NO:3. In another embodiment, the polypeptide has been isolated.

[0180] In a continuation of the fourth aspect, the present invention relates to animal feed or animal feed additives comprising one or more variants of SEQ ID NO:5, wherein the variant has protease activity and includes one or more substitutions, and / or deletions, and / or 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. In one embodiment, the number of positions including substitutions and / or deletions and / or insertions or any combination thereof in SEQ ID NO:5 is between 1 and 50, such as 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions including substitutions and / or deletions and / or insertions or any combination thereof in SEQ ID NO:5 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions and / or deletions and / or insertions in SEQ ID NO:5 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In yet another embodiment, the number of substitutions in SEQ ID NO:5 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15).

[0181] Amino acid alterations can be minor in nature, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions typically of 1–30 amino acids; small amino- or carboxyl-terminal extensions, such as amino-terminal methionine residues; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function, such as multihistidine sequences, antigenic epitopes, or binding domains.

[0182] Examples of conserved substitutions are found in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific reactivity are known in the art and are described, for example, by H. Neurath and R.R. Hill, 1979, in *The Proteins*, Academic Press, New York. Common substitutes are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0183] Alternatively, amino acid alterations have the property of changing the physicochemical properties of peptides. For example, amino acid alterations can improve the thermal stability of peptides, change substrate specificity, and alter the optimal pH.

[0184] Essential amino acids in peptides can be identified using methods 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 mutant molecule is tested to identify amino acid residues essential to the molecule's activity. See also Hilton et al., 1996, Journal of Biochemistry 271:4699-4708. Alternatively, mutations in amino acids at hypothetical contact sites can be combined with physical analysis of the structure, such as by techniques like NMR, crystallography, electron diffraction, or photoaffinity labeling, to determine the enzyme's active site or other biological interactions. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, Journal of Molecular Biology 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.

[0185] S1 family peptidases comprise a catalytic triad in the order His, Asp, Ser. Mutations in any amino acid of this catalytic triad will result in changes or loss of enzyme activity or substrate specificity. For example, the amino acids in the catalytic triad of S1 protease 1 isolated from *Haloxylon ammodendron* HTCC2649 (SEQ ID NO:5) are His-35, Asp-62, and Ser-148. Similarly, the amino acids in the catalytic triad of S1 protease 1 isolated from *Terracoccus* sp. (SEQ ID NO:14) are His-35, Asp-62, and Ser-149. And the amino acids in the catalytic triad of S1 protease 1 isolated from *Nocturia flavum* (SEQ ID NO:20) are His-35, Asp-62, and Ser-149.

[0186] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or truncation methods, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proceedings of the National Academy of Sciences of the United States of America (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).

[0187] Mutagenesis / reorganization methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenic peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenic 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.

[0188] The polypeptide can be a hybrid polypeptide, in which a region of one polypeptide is fused to the N-terminus or C-terminus of a region of another polypeptide.

[0189] The polypeptide can be a fusion polypeptide or a cleavable fusion polypeptide, wherein another polypeptide is fused at the N-terminus or C-terminus of the polypeptide of the present invention. Fusion polypeptides are generated by fusing a polynucleotide encoding another polypeptide with the polynucleotide of the present invention. Techniques for generating fusion polypeptides are known in the art and include linking the coding sequences encoding the polypeptides such that they are within a frame and that the expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using integrin technology, wherein the fusion polypeptide is generated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0190] Fusion peptides may further include a cleavage site between the two peptides. This site is cleaved upon secretion of the fusion protein, releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1 995, 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.

[0191] Carbohydrate molecules are typically attached to fungal-derived peptides during post-translational modifications. To aid in mass spectrometry analysis, the peptide can be incubated with an endoglucosidase to deglycosylate each N-linked site. For each deglycosylated N-linked site, an N-acetylglucosamine remains on the protein backbone.

[0192] In a fifth aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, wherein the polypeptide has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 of SEQ ID NO:11. In one embodiment, these peptides differ from the mature peptide of SEQ ID NO:11 by up to 50 amino acids, for example, between 1 and 50 amino acids, such as 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5 amino acids, or 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 amino acids. In one embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15).

[0193] In one embodiment, the polypeptide has been isolated. The animal feed or animal feed additive preferably comprises or consists of the amino acid sequence of SEQ ID NO:11 or its allelic variants; is a fragment having protease activity with a deletion of, for example, 30, 26, 22, 18, 13, 11, 8, or 5 amino acids from its N-terminus or C-terminus, or is a protease-active fragment comprising at least 174 amino acids, such as at least 178 amino acids, at least 182 amino acids, at least 186 amino acids, at least 191 amino acids, at least 196 amino acids, or at least 201 amino acids. In another embodiment, the animal feed or animal feed additive comprises or consists of the mature polypeptide of SEQ ID NO:11. In yet another embodiment, the animal feed or animal feed additive comprises or consists of amino acids 1 to 204 of SEQ ID NO:11.

[0194] In a continuation of the fifth aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, wherein the polypeptide has at least 65% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 70% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 75% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 80% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 81% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 82% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 83% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 84% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 85% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 86% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 87% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 88% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 89% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 90% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 91% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 92% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 93% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 94% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 95% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 96% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 97% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 98% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide has at least 99% sequence identity with SEQ ID NO:14. In one embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15).

[0195] In the continuation of the fifth aspect, these polypeptides differ from SEQ ID NO:14 by up to 50 amino acids, for example, between 1 and 50 amino acids, such as 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5 amino acids, or 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 amino acids. In one embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15).

[0196] In one embodiment, the polypeptide has been isolated. The animal feed or animal feed additive preferably comprises or consists of the amino acid sequence of SEQ ID NO:14 or its allelic variants; is a fragment having protease activity with a deletion of, for example, 30, 26, 22, 18, 13, 11, 8, or 5 amino acids from its N-terminus or C-terminus, or is a protease-active fragment comprising at least 174 amino acids, such as at least 178 amino acids, at least 182 amino acids, at least 186 amino acids, at least 191 amino acids, at least 196 amino acids, or at least 201 amino acids. In another embodiment, the animal feed or animal feed additive comprises or consists of SEQ ID NO:14. In yet another embodiment, the animal feed or animal feed additive comprises or consists of amino acids 1 to 204 of SEQ ID NO:14.

[0197] In a continuation of the fifth aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, these polypeptides being encoded by polynucleotides that hybridize under low-strict, medium-strict, medium-high-strict, high-strict, or very-high-strict conditions to: (i) the mature polypeptide coding sequence of SEQ ID NO:10, (ii) the mature polypeptide coding sequence of SEQ ID NO:12, or (iii) the full-length complement of (i) or (ii) (Sambrook et al., 1989, see above). In one embodiment, the polypeptide has been isolated.

[0198] In a continuation of the fifth aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, these polypeptides being encoded by a polynucleotide having at least 65%, for example, at least 70%, at least 75%, at least 80%, at least 85%, 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 coding sequence of SEQ ID NO:10. In another embodiment, the polypeptide has been isolated.

[0199] In a continuation of the fifth aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, these polypeptides being encoded by a polynucleotide having at least 65%, for example, at least 70%, at least 75%, at least 80%, at least 85%, 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 coding sequence of SEQ ID NO:12. In another embodiment, the polypeptide has been isolated.

[0200] In a continuation of the fifth aspect, the present invention relates to animal feed or animal feed additives comprising one or more variants of SEQ ID NO:14, wherein the variant has protease activity and comprises one or more substitutions, and / or deletions, and / or 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. In one embodiment, the number of positions including substitutions and / or deletions and / or insertions or any combination thereof in SEQ ID NO:14 is between 1 and 50, such as 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions including substitutions and / or deletions and / or insertions or any combination thereof in SEQ ID NO:14 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions and / or deletions and / or insertions in SEQ ID NO:14 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In yet another embodiment, the number of substitutions in SEQ ID NO:14 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO: 15). Examples of amino acid variations, conserved substitutions, and fusion peptides are described in a fourth aspect of the invention.

[0201] In a sixth aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, wherein the polypeptide has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 of SEQ ID NO:17. In one embodiment, these peptides differ from the mature peptide of SEQ ID NO:17 by up to 50 amino acids, for example, between 1 and 50 amino acids, such as 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5 amino acids, or 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 amino acids. In one embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15).

[0202] In one embodiment, the polypeptide has been isolated. The animal feed or animal feed additive preferably comprises or consists of the amino acid sequence of SEQ ID NO:17 or its allele variants; is a fragment having protease activity with a deletion of, for example, 30, 26, 22, 18, 13, 11, 8, or 5 amino acids from its N-terminus or C-terminus, or is a protease-active fragment comprising at least 174 amino acids, such as at least 178, at least 182, at least 186, at least 191, at least 196, or at least 201 amino acids. In another embodiment, the animal feed or animal feed additive comprises or consists of the mature polypeptide of SEQ ID NO:17. In yet another embodiment, the animal feed or animal feed additive comprises or consists of amino acids 1 to 204 of SEQ ID NO:17.

[0203] In a continuation of the sixth aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, wherein the polypeptide has at least 65% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 70% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 75% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 80% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 81% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 82% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 83% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 84% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 85% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 86% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 87% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 88% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 89% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 90% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 91% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 92% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 93% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 94% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 95% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 96% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 97% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 98% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide has at least 99% sequence identity with SEQ ID NO:20. In one embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15).

[0204] In the continuation of the sixth aspect, these polypeptides differ from SEQ ID NO:20 by up to 50 amino acids, for example, between 1 and 50 amino acids, such as 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5 amino acids, or 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 amino acids. In one embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15).

[0205] In one embodiment, the polypeptide has been isolated. The animal feed or animal feed additive preferably comprises or consists of the amino acid sequence of SEQ ID NO:20 or its allelic variants; is a fragment having protease activity with a deletion of, for example, 30, 26, 22, 18, 13, 11, 8, or 5 amino acids from its N-terminus or C-terminus, or is a protease-active fragment comprising at least 174 amino acids, such as at least 178 amino acids, at least 182 amino acids, at least 186 amino acids, at least 191 amino acids, at least 196 amino acids, or at least 201 amino acids. In another embodiment, the animal feed or animal feed additive comprises or consists of SEQ ID NO:20. In yet another embodiment, the animal feed or animal feed additive comprises or consists of amino acids 1 to 204 of SEQ ID NO:20.

[0206] In a continuation of the sixth aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, these polypeptides being encoded by polynucleotides that hybridize under low-strict, medium-strict, medium-high-strict, high-strict, or very-high-strict conditions with: (i) the mature polypeptide coding sequence of SEQ ID NO:16, or (ii) the full-length complement of (i) (Sambrook et al., 1989, see above). In one embodiment, the polypeptide has been isolated.

[0207] In a continuation of the sixth aspect, the present invention relates to animal feed or animal feed additives comprising one or more polypeptides having protease activity, these polypeptides being encoded by a polynucleotide having at least 65%, for example, at least 70%, at least 75%, at least 80%, at least 85%, 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 coding sequence of SEQ ID NO:16. In another embodiment, the polypeptide has been isolated.

[0208] In a continuation of the sixth aspect, the present invention relates to animal feed or animal feed additives comprising one or more variants of SEQ ID NO:20, wherein the variant has protease activity and comprises one or more substitutions, and / or deletions, and / or 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. In one embodiment, the number of positions including substitutions and / or deletions and / or insertions or any combination thereof in SEQ ID NO:20 is between 1 and 50, such as 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions including substitutions and / or deletions and / or insertions or any combination thereof in SEQ ID NO:20 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions and / or deletions and / or insertions in SEQ ID NO:20 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In yet another embodiment, the number of substitutions in SEQ ID NO:20 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO: 15). Examples of amino acid variations, conserved substitutions, and fusion peptides are described in a fourth aspect of the invention.

[0209] In a preferred embodiment, the polypeptides of the fourth, fifth, and sixth aspects of the present invention also include the characteristics of the first aspect of the present invention.

[0210] In a preferred embodiment, the polypeptides of the fourth, fifth, and sixth aspects of the present invention also include the characteristics of the second aspect of the present invention.

[0211] In a preferred embodiment, the polypeptides of the fourth, fifth, and sixth aspects of the present invention also include the characteristics of the third aspect of the present invention.

[0212] In a preferred embodiment, the polypeptides of the fourth, fifth, and sixth aspects of the present invention also include the characteristics of the first and second aspects of the present invention.

[0213] In a preferred embodiment, the polypeptides of the fourth, fifth, and sixth aspects of the present invention also include the characteristics of the first and third aspects of the present invention.

[0214] In a preferred embodiment, the polypeptides of the fourth, fifth, and sixth aspects of the present invention also include the characteristics of the second and third aspects of the present invention.

[0215] In a preferred embodiment, the polypeptides of the fourth, fifth, and sixth aspects of the present invention also include the characteristics of the first, second, and third aspects of the present invention.

[0216] characteristic

[0217] Acidity / Alkalinity Characteristics

[0218] In some embodiments of the invention, the proteases of the invention exhibit beneficial properties with respect to pH, such as acid stability and optimal pH. Activity across a broad range of physiological pH (e.g., from 4-7) covers both the gastrointestinal tract (crown pH 4-6; stomach pH in pigs can sometimes be as high as pH 5-6) and the small intestine (pH 6-7). One embodiment of the invention is an isolated polypeptide exhibiting improved protease activity at 40°C between pH 3 and 7, for example, at pH 3.0, pH 4.0, pH 5.0, pH 6.0, and / or pH 7.0, compared to protease 10R.

[0219] Temperature-Activity

[0220] The temperature-activity profile of the protease can be determined as described in Example 3. Activity at low temperatures (37°C–50°C) can be advantageous for protein digestion in animals.

[0221] In one embodiment, the present invention comprises a protease having a temperature activity profile at pH 7.0 with a relative activity of 0.10 or higher at 37°C, a relative activity of 0.40 or higher at 50°C, or a relative activity of 0.80 or higher at 60°C, compared to the activity of a protease at 60°C (Comparative Example 3).

[0222] Another embodiment of the invention is an isolated polypeptide that has improved protease activity at pH 7.0, for example, 60°C or below, such as 50°C or below, 37°C or below, or between 37°C and 60°C, or between 50°C and 60°C, or at 37°C, or at 50°C, or at 60°C, compared to protease 10R at pH 6.5.

[0223] thermal stability

[0224] Thermal stability can be determined as described in Example 5, i.e., by using DSC measurements to determine the denaturation temperature (T) of the purified protease protein. d Td indicates the thermal stability of the protein: T d The higher the value, the higher the thermal stability. Therefore, in a preferred embodiment, the protease of the present invention has a T value. d The T d T higher than the reference protein d T d It is determined from purified protease samples (preferably having a purity of at least 90% or 95%, as determined by SDS-PAGE).

[0225] In a preferred embodiment, such as by residual activity and denaturation temperature T d The provided thermal properties (e.g., heating stability, temperature stability, thermal stability, steam stability, and / or granulation stability) or other parameters of the proteases of the present invention are higher than corresponding values ​​(e.g., the residual activity or T of the proteases of SEQ ID NO:5 and / or SEQ ID NO:14). d More preferably, it is at least 101%, or at least 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or at least 110%. Even more preferably, the parameters of the protease of the present invention (such as residual activity or T) d The value is at least 120%, 130%, 140%, 150%, 160%, 170%, 180%, or at least 190% of the value of the protease in SEQ ID NO:5 and / or SEQ ID NO:14 and / or SEQ ID NO:20.

[0226] In yet another specific embodiment, the heat-stable protease of the present invention has a melting temperature T of at least 50°C. m (or denaturation temperature T) d The value of T was determined by differential scanning calorimetry (DSC) as described in Example 5 (i.e., at 20 mM sodium acetate, pH 4.0). In yet another specific embodiment, the T... mIt is at least 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃ or at least 100℃.

[0227] Steam stability

[0228] Steam stability can be determined, as described in Example 6, by determining the residual activity of the protease molecules after steam treatment at 85°C or 90°C for a short time.

[0229] Granulation stability

[0230] Granulation stability can be determined, as described in Example 7, by using enzyme granules premixed with the feed. The feed is conditioned to 95°C using steam in the mixer. After conditioning, the feed is pressurized into pellets and residual activity is determined.

[0231] Sources of peptides with protease activity

[0232] The protease-active polypeptides according to the invention can be obtained from any genus of microorganisms. For the purposes of this invention, as used herein in conjunction with the given sources, the term "obtained from" shall mean that the polypeptide encoded by the polynucleotide is produced by that source or by a strain in which a polynucleotide from that source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0233] The polypeptide can be a bacterial polypeptide. For example, the polypeptide can be a polypeptide with protease activity derived from a Gram-positive bacterium such as Actinobacteria or a Gram-negative bacterium such as Proteobacteria.

[0234] In one instance, the polypeptide is a protease from bacteria of the class Actinomycetes, such as those from the order Micrococciles, or from the family Mesocystaceae, or from the genus *Hypertricis*, *Terracoccus*, or *Knoellia*.

[0235] The strains of these taxa 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 (DSM), the Culture Collection of Fungi (CBS), and the Northern Research Center of the Patent Culture Collection of the Agricultural Research Institute (NRRL).

[0236] The probes described above can be used to identify and obtain the polypeptide from microorganisms isolated from other sources, including those from nature (e.g., soil, compost, water, etc.). Techniques for isolating microorganisms from natural habitats are well known in the art. The polynucleotide encoding the polypeptide can then be obtained by similarly screening the genome or cDNA library of another microorganism or a mixed DNA sample. Once a polynucleotide encoding a polypeptide is detected with this or these probes, the polynucleotide can be isolated or cloned using techniques well known to those skilled in the art (see, for example, Sambrook et al., 1989, above).

[0237] Polynucleotides

[0238] The present invention also relates to polynucleotides encoding the polypeptides of the present invention, as described herein. In one embodiment, the polynucleotide encoding the polypeptides of the present invention has been isolated.

[0239] Techniques for isolating or cloning polynucleotides are known in the art and include isolation from genomic DNA or cDNA, or combinations thereof. Polynucleotides can be cloned from genomic DNA, for example, by detecting cloned DNA fragments with shared structural features using well-known polymerase chain reaction (PCR) or antibody screening of expression libraries. 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). Polynucleotides can be cloned from strains of *Hypertricis*, *Terracoccus*, or *Knoellia*, or from related organisms of the *Micrococcus* genus, and therefore, for example, can be allelic or species variants of the polypeptide coding region of the polynucleotide.

[0240] Nucleic acid constructs

[0241] The present invention also relates to nucleic acid constructs comprising polynucleotides of the present invention operably linked to one or more control sequences, wherein the control sequences guide the expression of coding sequences in suitable host cells under conditions compatible with the control sequences.

[0242] The polynucleotide can be manipulated in many ways to facilitate polypeptide expression. Depending on the expression vector, manipulation of the polynucleotide prior to insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0243] The control sequence can be a promoter, i.e., a polynucleotide recognized by the host cell to express a polynucleotide encoding the polypeptide of the present invention. The promoter contains a transcriptional control sequence that mediates polypeptide expression. The promoter can be any polynucleotide exhibiting transcriptional activity in the host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0244] 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, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology). Microbiology 13:97-107), Escherichia coli lac operon, Escherichia coli trc promoter (Egon et al., 1988, Gene 69:301-315), Streptomyces agar hydrolase gene (dagA), and prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731) and tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 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.

[0245] Examples of suitable promoters for guiding the transcription of the nucleic acid constructs of this invention in filamentous fungal host cells are promoters derived from the following genes: Aspergillus nidulans acetamase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium moniliforme amyloglucosidase (WO 00 / 56900), Fusarium moniliforme Daria (WO 00 / 56900), Fusarium moniliforme Quinn (WO 00 / 56900). 00 / 56900), *Rhizopus mirtii* lipase, *Rhizopus mirtii* 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* translational elongation factor. The promoter, together with the NA2-tpi promoter (a modified promoter from an Aspergillus gene encoding neutral α-amylase, wherein the untranslated pre-progenitor has been replaced with an untranslated pre-progenitor from an Aspergillus gene encoding triose phosphate isomerase; non-limiting examples include a modified promoter from an Aspergillus niger gene encoding neutral α-amylase, wherein the untranslated pre-progenitor has been replaced with an untranslated pre-progenitor from an Aspergillus nidulans or Aspergillus oryzae gene encoding triose phosphate isomerase), and its mutant, truncated, and heterozygous promoters. Other promoters are described in U.S. Patent No. 6,011,147.

[0246] In yeast hosts, useful promoters are derived from genes targeting the following: *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 in Romanos et al., 1992, Yeast 8:423-488.

[0247] The control sequence can also be a transcription terminator recognized by the host cell to terminate transcription. The 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.

[0248] The preferred terminator for bacterial host cells is obtained from genes targeting the following: Bacillus clausti alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0249] Preferred terminators for filamentous fungal host cells are derived from the genes of: 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.

[0250] Preferred terminators for yeast host cells are derived from the genes of *Saccharomyces cerevisiae* enolase, *Saccharomyces cerevisiae* cytochrome C (CYC1), and *Saccharomyces cerevisiae* glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described above in Romanos et al., 1992.

[0251] The control sequence can also be a stabilizer region of mRNA downstream of the promoter and upstream of the gene coding sequence, which increases the expression of the gene.

[0252] Examples of suitable mRNA stable regions were obtained from the following: Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0253] This control sequence can also be a pre-translation, which is the untranslated region of an mRNA important for translation in the host cell. This pre-translation is operatively linked to the 5' end of the polynucleotide encoding the polypeptide. Any pre-translation that is functional in the host cell can be used.

[0254] Preferred precursors for use in filamentous fungal host cells were obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0255] Suitable precursors for yeast host cells were obtained from the genes of *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* 3-phosphoglycerate kinase, *Saccharomyces cerevisiae* gluten-factor, and *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0256] The control sequence can also be a polyadenylation sequence, a signaling sequence operatively linked to the 3' end of a polynucleotide and recognized by the host cell during transcription as an addition of polyadenylate residues to the transcribed mRNA. Any polyadenylation sequence that functions in the host cell can be used.

[0257] Preferred polyadenylated sequences for use in filamentous fungal host cells were obtained from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0258] The polyadenylated sequences useful for yeast host cells are described in Guo and Sherman, 1995, Molecular Cellular Biology, 15:5983-5990.

[0259] The control sequence can also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and guides the polypeptide into the cell's secretion pathway. The 5' end of the polynucleotide coding sequence can inherently contain a signal peptide coding sequence naturally linked to a segment of the coding sequence of the polypeptide within the translation reading frame. Alternatively, the 5' end of the coding sequence can contain a foreign signal peptide coding sequence relative to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, a foreign signal peptide coding sequence may be required. Alternatively, a foreign signal peptide coding sequence can simply replace the natural signal peptide coding sequence to enhance polypeptide secretion. However, any signal peptide coding sequence that guides the secretion pathway of the expressed polypeptide into the host cell can be used.

[0260] Effective signal peptide coding sequences for bacterial host cells are obtained from the genes of *Bacillus NCIB 11837* maltose amylase, *Bacillus licheniformis* subtilis protease, *Bacillus licheniformis* calc-lactamase, *Bacillus thermophilus* glutamyl-amylase, *Bacillus thermophilus* neutral proteases (nprT, nprS, nprM), and *Bacillus subtilis* prsA. Other signal sequences are described by Simonen and Palva, 1993, *Microbiological Reviews* 57:109-137.

[0261] The effective signal peptide coding sequences for filamentous fungal host cells are obtained from the genes of Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, Aspergillus oryzae cellulase, Aspergillus oryzae endoglucanase V, Aspergillus pubescens lipase, and Rhizopus oryzae aspartic protease.

[0262] Useful signal peptides for yeast host cells were obtained from the genes of Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences were described by Romanos et al. (1992, above).

[0263] 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. The propeptide-coding sequence can be obtained from the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Thermophilus laccase (WO 95 / 33836), Rhizopus oryzae aspartic protease, and Saccharomyces cerevisiae α-factor.

[0264] In the presence of both the signal peptide sequence and the propeptide sequence, the propeptide sequence is positioned immediately adjacent to the N-terminus of the polypeptide, and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[0265] It may also be desirable to add regulatory sequences that regulate the expression of polypeptides relative to the growth of the host cell. Examples of regulatory sequences are those that cause gene expression to be turned 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 systems can be used. In filamentous fungi, the Aspergillus niger glucosyl amylase promoter, the Aspergillus oryzae TAKA α-amylase promoter and the Aspergillus oryzae glucosyl amylase promoter, the Trichoderma reesei cellobiase I promoter and the Trichoderma reesei cellobiase 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 polypeptide will be operatively linked to the regulatory sequence.

[0266] expression carrier

[0267] The present invention also relates to recombinant expression vectors comprising the polynucleotide of the present invention, a promoter, and transcription and translation termination signals. Different nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction enzyme sites to allow insertion or substitution of the polynucleotide encoding the polypeptide at these sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located within the vector, such that the coding sequence is operatively linked to the suitable control sequence for expression.

[0268] 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. The vector can be a linear or closed circular plasmid.

[0269] The vector can be a self-replicating vector, that is, a vector existing as an extrachromosomal entity whose replication is independent of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any elements necessary to ensure self-replication. Alternatively, the vector can be one that, when introduced into the host cell, is integrated into the genome 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 (which together contain the total DNA of the genome to be introduced into the host cell), or transposons can be used.

[0270] 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, heavy metal resistance, or auxotrophic prototrophs, etc.

[0271] Examples of bacterial selective markers include the dal gene in Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance (such as resistance to ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline). Suitable markers for use in 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 carboxylamine 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. The amdS and pyrG genes of *Aspergillus nidus* or *Aspergillus oryzae*, and the bar gene of *Streptomyces hygroscopicus* are preferably used in *Aspergillus* cells. In *Trichoderma* cells, the adeA, adeB, amdS, hph, and pyrG genes are preferred.

[0272] 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.

[0273] 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.

[0274] 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 one or more precise locations on one or more chromosomes of the host cell genome via homologous recombination. To increase the likelihood of integration at precise locations, these integrative elements 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 increase the likelihood of homologous recombination. These integrative elements can be any sequence homologous to the target sequence in the host cell genome. Furthermore, these integrative elements can be non-coding or coding polynucleotides. On the other hand, the vector can integrate into the host cell genome via non-homologous recombination.

[0275] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. 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.

[0276] 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.

[0277] 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.

[0278] 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; WO 00 / 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.

[0279] One or more copies 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 an amplified copy of the selective marker gene, and thus additional copies of the polynucleotide, can be selected by culturing cells in the presence of a suitable selective reagent.

[0280] The procedures for connecting the elements described above 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.).

[0281] host cells

[0282] This 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 comprising 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 term "host cell" encompasses any progeny of a parent cell that differs from the parent cell due to mutations occurring during replication. The selection of the host cell depends largely on the gene encoding the polypeptide and its origin.

[0283] The host cell can be any cell that has been used to recombine and generate the polypeptide of the present invention, such as a prokaryotic cell or a eukaryotic cell.

[0284] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to: Bacillus, Clostridium, Enterococcus, Bacillus aeruginosa, Lactobacillus, Lactococcus, Marine Bacillus, 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.

[0285] 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.

[0286] The bacterial host cell can also be any streptococcal cell, including but not limited to: Streptococcus equina, Streptococcus pyogenes, Streptococcus mammae, and Streptococcus equine subsp. veterinaryis.

[0287] The bacterial host cell can also be any Streptomyces cell, including but not limited to: non-chromogenic Streptomyces, insecticidal Streptomyces, sky blue Streptomyces, gray Streptomyces and light blue Streptomyces cells.

[0288] DNA can be introduced into Bacillus cells via the following methods: protoplast transformation (see, for example, Chang and Cohen, 1979, Molecular Genetics and Genomics, 168:111-115), and competent cell transformation (see, for example, Young and Spizizen, 1961, Journal of Bacteriology, 81:823-829; or Dubnau and David Dubnau). Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221, electroporation (see, for example, Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, for example, Koehler and Thorne, 1987, Journal of Bacteriology 169:5271-5278). DNA can be introduced into E. coli cells via protoplast transformation (see, for example, Hanahan, 1983, Journal of Molecular Biology 166:557-580) or electroporation (see, for example, Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). DNA can be introduced into Streptomyces cells via protoplast transformation, electroporation (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, for example, Mazodier et al., 1989, Journal of Bacteriol. 171:3583-3585), or transduction (see, for example, Burke et al., 2001, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 98:6289-6294). DNA can be introduced into Pseudomonas cells by electroporation (see, for example, Choi et al., 2006, Journal of Microbiological Methods, 64:391-397) or conjugation (see, for example, Pinedo and Smets, 2005, Appl. Environ. Microbiol., 71:51-57).Introducing DNA into Streptococcus cells can be achieved through: native competent cells (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-207), electroporation (see, e.g., Buckley et al., 1999, Applied and Environmental Microbiology 65:3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0289] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0290] 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: Dictionary of the Fungi, Ainsworth and Bisby, 8th ed., 1995, CAB International, University Press, Cambridge, UK).

[0291] 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 class 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* (Skinner, Passmore, and Davenport, eds., Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0292] Yeast host cells can be cells from the genera *Candida*, *Hansenula*, *Kluyver*, *Pichia*, *Saccharomyces*, *Saccharomyces*, or *Yarrowia*, such as *Kluyveromyces lactis*, *Kluyveria*, *Saccharomyces cerevisiae*, *Saccharomyces sacchari*, *Saccharomyces davidiana*, *Saccharomyces douglas*, *Kluyver*, *Nordiya*, *Ovoyces*, or *Yarrowia lipolytica*.

[0293] The host cell of this fungus can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subphylum Eumycota and Oomycota (as defined above by Hawkesworth et al., 1995). 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) grows vegetatively through budding of single-celled cells, and carbon metabolism can be fermentation.

[0294] 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*, *Pleurotus ... or *Trichoderma*.

[0295] For example, the host cells of filamentous fungi can be *Aspergillus amblymorii*, *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 lucknowense*, and *Chrysosporium foetida*. merdarium), rent spores, Queensland golden spores (Chrysosporium queenslandicum), tropical golden spores, brown golden spores (Chrysosporium zonatum), gray-capped coprinus (Coprinus cinereus), hairy-skinned spores (Coriolushirsutus), rod-shaped spores (Fusarium), cereal spores (Fusarium), kuweiss spores (Fusarium), large-knife spores (Fusarium), grass spores (Fusarium), red spores (Fusarium), heterospores (Fusarium), albinofuss spores (Fusarium), acuminata spores (Fusarium), multibranched spores (Fusarium), pink spores (Fusarium), elderberry spores (Fusarium), skin-colored spores (Fusarium), pseudo-clastic spores (Fusarium), sulfur spores (Fusarium), round spores (Fusarium), pseudo-filamentous spores (Fusarium), patchy spores (Fusarium), specific humic molds (Fusarium), soft-haired humic molds (Fusarium), rice black molds (Mucor), thermophilic filamentous molds (Fusarium), rough spores (Nephrolepis), purpuric molds (Penicillium), Phanerochaete chrysosporium (Phanerochaete chrysosporium), radiata (Phlebia radiata), Pleurotus eryngii (Pleurotus) eryngii), terrestrial clostridium, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma corningensis, Trichoderma longibranchii, Trichoderma reesei, or green Trichoderma cells.

[0296] Fungal cells can be transformed through a process involving protoplast formation, protoplast transformation, and cell wall regeneration in a manner known per se. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP 238023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 81:1470-1474 and Christensen et al., 1988, Bio / Technology 6:1419-1422. Suitable methods for transforming Fusarium species are described in Maladier et al., 1989, 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, Journal of Bacteriology, 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences of the United States of America, 75:1920.

[0297] Method of generation

[0298] The present invention also relates to a method for producing the polypeptide of the invention, comprising (a) culturing cells under conditions favorable to the production of the polypeptide, the cells producing the polypeptide in their wild-type form; (b) optionally isolating the polypeptide; and (c) recovering the polypeptide. In one aspect, the cell is an Alternaria haematomycete cell. In another aspect, the cell is an Alternaria haematomycete HTCC2649 cell. In another aspect, the cell is a Terracoccus cell. In another aspect, the cell is a Terracoccus sp. 273MFTsu3.1 cell. In another aspect, the cell is a Knoellia cell. In another aspect, the cell is a Knoellia flavus cell.

[0299] The present invention also relates to a method for producing the polypeptide of the present invention, the method comprising (a) culturing a recombinant Bacillus spp. expression host cell containing a polynucleotide encoding the polypeptide of the present invention, the polynucleotide being operatively linked to one or more control sequences that direct the production of the polypeptide under conditions favorable to the production of the polypeptide; (b) optionally isolating the polypeptide; and (c) recovering the polypeptide.

[0300] In one embodiment, the host cell for the Bacillus expression is selected from the following list, which consists of the following items: Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Geobacillus stearothermophilus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, and Bacillus subtilis. Bacillus subtilis and Bacillus thuringiensis. In a preferred embodiment, the host cell for expression of the Bacillus genus is selected from the following list, which consists of the following items: Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis.

[0301] These host cells are cultured in a nutrient medium suitable for producing the polypeptide 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, said culture being carried out in a suitable medium and under conditions that allow for the expression and / or isolation of the polypeptide. This culture occurs using procedures known in the art in a suitable nutrient medium comprising 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 U.S. Center for Type Culture Collection). If the polypeptide is secreted into the nutrient medium, it can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

[0302] The peptide can be detected using methods known in the art that are specific to these 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 the peptide.

[0303] The polypeptide can be recovered using methods known in the art. For example, the polypeptide can be recovered from the nutrient medium through conventional procedures, including but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, fermentation broth containing the polypeptide is recovered.

[0304] Peptides can be purified by a variety of methods 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 (e.g., preparative isoelectric focusing), differential dissolution (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, edited by Janson and Ryden, VCH Publishing, New York, 1989) to obtain substantially pure peptides.

[0305] In terms of alternatives, the peptide is not recycled; instead, the host cell of the present invention expressing the peptide is used as the source of the peptide.

[0306] plant

[0307] This invention also relates to isolated plants, such as transgenic plants, plant parts, or plant cells, that include the polypeptides of this invention, thereby expressing and producing a recoverable amount of the polypeptide or domain. The polypeptide or domain can be recovered from the plant or plant part. Alternatively, the plant or plant part including the polypeptide or domain can be used as is to improve the quality of food or feed, such as improving nutritional value, palatability, and rheological properties, or disrupting anti-nutritional factors.

[0308] Genetically modified plants can be dicotyledonous (dicotyledonous plants) or monocotyledonous (monocotyledonous plants). Examples of monocotyledonous plants are grasses, such as meadow grass (bluegrass, Kentucky bluegrass); forage grasses, such as fescue (Festuca) and ryegrass (Lolium); temperate grasses, such as creeping bentgrass (Agrostis); and cereals, such as wheat, oats, rye, barley, rice, sorghum, and corn.

[0309] Examples of dicotyledonous plants include tobacco, legumes (such as lupins, potatoes, sugar beets, peas, beans, and soybeans), and cruciferous plants (such as cauliflower, rapeseed, and the closely related model organism Arabidopsis thaliana).

[0310] Examples of plant parts include stems, callus, leaves, roots, fruits, seeds, and tubers, as well as individual tissues that include these parts, such as epidermis, mesophyll, parenchyma, vascular tissue, and meristematic tissue.

[0311] Plant cells and specific plant cell compartments (such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes, and cytoplasm) are also considered plant parts.

[0312] Also included within the scope of this invention are such plants, plant parts, and offspring of plant cells.

[0313] Transgenic plants or plant cells expressing the polypeptide or domain can be constructed using methods known in the art.

[0314] The present invention also relates to a method for generating the polypeptide or domain of the present invention, the method comprising: (a) culturing a transgenic plant or plant cell comprising a polynucleotide encoding the polypeptide or domain under conditions conducive to the generation of the polypeptide or domain, and (b) recovering the polypeptide or domain.

[0315] Fermentation broth preparations or cell compositions

[0316] The present invention also relates to fermentation broth formulations or cell compositions comprising the polypeptides of the present invention. The fermentation broth product further comprises additional components used in the fermentation process, such as cells (including host cells containing genes encoding the polypeptides of the present invention, which are used to produce the polypeptides of interest), cell debris, biomass, fermentation medium, and / or fermentation products. In some embodiments, the composition is a cell-killing whole culture medium comprising one or more organic acids, killed cells and / or cell debris, and a culture medium.

[0317] As used herein, the term "fermentation broth" refers to a preparation produced by cell fermentation that undergoes little or no recovery and / or purification. For example, fermentation broth is produced when a microbial culture is incubated to saturation under carbon-limited conditions that allow protein synthesis (e.g., expression by enzymes of the host cell) and secretion of proteins into the cell culture medium. Fermentation broth may contain unfractionated or fractionated contents of the fermentation material obtained at the end of fermentation. Typically, fermentation broth is unfractionated and includes used culture medium and cell debris remaining after, for example, removal of microbial cells (e.g., filamentous fungal cells) by centrifugation. In some embodiments, fermentation broth contains used cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0318] In one embodiment, the fermentation broth formulation and cell composition comprise a first organic acid component (comprising at least one 1-5 carbon organic acid and / or its salt) and a second organic acid component (comprising at least one 6- or more carbon organic acid and / or its salt). In a specific embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, its salt, or a mixture of two or more of the foregoing; and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, its salt, or a mixture of two or more of the foregoing.

[0319] In one aspect, the composition comprises one or more organic acids and optionally further comprises killed cells and / or cell debris. In one embodiment, these killed cells and / or cell debris are removed from the cell-killing whole culture medium to provide a composition free of these components.

[0320] These fermentation broth formulations or cell compositions may further include preservatives and / or antimicrobial (e.g., bacteriostatic) agents, including but not limited to sorbitol, sodium chloride, potassium sorbate, and other agents known in the art.

[0321] The cell-killing whole culture or composition may contain the ungraded contents of the fermentation material obtained at the end of fermentation. Typically, the cell-killing whole culture or composition includes used culture medium and cell debris present after microbial cells (e.g., filamentous fungal cells) have been grown to saturation and incubated under carbon-limited conditions to allow protein synthesis. In some embodiments, the cell-killing whole culture or composition includes used cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, methods known in the art can be used to permeate and / or lyse the microbial cells present in the cell-killing whole culture or composition.

[0322] The whole culture medium or cell composition described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture medium components, and / or one or more insoluble enzymes. In some embodiments, insoluble components may be removed to provide a clear liquid composition.

[0323] The whole-liquid formulations and cell compositions of the present invention can be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0324] Composition

[0325] 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.

[0326] 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 include the formulation agents described below. Alternatively, these compositions may include 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.

[0327] In one embodiment, the composition comprises a polypeptide from the third aspect of the invention and optionally a formulation agent. In one embodiment, the composition comprises a polypeptide from the fourth aspect of the invention and optionally a formulation agent. In one embodiment, the composition comprises a polypeptide from the fifth aspect of the invention and optionally a formulation agent. In one embodiment, the composition comprises a polypeptide from the sixth aspect of the invention and optionally a formulation agent.

[0328] formulation

[0329] The enzymes of the present invention can be formulated as liquids or solids. For liquid formulations, the formulation may include polyols (e.g., glycerol, ethylene glycol, or propylene glycol), salts (e.g., sodium chloride, sodium benzoate, potassium sorbate), or sugars or sugar derivatives (e.g., dextrin, glucose, sucrose, and sorbitol). Thus, in one embodiment, the composition is a liquid composition comprising the polypeptide 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.

[0330] For solid formulations, the formulation may be, for example, in the form of granules, spray-dried powder, or agglomerates. Formulating agents may include salts (organic or inorganic zinc, sodium, potassium, or calcium 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 sugars or sugar derivatives (such as, for example, sucrose, dextrin, glucose, lactose, sorbitol).

[0331] In one embodiment, the solid composition is in particulate form. The particulates may have a matrix structure in which the components are homogeneously mixed. However, the particulates 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 inert particles (containing the protease of the invention, optionally combined with one or more other enzymes applied thereto).

[0332] 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.

[0333] Salt coatings are typically at least 1 μm thick and can be a specific 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.

[0334] 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 containing the protease of the present invention, and a salt coating.

[0335] In another embodiment, the formulation agent 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 agent 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.

[0336] Animal feed and animal feed additives

[0337] This invention also relates to animal feed compositions and animal feed additives. The animal feed compositions or diets have a relatively high protein content. Characteristics of poultry and pig diets are shown in columns 2-3 of Table B in 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.

[0338] The animal feed composition according to the invention has a crude protein content of 50-800 g / kg, and further contains at least one or more proteases as described herein.

[0339] In addition, or alternatively (regarding the crude protein content mentioned above), the animal feed composition of the present invention has a metabolizable energy content 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.

[0340] In a specific embodiment, the contents of metabolizable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and / or lysine fall within any one of WO 01 / 58275, Table B, ranges 2, 3, 4, or 5 (R.2-5).

[0341] 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.).

[0342] Metabolizable energy can be calculated from NRC publication *Nutrient Requirements in Swine*, Ninth Reprint 1988, National Research Council, Department of Agriculture, Animal Nutrition Society, Swine Nutrition Division. National Academy of Sciences Press, Washington, D.C., pp. 2–6, and the *European Table of Energy Values ​​for Poultry Feed-stuffs*, Spelderholt Poultry Research and Extension Center, 7361DA Beckbergen, Netherlands, Grafisch bedrijf Ponsen & Looijen, Wageningen. ISBN 90-71463-12-5.

[0343] The dietary content of calcium, available phosphorus, and amino acids in a complete animal diet is calculated based on feed tables (such as Feed Table (Veevoedertabel) 1997, Chemical Composition Data (gegevens over chemische samenstelling), Digestibility and Nutritional Value of Feed (verteerbaarheid en voederwaarde van voedermiddelen), Central Veevoederbureau, Runderweg 6,8219pk Leylistard, ISBN 90-72839-13-7).

[0344] In a specific embodiment, the animal feed composition of the present invention comprises at least one plant protein as defined above.

[0345] 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, typically in an amount of 0-30%.

[0346] In a further specific embodiment, the animal feed composition of the present invention comprises 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.

[0347] The animal feed may include plant-based proteins. In specific embodiments, these plant-based proteins have a protein content of at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w / w). The plant-based proteins 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.

[0348] In one specific embodiment, the plant protein source is material from one or more plants of the Fabaceae family, such as soybean, lupin, pea, or bean. In another specific embodiment, the plant protein source is material from one or more plants of the Chenopodiaceae family, such as beet, sugar beet, spinach, or quinoa. Other examples of plant protein sources are rapeseed and cabbage. In 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.

[0349] Animal diets can be formulated, for example, into pastes (non-pelleted) or pellets. Typically, the milled feed is mixed and adequate amounts of essential vitamins and minerals are added according to the instructions for these types discussed. Enzymes are added in the form of solid or liquid enzyme formulations. For example, for pastes, solid or liquid enzyme formulations may be added before or during the ingredient mixing step. For pelleted feeds, the (solid or liquid) protease / enzyme preparation may also be added before or during the feed ingredient step. Typically, liquid protease / enzyme preparations include the protease of the present invention, optionally accompanied by a polyol (e.g., glycerol, ethylene glycol, or propylene glycol), and are added after the pelleting step, for example, by spraying the liquid formulation onto pellets. Enzymes can also be incorporated into feed additives or premixes.

[0350] 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.

[0351] In one embodiment, the composition includes one or more additional enzymes. In one embodiment, the composition includes one or more microorganisms. In one embodiment, the composition includes one or more vitamins. In one embodiment, the composition includes one or more minerals. In one embodiment, the composition includes one or more amino acids. In one embodiment, the composition includes one or more other feed ingredients.

[0352] In another embodiment, the composition comprises one or more polypeptides of the present invention, one or more formulation agents, and one or more additional enzymes. In one embodiment, the composition comprises one or more polypeptides of the present invention, one or more formulation agents, and one or more microorganisms. In one embodiment, the composition comprises one or more polypeptides of the present invention, one or more formulation agents, 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 formulation agents, and one or more amino acids. In one embodiment, the composition comprises one or more polypeptides of the present invention, one or more formulation agents, and one or more other feed ingredients.

[0353] In another embodiment, the composition comprises one or more polypeptides of the present invention, one or more formulation agents, and one or more components selected from the following list, which consists of: 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.

[0354] 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.

[0355] Currently, it is considered to administer the protease at 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 (ppm) of protease protein per kg of feed.

[0356] 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.

[0357] In one specific 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 specifically 0.05% to 5.0% or 0.2% to 1.0% (% refers to g additive / 100g feed). Specifically, the same applies to premixes.

[0358] 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).

[0359] Other enzymes

[0360] In another embodiment, the compositions described herein optionally include one or more enzymes. Enzymes can be classified based on the Enzyme Nomenclature manual from NC-IUBMB, 1992, and can also be found on the ENZYME website: http: / / www.expasy.ch / enzyme / . ENZYME is an information repository for 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 A. The ENZYME database, 2000, Nucleic Acids Res 28:304-305). The IUB-MB enzyme nomenclature is based on their substrate specificity and occasionally on their molecular mechanisms; this classification does not reflect the structural characteristics of these enzymes.

[0361] Henrissat et al. describe another classification of certain glycoside hydrolases (e.g., endoglucanases, xylanases, galactanases, mannanases, glucanases, lysozymes, and galactosidases) in “The carbohydrate-active enzymes database (CAZy), Nucleic Acids Res. (January 1, 2014)” 42(D1):D490-D495; see also www.cazy.org.

[0362] Therefore, the compositions of the present invention may further comprise at least one other enzyme selected from the group consisting of: phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); α-galactosidase (EC 3.2.1.22); protease (EC 3.4); phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase, such as, for example, α-amylase (EC 3.2.1.1); lysozyme (EC 3.2.1.1); and other enzymes. 3.2.1.17); arabinofuranase (EC 3.2.1.55); β-xylosidase (EC 3.2.1.37); acetylxylan esterase (EC 3.1.1.72); ferulic acid esterase (EC 3.1.1.73); cellulase (EC 3.2.1.4); cellobiose hydrolase (EC 3.2.1.91); β-glucosidase (EC 3.2.1.21); amylopectinase (EC 3.2.1.41) and β-glucanase (EC 3.2.1.4 or EC 3.2.1.6), or any combination thereof.

[0363] In one specific 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 Blue (AB Enzymes), (Huvepharma) XP (Verenium / DuPont) and PHY (DuPont). Other preferred phytases include those described, for example, WO 98 / 28408, WO 00 / 43503, and WO 03 / 066847.

[0364] In one specific embodiment, the composition of the present invention includes xylanase (EC 3.2.1.8). Examples of commercially available xylanases include... WX and G2 (DSM Nutritional Products) XT and Barley (AB Vista), (Verenium) X (Huvepharma) and XB (xylanase / β-glucanase, DuPont).

[0365] In one specific embodiment, the composition of the present invention comprises a protease (EC 3.4). Examples of commercially available proteases include... ProAct (DSM Nutritional Products).

[0366] microorganism

[0367] In one embodiment, the animal feed composition further comprises one or more additional microorganisms. In a specific embodiment, the animal feed composition further comprises bacteria from one or more genera including: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Bacillus, Propionibacterium, Bifidobacterium, Clostridium, and Megasphaera, or any combination thereof.

[0368] In a preferred embodiment, the animal feed composition further comprises bacteria from one or more strains of the following: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circumflex, Enterococcus faecalis, Enterococcus spp. and Pediococcus spp., Lactobacillus spp., Bifidobacterium spp., Lactobacillus acidophilus, Pediococcus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillus farciminus, Lactobacillus rhamnosus, Clostridium butyricum, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Lactobacillus salivarius. ssp. salivarius), Megasphaera elsdenii, Propionibacterium.

[0369] In a more preferred embodiment, the animal feed composition further comprises bacteria from one or more of the following strains of Bacillus subtilis: 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); and BS 278 (NRRL B-50634).

[0370] The bacterial count for each bacterial strain in this animal feed composition is 1 x 10^6. 4 and 1x 10 14 The CFU / kg dry matter content is preferably between 1 x 10. 6 and 1x 10 12 Between CFU / kg of dry matter, and more preferably 1 x 10 7 Up to 1x10 11 Between CFU / kg dry matter. In a more preferred embodiment, the bacterial count of each bacterial strain in the animal feed composition is 1 x 10⁻⁶. 8 and 1x 10 10 Between CFU / kg of dry matter.

[0371] The bacterial count for each bacterial strain in this animal feed composition is 1 x 10^6. 5 and 1x 10 15 Between CFU / animal / day, preferably 1 x 10 7 and 1x 10 13 Between CFU / animal / day, and more preferably 1x10 8 and 1x 10 12 Between CFU / animal / day. In a more preferred embodiment, the bacterial count of each bacterial strain in the animal feed composition is 1 x 10⁻⁶. 9 and 1x 10 11 CFU / Animal / Between Heaven.

[0372] In another embodiment, the one or more bacterial strains exist in a stable spore form.

[0373] premix

[0374] In one embodiment, the animal feed may include a premix, which may include, for example, vitamins, minerals, enzymes, amino acids, preservatives, antibiotics, other feed ingredients, or any combination thereof mixed into the animal feed.

[0375] Vitamins and minerals

[0376] In another embodiment, the animal feed may include 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 include one or more minerals, such as one or more trace minerals and / or one or more macro minerals.

[0377] Typically, fat-soluble vitamins and water-soluble vitamins, along with trace minerals, form a portion of what is known as a premix intended to be added to feed, while large amounts of minerals are usually added separately to the feed.

[0378] Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, such as vitamin K3.

[0379] 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.

[0380] Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, and zinc.

[0381] Non-limiting examples of minerals include calcium, magnesium, potassium, and sodium.

[0382] Table A of WO 01 / 58275 lists the nutritional requirements for these components (using poultry and piglets / pigs as examples). Nutritional requirements refer to the specified concentrations of these components that should be provided in the diet.

[0383] In alternative embodiments, 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 refers to any one or more of one, two, three, four, etc., up to all thirteen, or up to all fifteen individual components. More specifically, the 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.

[0384] In yet another embodiment, the animal feed additive of the present invention comprises at least one of the following vitamins, preferably such that its concentration in the feed falls within the range defined in Table 1 below (for piglet diets and broiler chicken diets, respectively).

[0385] Table 1: General Vitamin Recommendations

[0386] Vitamins Piglet Diet Broiler Chicken Diet Vitamin A 10,000-15,000 IU / kg feed 8-12,500 IU / kg feed Vitamin D3 1800-2000 IU / kg feed 3000-5000 IU / kg feed Vitamin E 60-100mg / kg feed 150-240mg / kg feed Vitamin K3 2-4 mg / kg feed 2-4 mg / kg feed Vitamin B1 2-4 mg / kg feed 2-3 mg / kg feed Vitamin B2 6-10mg / kg feed 7-9 mg / kg feed Vitamin B6 4-8 mg / kg feed 3-6 mg / kg feed Vitamin B12 0.03-0.05 mg / kg feed 0.015-0.04 mg / kg feed Niacin (vitamin B3) 30-50mg / kg feed 50-80mg / kg feed pantothenic acid 20-40mg / kg feed 10-18 mg / kg feed folic acid 1-2 mg / kg feed 1-2 mg / kg feed Biotin 0.15-0.4 mg / kg feed 0.15-0.3 mg / kg feed choline chloride 200-400mg / kg feed 300-600mg / kg feed

[0387] amino acids

[0388] The compositions of the present invention may also include one or more amino acids. Examples of amino acids for use in animal feed are lysine, alanine, β-alanine, threonine, methionine, and tryptophan.

[0389] Other feed ingredients

[0390] The compositions of the present invention may further include colorants, stabilizers, growth-improving additives and flavoring compounds / seasonings, polyunsaturated fatty acids (PUFAs); reactive oxygen species, antimicrobial peptides and antifungal peptides.

[0391] Examples of colorants are carotenoids, such as beta-carotene, astaxanthin, and lutein.

[0392] Examples of flavoring compounds / seasonings are methoxycresol, anethole, decadecyl, undecyl and / or dodecyl lactone, ionone, irisone, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin, and tannins.

[0393] Examples of antimicrobial peptides (AMPs) are CAP18, leucocin A, tritrpticin, protegrin-1, thanatin, defensin, lactoferrin, lactoferrin peptide, and oxispirin such as novispirin (Robert Lehrer, 2000), plectasins, and statins, including the 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.

[0394] Examples of antifungal peptides (AFPs) are peptides from Aspergillus giganteus and Aspergillus niger, along with variants and fragments that retain antifungal activity, as disclosed in WO 94 / 01459 and WO 02 / 090384.

[0395] 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.

[0396] Examples of types of reactive oxygen species include chemicals such as perborates, persulfates, or percarbonates; and enzymes such as oxidases, oxygenases, or synthases.

[0397] 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.

[0398] use

[0399] The present invention also relates to methods for using polypeptides or combinations thereof having protease activity (for example, for animal feed).

[0400] Uses in animal feed

[0401] 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.

[0402] 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.

[0403] 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.

[0404] 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.

[0405] In one specific 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 specific 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.

[0406] 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 "correct dosage" specifically refers to the goal of obtaining consistent and constant results, and the ability to optimize the dosage based on the desired effect.

[0407] 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 products.

[0408] This protease preparation can be (a) added directly to feed, or (b) it can be 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.

[0409] Specifically, protease preparations with the purity of the aforementioned order 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 differences between batches.

[0410] These types of protease products can certainly be mixed with other enzymes.

[0411] 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.

[0412] As used herein, the term plant-based protein refers to any compound, composition, formulation, or mixture comprising at least one protein derived from or derived from a plant, wherein said protein includes modified proteins or protein derivatives. In specific embodiments, the protein content of these plant-based proteins is at least 10%, 20%, 30%, 40%, 50%, or 60% (w / w).

[0413] 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.

[0414] In one specific embodiment, the plant-based protein source is material from one or more plants of the Fabaceae family (e.g., soybean, lupin, pea, or broad bean).

[0415] In another specific embodiment, the plant protein source is material derived from one or more plants of the Chenopodiaceae family, such as beets, sugar beets, spinach, or Quinoa.

[0416] Other examples of plant-based protein sources include rapeseed, sunflower seed, cottonseed, and cabbage.

[0417] Soybeans are a preferred source of plant-based protein.

[0418] Other examples of plant-based protein sources include cereals such as barley, wheat, rye, oats, corn, rice, black wheat, and sorghum.

[0419] In specific embodiments of the treatment process, the protease(s) discussed affect (or exert a hydrolytic or degrading effect on) these proteins, such as plant proteins or protein sources. 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(s) discussed. 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.

[0420] In another specific embodiment of the processing procedure 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 for it, 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.

[0421] 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.

[0422] 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, protein and / or amino acid digestibility 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.

[0423] 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.

[0424] Preparation method

[0425] In one embodiment, the present invention also relates to a method for preparing animal feed or feed additives, the method comprising preparing an animal feed or feed additive comprising animal feed and a protease of the first aspect of the present invention.

[0426] In one embodiment, the present invention also relates to a method for preparing animal feed or feed additives, the method comprising preparing an animal feed or feed additive comprising animal feed and a protease of the second aspect of the present invention.

[0427] In one embodiment, the present invention also relates to a method for preparing animal feed or feed additives, the method comprising preparing an animal feed or feed additive comprising animal feed and a protease of the third aspect of the present invention.

[0428] In one embodiment, the present invention also relates to a method for preparing animal feed or feed additives, the method comprising preparing an animal feed or feed additive comprising animal feed and a protease of the fourth aspect of the present invention.

[0429] In one embodiment, the present 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 the protease of the fifth aspect of the present invention.

[0430] In one embodiment, the present invention also relates to a method for preparing animal feed or feed additives, the method comprising preparing an animal feed or feed additive comprising animal feed and a protease of the sixth aspect of the present invention.

[0431] Nucleic acid constructs, expression vectors, recombinant host cells, and methods for producing proteases

[0432] The present invention also relates to nucleic acid constructs, expression vectors and recombinant host cells comprising such polynucleotides encoding the protease of the present invention.

[0433] The present invention also relates to a method for producing a protease, comprising (a) culturing a recombinant host cell containing such a polynucleotide; and (b) recovering the protein.

[0434] The protein can be native or heterologous to the host cell. The term "protein" here does not refer to a specific length of encoded product and, therefore, encompasses peptides, oligopeptides, and proteins. The term "protein" also encompasses two or more polypeptides that combine to form the encoded product. These proteins also include hybrid polypeptides and fusion polypeptides.

[0435] Preferably, the protein is a protease. The gene can be obtained from any prokaryotic, eukaryotic, or other source.

[0436] Preferred embodiments of the present invention

[0437] Preferred embodiments of the invention are described in the following set of items.

[0438] 1. An animal feed or animal feed additive comprising one or more polypeptides having protease activity, wherein the polypeptide is selected from the group consisting of the following items:

[0439] (a) A polypeptide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 of SEQ ID NO:2;

[0440] (b) A polypeptide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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:5;

[0441] (c) A polypeptide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 of SEQ ID NO:11;

[0442] (d) A polypeptide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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:14;

[0443] (e) A polypeptide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 of SEQ ID NO:17;

[0444] (f) A polypeptide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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:20;

[0445] (g) A polypeptide encoded by a polynucleotide that hybridizes with the following under low-strict, medium-strict, medium-high-strict, high-strict, or very high-strict conditions:

[0446] (i) The mature polypeptide coding sequence of SEQ ID NO:1;

[0447] (ii) The mature polypeptide coding sequence of SEQ ID NO:3;

[0448] (iii) The mature polypeptide coding sequence of SEQ ID NO:10;

[0449] (iv) The mature polypeptide coding sequence of SEQ ID NO:12;

[0450] (v) The mature polypeptide coding sequence of SEQ ID NO:16; or

[0451] (vi)(i), (ii), (iii), (iv), or (v) full-length complements;

[0452] (h) A polypeptide encoded by a polynucleotide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 coding sequence of SEQ ID NO:1;

[0453] (i) A polypeptide encoded by a polynucleotide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 coding sequence of SEQ ID NO:3;

[0454] (j) A polypeptide encoded by a polynucleotide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 coding sequence of SEQ ID NO:10;

[0455] (k) A polypeptide encoded by a polynucleotide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 coding sequence of SEQ ID NO:12;

[0456] (l) A polypeptide encoded by a polynucleotide having at least 65%, for example at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 coding sequence of SEQ ID NO:16;

[0457] (m) A variant of SEQ ID NO:5, SEQ ID NO:14, or SEQ ID NO:20, or a mature polypeptide of SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:17, 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; and

[0458] (n) A fragment of a (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), or (m) polypeptide having protease activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids.

[0459] 2. Animal feed or animal feed additive as described in Project 1, wherein the polypeptide is obtained from or can be obtained from the order Micrococciles.

[0460] 3. Animal feed or animal feed additive as described in any one of items 1 to 2, wherein the polypeptide is obtained from or can be obtained from the family Mesophyceae.

[0461] 4. Animal feed or animal feed additives as described in any one of items 1 to 3, wherein:

[0462] (a) The polypeptide exhibits at pH 4 at an activity at least twice that of the soybean-corn flour protein, for example at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3 times, at least 3.25 times, at least 3.5 times, at least 3.75 times, or at least 4 times greater than that of the protein 10R (SEQ ID NO:8) at the same pH.

[0463] (b) The polypeptide exhibits at least twice the activity of protease 10R (SEQ ID NO:8) at the same pH at pH 5, for example, at least 2.25 times, at least 2.5 times, at least 2.75 times, or at least 3 times the activity of the protein in soybean-corn flour; and

[0464] (c) Compared with protease 10R (SEQ ID NO:8) at the same pH, the polypeptide has at least 50% activity against soybean-corn flour at pH 7, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., at least the same), or at least 105% activity.

[0465] 5. Animal feed or animal feed additives as described in any one of items 1 to 4, wherein:

[0466] (a) Compared with its activity at pH 7, the peptide has at least 25% activity against soybean-corn flour at pH 4, for example at least 30%, at least 35%, or at least 40% activity;

[0467] (b) Compared to its activity at pH 7, the peptide exhibits at least 45% activity against soybean-corn flour at pH 5, for example, at least 50%, at least 55%, at least 60%, or at least 65% activity; and

[0468] (c) Compared with protease 10R (SEQ ID NO:8) at the same pH, the polypeptide has at least 50% activity against soybean-corn flour at pH 7, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., at least the same), or at least 105% activity.

[0469] 6. The animal feed or animal feed additive as described in any one of items 1 to 5, wherein the polypeptide comprises a mature polypeptide of SEQ ID NO:5, SEQ ID NO:14 or SEQ ID NO:20, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17 or SEQ ID NO:19, or amino acids 1 to 203 of SEQ ID NO:2, amino acids 1 to 203 of SEQ ID NO:4, amino acids 1 to 203 of SEQ ID NO:5, amino acids 1 to 204 of SEQ ID NO:11, amino acids 1 to 204 of SEQ ID NO:13, amino acids 1 to 204 of SEQ ID NO:14, amino acids 1 to 204 of SEQ ID NO:17, amino acids 1 to 204 of SEQ ID NO:19 or amino acids 1 to 204 of SEQ ID NO:20, or is composed of the like.

[0470] 7. Animal feed or animal feed additive containing one or more polypeptides with protease activity, wherein:

[0471] (a) The polypeptide is a serine protease of the S1 family of peptidases;

[0472] (b) The polypeptide exhibits at pH 4 at an activity at least twice that of the soybean-corn flour protein, for example at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3 times, at least 3.25 times, at least 3.5 times, at least 3.75 times, or at least 4 times greater than that of the protein 10R (SEQ ID NO:8) at the same pH.

[0473] (c) The polypeptide exhibits at least twice the activity of protease 10R (SEQ ID NO:8) at the same pH at pH 5, for example, at least 2.25 times, at least 2.5 times, at least 2.75 times, or at least 3 times the activity of protease 10R (SEQ ID NO:8) at the same pH; and

[0474] (d) Compared with protease 10R (SEQ ID NO:8) at the same pH, this polypeptide has at least 50% activity against soybean-corn flour at pH 7, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., at least the same), or at least 105% activity.

[0475] 8. The animal feed or animal feed additive as described in Item 7, wherein the polypeptide has less than 20 times, for example less than 15 times, less than 10 times, less than 9 times, less than 8 times the activity of protease 10R (SEQ ID NO:8) against soybean-corn flour at pH 4, and less than 20 times, for example less than 15 times, less than 10 times, less than 9 times, less than 8 times the activity of protease 10R (SEQ ID NO:8) against soybean-corn flour at pH 5.

[0476] 9. The animal feed or animal feed additive as described in any one of items 7 or 8, wherein the polypeptide has less than 200% activity against soybean-corn flour at pH 7, for example less than 180%, less than 170%, less than 160%, less than 150%, less than 140%, less than 130%, or less than 125% activity compared to protease 10R (SEQ ID NO: 8) at the same pH.

[0477] 10. An animal feed or animal feed additive containing one or more polypeptides with protease activity, wherein:

[0478] (a) The polypeptide is a serine protease of the S1 family of peptidases;

[0479] (b) Compared with its activity at pH 7, the peptide has at least 25% activity against soybean-corn flour at pH 4, for example at least 30%, at least 35%, or at least 40% activity;

[0480] (c) Compared to its activity at pH 7, the peptide exhibits at least 45% activity against soybean-corn flour at pH 5, for example, at least 50%, at least 55%, at least 60%, or at least 65% activity; and

[0481] (d) Compared with protease 10R (SEQ ID NO:8) at the same pH, this polypeptide has at least 50% activity against soybean-corn flour at pH 7, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., at least the same), or at least 105% activity.

[0482] 11. The animal feed or animal feed additive as described in item 10, wherein the polypeptide has less than 100% activity against soybean-corn flour at pH 4, for example less than 90%, less than 80%, less than 75%, or less than 70%, compared to its activity at pH 7, and has less than 95% activity against soybean-corn flour at pH 5, for example less than 90%, less than 85%, or less than 80%, compared to its activity at pH 7.

[0483] 12. The animal feed or animal feed additive as described in any one of items 10 or 11, wherein the polypeptide has less than 200% activity against soybean-corn flour at pH 7, for example less than 180%, less than 170%, less than 160%, less than 150%, less than 140%, less than 130%, or less than 125% activity compared to protease 10R (SEQ ID NO:8) at the same pH.

[0484] 13. Animal feed or animal feed additive as described in any one of items 7 to 12, wherein the polypeptide is obtained from or can be obtained from the order Micrococciles.

[0485] 14. Animal feed or animal feed additive as described in any one of items 7 to 12, wherein the polypeptide is obtained from or can be obtained from the family Mesophyceae.

[0486] 15. An animal feed or animal feed additive containing one or more polypeptides with protease activity, wherein:

[0487] (a) The polypeptide is a serine protease of the S1 family of peptidases; and

[0488] (b) The polypeptide is obtained from or can be obtained from the family Mesocystaceae.

[0489] 16. Animal feed or animal feed additive as described in any one of items 1 to 15, wherein the polypeptide comprises one or more motifs VCG[E / Q]KVGQP (SEQ ID NO:15).

[0490] 17. The animal feed or animal feed additive as described in any one of items 1 to 16, having a crude protein content of 50 to 800 g / kg.

[0491] 18. The animal feed or animal feed additive as described in any one of items 1 to 17, further comprising one or more components selected from the following list, which consists of the following items:

[0492] One or more other enzymes;

[0493] One or more microorganisms;

[0494] One or more vitamins;

[0495] One or more minerals;

[0496] One or more amino acids; and

[0497] One or more other feed ingredients.

[0498] 19. Animal feed or animal feed additive as described in item 18, wherein these additional enzymes are 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.

[0499] 20. Animal feed or animal feed additive as described in Item 18, wherein the one or more microorganisms are selected from the group consisting of: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circumflex, Bifidobacterium bifidum, Bifidobacterium animalis, Carnobacterium sp., Clostridium butyricum, Clostridium, Enterococcus faecalis, Enterococcus, Lactobacillus, Lactobacillus acidophilus, Lactobacillus farciminus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactococcus lactis, Leuconostoc, Megasphaera elsdenii, Megasphaera sp.), Pediococcus acidilactici, Pediococcus spp., Propionibacterium thoenii, Propionibacterium spp., and Streptococcus spp. or any combination thereof.

[0500] 21. Use of animal feed or animal feed additives as described in any one of items 1 to 20:

[0501] In the preparation of compositions for use in animal feed;

[0502] Used to improve the nutritional value of animal feed;

[0503] Used to increase the digestible and / or soluble protein in animal feed;

[0504] Used to increase the degree of protein hydrolysis in animal diets;

[0505] Used to improve one or more performance parameters in animals; and / or

[0506] Used for processing proteins.

[0507] 22. A method for preparing animal feed, comprising mixing an animal feed additive as described in any one of items 1 to 20 with at least one protein or protein source.

[0508] 23. A method for improving the nutritional value of animal feed, wherein an animal feed or animal feed additive as described in any one of items 1 to 20 is added to the feed.

[0509] 24. A method for processing protein, comprising the step of adding an animal feed or animal feed additive as described in any one of items 1 to 20 to at least one protein or protein source.

[0510] 25. A method for increasing the digestibility and / or solubility of protein, comprising mixing an animal feed additive as described in any one of claims 1 to 20 with at least one protein or protein source.

[0511] 26. A method for improving one or more performance parameters in an animal, comprising administering an animal feed or animal feed additive as described in any one of items 1 to 20 to one or more animals.

[0512] 27. The use as described in item 21 or the method as described in item 26, wherein the performance parameters are selected from the following list, which consists of the following items: body weight gain (BWG), European Production Efficiency Factor (EPEF), and feed conversion ratio (FCR).

[0513] 28. A method for producing a polypeptide, the method comprising:

[0514] (a) Culturing recombinant Bacillus expression host cells containing a polynucleotide encoding the polypeptide shown in Item 1, the polynucleotide being operatively linked to one or more control sequences that direct the production of the polypeptide under conditions favorable to its production; and

[0515] (b) The polypeptide was recovered.

[0516] Example

[0517] strain

[0518] The S1 protease 1 from the genus *Janibacter* was identified in the published genome sequence of *Janibacter* strain HTCC2649, as described in Thrash, JC, Cho, JC, Bertagnolli, AD, Ferrira, S., Johnson, J., Vergin, KL, and Giovannini, SJ, “Genome sequence of the Marine Janibacter Sp. Strain HTCC2649”, 2011, *Journal of Bacteriology* 193:584-585. The DNA used in this paper was synthesized as described in Example 1. According to that article, the strain was isolated from water collected at a depth of 10 meters at Hydroelectric Power Station S, 12 miles southeast of Bermuda.

[0519] The S1 protease 1 from the genus *Terracoccus* sp. 273MFTsu3.1 was identified in the genome sequence (from JGI http: / / genome.jgi.doe.gov / (taxonomic unit id 2522125155, JGI project id 1000316)). The DNA used in this paper was synthesized as described in Example 10.

[0520] The S1 protease 1 from *Norris flavus* TL1 was identified in the genome sequence of *Norris flavus* (submitted to the EMBL / GenBank / DDBJ database by W. Zhu and G. Wang in August 2013). *Norris flavus* TL1 was isolated from pig manure at a pig farm of Huazhong Agricultural University in Wuhan, People's Republic of China, according to Xiang Yu, Yan Du, and Gejiao Wang, *International Journal of Systematic and Evolutionary Microbiology* (2012), 62, 384-389. The DNA used in this paper was synthesized as described in Example 14.

[0521] Protein assay

[0522] 1) Suc-AAPF-pNA assay:

[0523] pNA substrate: Suc-AAPF-pNA (Bachem L-1400).

[0524] Temperature: Room temperature (25℃)

[0525] Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl2, 150 mM KCl, 0.01% Triton X-100, adjusted to pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 with HCl or NaOH.

[0526] Mix 20 μl of protease (diluted in 0.01% Triton X-100) with 100 μl of assay buffer. The assay can be initiated by adding 100 μl of pNA substrate (50 mg, dissolved in 1.0 mL DMSO and further diluted 45-fold with 0.01% Triton X-100). Monitor OD. 405 The increase is used as a measure of protease activity.

[0527] 2) Protazyme AK assay:

[0528] Substrate: Protazyme AK tablets (cross-linked and stained casein; from Megazyme)

[0529] Temperature: Controlled (measured temperature).

[0530] Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl2, 150 mM KCl, 0.01% Triton X-100, adjusted to pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 6.5, 7.0, 8.0, 9.0, 10.0, and 11.0 with HCl or NaOH.

[0531] Suspend Protazyme AK tablets in 2.0 mL of 0.01% Triton X-100 with gentle agitation. Disperse 500 μL of this suspension and 500 μL of assay buffer in an Eppendorf tube and place on ice. Add 20 μL of protease sample (diluted in 0.01% Triton X-100). Initiate the assay by transferring the Eppendorf tube to an Eppendorf thermostat set to the assay temperature. Incubate the tube on the Eppendorf thermostat at the highest shaking rate (1400 rpm) for 15 minutes. Stop the incubation by transferring the tube back to an ice bath. Subsequently, centrifuge the tube in an ice-cold centrifuge for several minutes and transfer 200 μL of the supernatant to a microtiter plate. Read the OD. 650 As a measure of protease activity, a blank buffer (instead of the enzyme) is included in the assay.

[0532] 3) Determination of o-phthalaldehyde (OPA):

[0533] This assay detects primary amines and thus measures the difference in absorbance between a protease-treated sample and a control sample by the cleavage of peptide bonds by a protease. The assay is essentially performed according to Nielsen et al. (Nielsen, PM; Petersen, D; Dampmann, C., Improved method for determining food protein degree of hydrolysis, J Food Sci, 2001, 66:642-646).

[0534] Filter 0.5 ml of sample through a 100 kDa Microcon centrifuge filter (60 min, 11,000 rpm, 5 °C). Dilute these samples approximately (e.g., 10-fold, 50-fold, or 100-fold) in deionized water and load 25 μL of each sample into a 96-well microtiter plate (5 replicates). Dispense 200 μL of OPA reagent (100 mM disodium tetraborate decahydrate, 3.5 mM sodium dodecyl sulfate (SDS), 5.7 mM dithiothreitol (DDT), 6 mM phthalaldehyde) into all wells, shake the plate (10 sec, 750 rpm), and measure the absorbance at 340 nm.

[0535] Example 1: Expression of S1 protease 1 from *Hypericum* strain HTCC2649

[0536] Based on the publicly disclosed nucleotide sequence identified as SEQ ID NO:1, a codon-optimized synthetic gene with SEQ ID NO:3 was synthesized by GeneArt (GENEART AG BioPark, 11 Josef-Engert-Str., 93053, Regensburg, Germany). The synthetic gene was subcloned into a Bacillus expression vector using ClaI and MluI restriction enzyme sites, as described in WO 2012 / 025577. The S1 protease 1 was expressed using a Bacillus clausti secretion signal (with the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA, SEQ ID NO:6) instead of the native secretion signal. The expression plasmid was transformed into Bacillus subtilis. The expression cassette was integrated into the pectin lyase locus via homologous recombination. Transformants were selected on LB plates supplemented with 6 μg chloramphenicol per ml. A recombinant Bacillus subtilis clone containing the integrated expression construct was selected and designated as S1 protease 1 from *Haloxylon ammodendron* HTCC2649. It was cultured on a rotating shaker in 500 mL Erlenmeyer flasks with baffles, each containing 100 mL of yeast extract-based medium. The clone was cultured at 30°C for 3 days. The enzyme, containing the supernatant, was harvested and purified as described in Example 2.

[0537] Example 2: Purification of S1 protease 1 from *Haloxylon ammodendron* HTCC2649

[0538] The culture medium was centrifuged (20000x g, 20 min) and the supernatant was carefully decanted to separate it from the precipitate. The supernatant was filtered through a Nalgene 0.2 μm filter unit to remove any remaining Bacillus host cells. The 0.2 μm filtrate was transferred to a G25 dextran gel column (GE Healthcare) containing 10 mM MES / NaOH, 100 mM H3BO3, and 2 mM CaCl2 (pH 5.5). The enzyme transferred from the G25 dextran gel was applied to an SP-agarose gel FF column (GE Healthcare) equilibrated in 20 mM MES / NaOH (pH 5.5). After thoroughly washing the column with equilibration buffer, the protease was eluted using a linear gradient between the equilibration buffer and 20 mM MES / NaOH, 0.5 mM CaCl2, and 0.5 M NaCl (pH 5.5) for more than five column volumes. The main peak from an SP-agarose gel FF column (containing S1 protease 1 from Alternaria alternata) was merged, and solid ammonium sulfate was added to a final ammonium sulfate concentration of 1.6 M (NH4)2SO4. The ammonium sulfate-adjusted merged product was applied to a phenyl-agarose gel FF highly substituted column (from GE Healthcare), which was equilibrated in 10 mM MES / NaOH, 100 mM H3BO3, 2 mM CaCl2, and 1.6 M (NH4)2SO4 (pH 6.0). After thoroughly washing the column with equilibration buffer, the protease was eluted with a linear gradient of equilibration buffer and 10 mM MES / NaOH, 100 mM H3BO3, 10 mM MES, 2 mM CaCl2 (pH 6.0) + 25% 2-propanol for more than five column volumes. The main peak from a phenyl-agarose gel FF column (containing S1 protease 1 from *Hypericum fasciatus*) was merged and transferred to a G25 dextran gel column (GE Healthcare) in 20 mM CH3COOH / NaOH, 1 mM CaCl2 (pH 4.5). The enzyme transferred from the G25 dextran gel was then applied to an SP-agarose gel FF column (GE Healthcare) equilibrated in 20 mM MES / NaOH, 1 mM CaCl2 (pH 4.5). After thoroughly washing the column with equilibration buffer, the protease was eluted with a linear NaCl gradient (0-->0.5 M) in the same buffer for more than four column volumes. The protease activity from the column fraction was analyzed (measured using Protazyme AK at pH 7) and further analyzed by SDS-PAGE. Fractions showing only one band on a Coomassie-stained SDS-PAGE gel were merged, and the pH was adjusted to 5.8 with 3% NaOH. The condensate from the SP-agarose gel FF column was a purified formulation and used for further characterization.

[0539] Example 3: Characterization of S1 protease 1 (SEQ ID NO:5) from *Haloxylon ammodendron* HTCC2649

[0540] The Protazyme AK assay was used to obtain pH-activity and pH-stability profiles (residual activity after 2 hours at the indicated pH). For the pH-stability profile, the protease was diluted 8x in different assay buffers to achieve the pH values ​​of those buffers and then incubated at 37°C for 2 hours. After incubation, the pH of the protease incubation was adjusted to the same pH as before the residual activity assay by dilution in a pH 8.0 assay buffer. The temperature-activity profile at pH 7.0 was obtained using the Protazyme AK assay.

[0541] The results are shown in Tables 2-4 below. Data for protease 10R (SEQ ID NO:8) are included in these tables. For Table 2, activity is relative to the optimal pH of the enzyme. For Table 3, activity is relative to the residual activity of the samples held under stable conditions (5°C, pH 8.0 or pH 9.0). For Table 4, activity is relative to the optimal temperature of the enzyme at pH 7.0 or pH 6.5. pH-activity and pH-stability profiles for protease 10R were obtained using the Suc-AAPF-pNA assay, and temperature-activity profiles were obtained using the Protazyme AK assay at pH 6.5.

[0542] Table 2: pH-activity curves at 25°C as determined by kinetic Suc-AAPF-pNA assay

[0543]

[0544] Table 3: pH-stability curves determined using kinetic Suc-AAPF-pNA assays (after 2 hours at 37°C) (residual activity)

[0545]

[0546] Table 4: Temperature activity at pH 7.0 or pH 6.5 as determined by Protazyme AK assay. curve

[0547]

[0548] Compared with the data for protease 10R, the pH-activity and pH-stability curves (residual activity after 2 hours at 37°C) of S1 protease 1 from *Hypertricis* HTCC2649 against the substrate of protazyme AK, as well as the temperature activity curve against protazyme AK at pH 7.0, are also shown below. Figure 1-3 .

[0549] Other characteristics of S1 protease 1 (SEQ ID NO:5) from *Hypericum* HTCC2649

[0550] Inhibitor: PMSF.

[0551] The N-terminal sequence is determined to be: ANVYGGQ.

[0552] For example, the relative molecular weight determined by SDS-PAGE is approximately M. r =24kDa.

[0553] The molecular weight determined by complete molecular weight analysis is 20406.3 Da.

[0554] Mature sequences (from Edman N-terminal sequencing data and complete MS data):

[0555] ANVYGGQQIEFSGYVCSLGFNATKAGAPVFITAGHCGEGYQTFSKNGTTLGKTQAFSFPGNDYAYSTLASSWTGIGAVDLWTGSARAVTGSSNAAVGTAICKS GRTTYWTCGSVQAKNVTVNYDNGDGTTSSVSGLTKSNTCTEGGDSGGSWMAGNLAQGVTSGGAGYGSSGVCGEKVGQPNIAYFQPVGEILSAYGLTLKTA(SEQ ID NO:5)

[0556] The calculated molecular weight from this mature sequence is 20406.2 Da.

[0557] Example 4: Soybean-Corn Flour Activity Assay

[0558] The activity profile of the protease at pH 3–7 was obtained using soybean-corn flour as a substrate for endpoint determination.

[0559] Substrate: From the Danish Technological Institute, Gl. 1,6092 Stenderup obtains a mixture of soybean flour and corn flour at a ratio of 30:70. Commercial raw materials are milled ( 2mm sieve), mix for 10 minutes, then grind ( Mix for 10 minutes after passing through a 1mm sieve. Sieving analysis using a JEL 200 rotary sieve (J. Engelsmann AG, Ludwigshafen, Germany) showed that 1% of the particles were >500 micrometers and approximately 82% were <212 micrometers.

[0560] Assay buffer: Prepare five buffer solutions containing 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CAPS, 12 mM CaCl2, 150 mM KCl, and 0.01% Triton X-100, and adjust the pH to one of the following values ​​using HCl or NaOH: after mixing the soybean-corn flour substrate (1 g) with the assay buffer (10 mL), give a slurry with a final pH of one of the following: 3.0, 4.0, 5.0, 6.0, and 7.0.

[0561] Before adding the protease, mix the substrate slurry (2 mL) for 30 min and incubate at 40 °C (500 rpm) for 3 h. Dissolve the protease (200 mg enzyme protein / kg dry matter) in 100 μl of 100 mM sodium acetate buffer (9.565 g / L NaOAc, 1.75 g / L acetic acid, 5 mM CaCl2, 0.01% BSA, 0.01% Tween 20, pH 6.0) and add the protease. Centrifuge the sample (10 min, 4000 rpm, 0 °C) and analyze the collected supernatant using o-phthalaldehyde (OPA).

[0562] The results are shown in Table 5 and Figure 4 The proteolytic activity of S1 protease 1 from *Haloxylon ammodendron* HTCC2649 on soybean-corn flour increased with increasing pH from pH 3 to pH 7, and its activity was significantly higher than that of protease 10R across the entire pH range of 3–7, particularly in the pH range of 4–6. These data suggest that S1 protease 1 from *Haloxylon ammodendron* HTCC2649 has the potential to be more efficient in protein hydrolysis, for example in ruminants along with monogastric animals; for example in the crop of broilers with pH typically between 4 and 6, and in the stomach of pigs with pH varying from approximately 2 to 6 (depending on factors such as feed, gastric region, and time post-feeding).

[0563] Table 5: Protease activity (OD) of soybean-corn flour at pH 3.0, 4.0, 5.0, 6.0 and 7.0 340 x dilution factor son)

[0564]

[0565] Example 5: Thermal stability

[0566] Desalt aliquots of the protease protein sample (purified as described in, for example, Examples 2, 9, or 11) or change the buffer to 20 mM sodium acetate, pH 4.0 using a pre-packed PD-10 column, or dialyze 2 x 500 ml of 20 mM sodium acetate, pH 4.0 at 4°C in a 2–3 h step, followed by an overnight step. Filter the sample 0.45 μm and dilute with buffer to approximately 2 A280 units. Use this dialysate as a reference in differential scanning calorimetry (DSC). Degas the samples using a vacuum aspirator and stir for approximately 10 minutes.

[0567] DSC scans were performed on a MicroCal VP-DSC at a constant scan rate of 1.5℃ / min from 20℃ to 90℃. Data processing was performed using MicroCal Origin software (version 4.10), and the denaturation temperature T was... d (also known as melting temperature T) m ) is defined as the temperature at the peak tip of the autocorrelation curve.

[0568] Example 6: Steam stability

[0569] The residual activity of the protease after steam treatment can be evaluated using the following assay.

[0570] In these experiments, modified settings were used so that the steam was supplied from a steam generator and introduced into the chamber. When the temperature reached approximately 93°C–94°C, the samples, placed on a plate, were inserted into the chamber via a drawer. After insertion, the temperature was reduced by 4°C. Incubation was performed for 30 seconds while maintaining a constant temperature of approximately 90°C. Afterward, the plate was quickly removed from the chamber, the samples were placed on ice for resuspending, and protease activity was assessed using assays such as Suc-AAPF-pNA or o-phthalaldehyde (OPA). Residual activity was calculated by comparing each enzyme sample with similar samples that had not undergone steam treatment.

[0571] Example 7: Granulation Stability Test

[0572] Enzyme granulation was performed as described in U.S. Patent No. 4,106,991, Example 1. The obtained particles were dried in a fluidized bed to a moisture content of less than 1% and sieved to obtain a product with particles ranging from 250 μm to 850 μm. Finally, the product was coated with palm oil and calcium carbonate as described in U.S. Patent No. 4,106,991, Example 22.

[0573] In a small horizontal mixer, approximately 50g of enzyme granules are premixed with 10kg of feed for 10 minutes. This premix is ​​then mixed with 90kg of feed for 10 minutes in a large horizontal mixer. The feed is then transferred from the mixer to a regulator (a series mixer with steam injection) at a rate of approximately 300kg / hour. The regulator heats the feed to 95°C (measured at the exhaust port) by injecting steam. The residence time in the regulator is 30 seconds. The feed is then transferred from the regulator to a Simon Heesen press equipped with 3.0 x 35mm horizontal dies and compressed into pellets approximately 15mm in length. After compression, these pellets are placed in a cooler and cooled for 15 minutes.

[0574] The Suc-AAPF-pNA assay was used to measure protease activity before pelleting and after pelleting in the feed pellets. Pelletization stability was determined by comparing the protease activity in pelleted feed with that in non-pelleted feed.

[0575] Example 8: Expression of four variants of S1 protease 1 from the genus *Haloxylon ammodendron*

[0576] Four variants of S1 protease 1 from the genus *Haloxylon ammodendron* were prepared, each containing a single amino acid change. The amino acid changes were as follows: S68N, T71N, T87Q, and S90T (numbered based on SEQ ID NO:5). The four variants were constructed by incorporating the changes into the WT DNA sequence (SEQ ID NO:1) using PCR with the original construct DNA from Example 1 as a template and primers containing the DNA changes. Two DNA fragments were prepared for each construct, each fragment covering a portion of the gene and the upstream or downstream flanking region described in Example 1. The two fragments were fused together by SOE PCR to assemble the two fragments into a linear vector construct. Equal fractions of this SOE PCR product were transformed into *Bacillus subtilis*. Transformants were selected on LB plates supplemented with 6 μg chloramphenicol per ml. For each variant, a recombinant clone with a confirmed correct sequence containing a single amino acid change was selected for fermentation in liquid culture. The supernatant containing the enzyme was harvested and the four variant enzymes were purified as described in Example 9.

[0577] Example 9: Purification of S1 protease 1 from a variant of the genus *Haloxylon ammodendron*

[0578] Each variant was purified using the following procedure:

[0579] The culture medium was centrifuged (20000 x g, 20 min) and the supernatant was carefully decanted from the precipitate. The supernatant was filtered through a Nalgene 0.2 μm filter unit to remove any remaining Bacillus host cells. The filtrate was mixed 1:1 with 3.0 M (NH4)2SO4 to give a final ammonium sulfate concentration of 1.5 M (NH4)2SO4. The ammonium sulfate-adjusted filtrate was applied to a decyl-agarose column (from UpFront Chromatography) equilibrated in 5 mM MES / NaOH, 50 mM H3BO3, 1 mM CaCl2, and 1.5 M (NH4)2SO4 (pH 6.0). After thoroughly washing the column with equilibration buffer, the protease was gradually eluted with 10 mM MES / NaOH, 100 mM H3BO3, and 2 mM CaCl2 (pH 6.0) containing 30% 2-propanol. Elution peaks containing S1 protease 1 from variants of *Hypericum* were collected and transferred to a G25 dextran gel column (GE Healthcare) in 20 mM CH3COOH / NaOH, 1 mM CaCl2 (pH 4.5). The variant transferred from the G25 dextran gel was then applied to an SP-agarose gel FF column (GE Healthcare) equilibrated in 20 mM CH3COOH / NaOH, 1 mM CaCl2 (pH 4.5). After thorough washing of the column with equilibration buffer, the protease was eluted with a linear NaCl gradient (0-->0.5 M) in the same buffer for more than four column volumes. The protease activity of the fractions from the column was analyzed (measured using Protazyme AK at pH 7) and the major activity peak fraction was further analyzed by SDS-PAGE. Fractions showing only one band on Coomassie-stained SDS-PAGE gels were pooled as a purified formulation for further characterization.

[0580] Example 10: Expression of S1 protease 1 from *Terracoccus* sp.

[0581] Based on the nucleotide sequence identified as SEQ ID NO:10, a codon-optimized synthetic gene with SEQ ID NO:12 was synthesized by GeneArt (GENEART AG BioPark, 11 Josef-Engert-Str., 93053, Regensburg, Germany). The synthetic gene was subcloned into a Bacillus expression vector using ClaI and MluI restriction enzyme sites, as described in WO 2012 / 025577. The S1 protease 1 was expressed using a Bacillus clauri secretion signal (with the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA, SEQ ID NO:6) instead of the native secretion signal. The expression plasmid was transformed into Bacillus subtilis. The expression cassette was integrated into the pectin lyase locus via homologous recombination. Transformants were selected on LB plates supplemented with 6 μg chloramphenicol per ml. A recombinant Bacillus subtilis clone containing the integrated expression construct was selected and designated as S1 protease 1 from *Terracoccus* sp. It was cultured on a rotating shaker in 500 mL Erlenmeyer flasks with baffles, each containing 100 mL of yeast extract-based medium. The clone was cultured at 30°C for 3 days. The enzyme containing the supernatant was harvested and purified as described in Example 9.

[0582] Example 11: Purification of S1 protease 1 from *Terracoccus* sp.

[0583] The culture medium was centrifuged (20000x g, 20 min), and the supernatant was carefully decanted from the precipitate. The supernatant was filtered through a Nalgene 0.2 μm filter unit to remove any remaining Bacillus host cells. The pH of the 0.2 μm filtrate was adjusted to pH 8.0 with 3% NaOH, and this solution was applied to a MEP Hypercel column (from Pall Corporation) equilibrated in 20 mM Tris / HCl, 1 mM CaCl2 (pH 8.0). After thoroughly washing the column with equilibration buffer, the protease was eluted stepwise with 20 mM CH3COOH / NaOH, 1 mM CaCl2 (pH 4.5). The elution peak containing S1 protease 1 from *Terracoccus* sp. was collected and diluted 3x with demineralized water to reduce conductivity. The solution was pH adjusted to 4.5 and applied to a SOURCE 30S column (from GE Healthcare) equilibrated in 20 mM CH3COOH / NaOH, 1 mM CaCl2 (pH 4.5). After thoroughly washing the column with equilibration buffer, the protease was eluted with a linear gradient of equilibration buffer and 20 mM CH3COOH / NaOH, 1 mM CaCl2, 0.5 M NaCl (pH 4.5) for more than five column volumes. The protease activity of the fraction from the column was analyzed (using Suc-AAPF-pNA assay at pH 8), and the major activity peaks were pooled. Finally, the pH was adjusted to 5.0 with 3% NaOH. The pH-adjusted pooled product was a purified formulation used for further characterization.

[0584] Example 12: S1 protease 1 (SEQ ID NO:14) from *Terracoccus* sp.

[0585] Characterization of S1 protease 1 from variants of the genus *Haloxylon*

[0586] pH-activity and pH-stability profiles of S1 protease 1 from *Terracoccus* sp. were obtained using the Suc-AAPF-pNA assay. pH-activity and pH-stability profiles of S1 protease 1 from *Hypericum* variants were obtained using the Protazyme AK assay. For the pH-stability profile, the protease was diluted 8–10x in different assay buffers to achieve the desired pH and then incubated at 37°C for 2 hours. After incubation, the pH of the protease was adjusted to the same level as before the residual activity assay by dilution in a pH 8.0 assay buffer. Temperature-activity profiles at pH 7.0 were obtained using the Protazyme AK assay.

[0587] The results are shown in Tables 6-8 below. Data for protease 10R are included in these tables. For Table 6, activity is relative to the enzyme's optimal pH. For Table 7, activity is relative to the residual activity of the samples held under stable conditions (5°C, pH 8.0 or pH 9.0). For Table 8, activity is relative to the enzyme's optimal temperature at pH 7.0 or pH 6.5. pH-activity and pH-stability profiles for protease 10R were obtained using the Suc-AAPF-pNA assay, and temperature-activity profiles were obtained using the Protazyme AK assay at pH 6.5.

[0588] Table 6: pH-activity curves

[0589]

[0590]

[0591] Table 7: pH-stability curves (residual activity after 2 hours at 37°C)

[0592]

[0593] Table 8: Temperature activity curves at pH 7.0 or pH 6.5

[0594]

[0595]

[0596] Other characteristics of S1 protease 1 from the genus *Terracoccus* sp.

[0597] Inhibitor: PMSF.

[0598] The N-terminal sequence is determined to be: ANVYGGQ.

[0599] For example, the relative molecular weight determined by SDS-PAGE is approximately M. r =24kDa.

[0600] The molecular weight determined by complete molecular weight analysis is 20603.4 Da ((M+H)). + ).

[0601] Mature sequences (from Edman N-terminal sequencing data and complete MS data):

[0602] ANVYGGQQIEFSGYVCSLGFNATRGGAPVFVTAGHCGEGYQTFSKGGTTLGSTQAYSFPGNDYAYSTLTSSWTGVGAVDLYDGVNARRVSGYSNAPVGTAICKS GRTTGWTCGSVQAKNVTVNYSNADGSTSTVSGLTKSNTCTEGGDSGGSWMASTSAQGVTSGGAGYGANSVCGQKVGQPNIAYFQPVDEIVSAYGLTLKTS(SEQ ID NO:14)

[0603] The calculated molecular weight from this mature sequence is 20601.2 Da ((M+H)). + The calculated molecular weight of the peak is 20602.2 Da.

[0604] Example 13: Soybean-Corn Flour Activity Assay

[0605] The endpoint assay, as used in Example 4, with soybean-corn flour as the substrate, was used to obtain the activity profile of the protease at pH 3–7.

[0606] The results are shown in Table 9 and Figure 5 The proteolytic activity of S1 protease 1 from the *Haloxylon ammodendron* variant, together with that from *Terracoccus*, on soybean-corn flour increased with increasing pH from pH 3 to pH 7, and was significantly higher than that of protease 10R across the entire pH range of 3–7, particularly in the pH range of 4–6. These data suggest that S1 protease 1 from the *Haloxylon ammodendron* variant, together with that from *Terracoccus*, has the potential to be more efficient in protein hydrolysis, for example in ruminants and monogastric animals; such as in the crop of broilers typically at pH between 4 and 6, and in the stomach of pigs at pH varying from approximately 2 to 6 (depending on factors such as feed, gastric region, and time post-feeding).

[0607] Furthermore, S1 protease 1 from the variants S68N and T71N of Alternaria shares a very similar pH-activity profile with soybean-corn flour at 40°C with S1 protease 1 from the genus Terracoccus, and is significantly different from other proteases known in the art.

[0608] Table 9: Protease activity (OD) of soybean-corn flour at pH 3.0, 4.0, 5.0, 6.0 and 7.0 340 x dilution factor son)

[0609]

[0610] Example 14: Expression of S1 protease 1 from Norfolk's bacterium TL1

[0611] S1 protease 1 from *Nocturia flavum*, with the sequence SEQ ID NO:17 (SWISSPROT: A0A0A0JF07), was expressed as a codon-optimized synthetic gene (SEQ ID NO:18), in which the *Bacillus clausti* secretion signal replaced the native secretion signal as described in Example 10, giving the amino acid sequence SEQ ID NO:19. This gene was synthesized by GeneArt (GENEART AG BioPark, 11 Josef-Engert-Str., 93053, Regensburg, Germany). It was cloned and expressed as described in Example 10 for S1 protease 1 from *Terracoccus* sp.

[0612] Example 15: Purification of S1 protease 1 from Noorhizium anisopliae TL1

[0613] The culture medium was centrifuged (20000xg, 20 min), and the supernatant was carefully decanted from the precipitate. The supernatant was filtered through a Nalgene 0.2 μm filter unit to remove any remaining Bacillus host cells. The pH of the 0.2 μm filtrate was adjusted to pH 8.0 with 3M Tris base, and the pH-adjusted filtrate was applied to an MEP Hypercel column (Pall Corporation) equilibrated in 20 mM Tris / HCl and 1 mM CaCl2 (pH 8.0). After thoroughly washing the column with equilibration buffer, the protease was eluted stepwise with 20 mM CH3COOH / NaOH and 1 mM CaCl2 (pH 4.5). The elution peak containing S1 protease 1 from Noorhiza flavum TL1 was collected and diluted 3x with demineralized water to reduce conductivity. The solution was pH adjusted to 4.5 and applied to a SOURCE 30S column (from GE Healthcare) equilibrated in 20 mM CH3COOH / NaOH, 1 mM CaCl2 (pH 4.5). After thoroughly washing the column with equilibration buffer, the protease was eluted with a linear gradient of equilibration buffer and 20 mM CH3COOH / NaOH, 1 mM CaCl2, 0.5 M NaCl (pH 4.5) for more than five column volumes. The protease activity of the fraction from the column was analyzed (using Suc-AAPM-pNA assay at pH 8), and the major activity peaks were pooled. Finally, the pH was adjusted to 5.5 with 3% NaOH. The pH-adjusted pooled product was a purified formulation used for further characterization.

[0614] Example 16: Characterization of S1 protease 1 from Noorhizium anisopliae TL1 (SEQ ID NO:20)

[0615] pH-activity and pH-stability profiles of S1 protease 1 from *Nocturia flavum* TL1 were obtained using the Suc-AAPM-pNA assay. For the pH-stability profile, the protease was diluted 10x in different assay buffers to achieve the desired pH and then incubated at 37°C for 2 hours. After incubation, the pH of the protease was adjusted to the same level as before the residual activity assay by dilution in a pH 9.0 assay buffer. Temperature-activity profiles at pH 7.0 were obtained using the Protazyme AK assay.

[0616] The results are shown in Tables 10-12 below. Data for protease 10R are included in these tables. For Table 10, activity is relative to the enzyme's optimal pH. For Table 11, activity is relative to the residual activity of samples held at stable conditions (5°C, pH 9.0). For Table 12, activity is relative to the enzyme's optimal temperature at pH 7.0 or pH 6.5. pH-activity and pH-stability profiles for protease 10R were obtained using the Suc-AAPF-pNA assay, and temperature-activity profiles were obtained using the Protazyme AK assay at pH 6.5.

[0617] Table 10: pH-activity curves

[0618]

[0619] Table 11: pH-stability curves (residual activity after 2 hours at 37°C)

[0620]

[0621]

[0622] Table 12: Temperature activity curves at pH 7.0 or pH 6.5

[0623]

[0624] Other characteristics of S1 protease 1 from Knollella flavonoids TL1

[0625] Inhibitor: PMSF.

[0626] The N-terminal sequence is determined to be: ANVYGGQ.

[0627] For example, the relative molecular weight determined by SDS-PAGE is approximately M. r =24kDa.

[0628] The molecular weight determined by complete molecular weight analysis is 20697.8 Da.

[0629] Mature sequences (from Edman N-terminal sequencing data and complete MS data):

[0630] ANVYGGQQIEFSGYVCSLGFNATKSGTPVFITAGHCAEGNQTFTRNGTTLGTTRGWSFPGNDYAYSSLTSSWTGIGAVDLWNGTSARSVTGSSNAAVGTAICKS GRTTGWTCGSVQTKNVTVNYNNGDGTYSTVSGLTKSNTCTEGGDSGGSWMAGNLAQGVTSGGAGYGSNGVCGQKVGQPNIAYFQPIGEILSVYGLTLKTA(SEQ ID NO:20)

[0631] The calculated molecular weight from this mature sequence is 20698.4 Da.

[0632] Example 17: Soybean-Corn Flour Activity Assay

[0633] The endpoint assay, as used in Example 4, with soybean-corn flour as the substrate, was used to obtain the activity profile of the protease at pH 3–7.

[0634] The results are shown in Table 13. The proteolytic activity of S1 protease 1 from *Norgestrelia flavus* on soybean-corn flour increased with increasing pH from pH 3 to pH 7, and its activity was significantly higher than that of protease 10R across the entire pH range of 3–7, particularly in the pH range of 4–6. These data suggest that S1 protease 1 from *Norgestrelia flavus* has the potential to be more efficient in protein hydrolysis, for example in ruminants and monogastric animals; for example in the crop of broilers with pH typically between 4 and 6, and in the stomach of pigs with pH varying from about 2 to 6 (depending on factors such as feed, gastric region, and time after feeding).

[0635] Table 13: Protease activity (OD) of soybean-corn flour at pH 3.0, 4.0, 5.0, 6.0 and 7.0 340 x dilution factor son)

[0636]

[0637] 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 contemplated to be within the scope of the invention. In fact, various modifications to the invention, other than 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. sequence list <110> Novozymes A / S <120> Peptides with serine protease activity and the polynucleotides encoding them, and their applications in animal feed. <130> 13078-WO-PCT <150> EP14191691.6 <151> 2014-11-04 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 1212 <212> DNA <213> Janibacter sp. HTCC2649 <220> <221> CDS <222> (1)..(1209) <220> <221> signal peptide <222> (1)..(90) <220> <221> Mature peptides <222> (601)..(1209) <400> 1 atg tct cgc aga agt ctc acc gtc ctg gcc gga acc ctc tcg gcc 45 Met Ser Arg Arg Ser Leu Thr Val Leu Ala Gly Thr Leu Ser Ala -200 -195 -190 gct gca gca gca acc gcc ctc tgc gtc gcg ccc gcc gcg aac gct 90 Ala Ala Ala Ala Thr Ala Leu Cys Val Ala Pro Ala Ala Asn Ala -185 -180 -175 gcg aac cct ggc ccg agc ggc ccg agc agc ccg agc acc gga ccc 135 Ala Asn Pro Gly Pro Ser Gly Pro Ser Ser Pro Ser Thr Gly Pro ‑170 ‑165 ‑160 ctg gct gtc gac tcc ggt gac tcc gtc gcc gag atg tcg gcg cag 180 Leu Ala Val Asp Ser Gly Asp Ser Val Ala Glu Met Ser Ala Gln ‑155 ‑150 ‑145 tgg ctc gcg acg gag gag ggc ctg agc ctc gag acc gcc cgt gac 225 Trp Leu Ala Thr Glu Glu Gly Leu Ser Leu Glu Thr Ala Arg Asp ‑140 ‑135 ‑130 cga gtg gcc gct cag gag ggg ttg tcg cgc acg gcg acg tcg ctg 270 Arg Val Ala Ala Gln Glu Gly Leu Ser Arg Thr Ala Thr Ser Leu ‑125 ‑120 ‑115 gag acg tcg ctc ggc gcc aag gcc gtc ggc acg tgg atc gac cag 315 Glu Thr Ser Leu Gly Ala Lys Ala Val Gly Thr Trp Ile Asp Gln ‑110 ‑105 ‑100 gcg acg ggc gtg ctc cac gtc aac gtc acc gac gcc gcc gcc gcg tcc 363 Ala Thr Gly Val Leu His Val Asn Val Thr Asp Ala Ala Ala Ala Ser ‑95 ‑90 ‑85 ‑80 acg gtg cgt tcc gcc ggg gcg agc gcc cgt gtc gtc agc gcc gac aag 411 Thr Val Arg Ser Ala Gly Ala Ser Ala Arg Val Val Ser Ala Asp Lys ‑75 ‑70 ‑65 tcc cgg ctc gcc gcc tcc gag aag gcc gcc acc acc gtc gcc ggc aag 459 Ser Arg Leu Ala Ala Ser Glu Lys Ala Ala Thr Thr Val Ala Gly Lys ‑60 ‑55 ‑50 gac acc atc gcg tcc tac gtc gac ccg gtc acc aac aag gtc atc ctc 507 Asp Thr Ile Ala Ser Tyr Val Asp Pro Val Thr Asn Lys Val Ile Leu ‑45 ‑40 ‑35 acg gtg ccc gcg gat cgc gtc gag gcg acc cgc gcc aag atc gcc gac 555 Thr Val Pro Ala Asp Arg Val Glu Ala Thr Arg Ala Lys Ile Ala Asp ‑30 ‑25 ‑20 ccg tcc gtc acg gtc gag ggc acg cag gcc aag gtc tcc acc cag gcc 603 Pro Ser Val Thr Val Glu Gly Thr Gln Ala Lys Val Ser Thr Gln Ala ‑15 ‑10 ‑5 ‑1 1 aac gtc tac ggc ggc cag cag atc gag ttc agc ggc tac gtc tgc tcg 651 Asn Val Tyr Gly Gly Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys Ser 5 10 15 ctc ggc ttc aac gcc acc aag gcc ggc gcc ccg gtc ttc atc acg gcc 699 Leu Gly Phe Asn Ala Thr Lys Ala Gly Ala Pro Val Phe Ile Thr Ala 20 25 30 ggc cac tgc ggc gag ggc tac cag acc ttc tcc aag aac ggc acg acc 747 Gly His Cys Gly Glu Gly Tyr Gln Thr Phe Ser Lys Asn Gly Thr Thr 35 40 45 ctg ggc aag aca cag gcc ttc tcg ttc ccc ggc aac gac tac gcc tac 795 Leu Gly Lys Thr Gln Ala Phe Ser Phe Pro Gly Asn Asp Tyr Ala Tyr 50 55 60 65 tcg acc ctc gcg tcg agc tgg acc ggc atc ggc gcg gtc gac ctg tgg 843 Ser Thr Leu Ala Ser Ser Trp Thr Gly Ile Gly Ala Val Asp Leu Trp 70 75 80 acc ggc tcc gca cgg gcg gtg acg ggg tcg agc aac gcc gcc gtc ggc 891 Thr Gly Ser Ala Arg Ala Val Thr Gly Ser Ser Asn Ala Ala Val Gly 85 90 95 acc gcg atc tgc aag tcc ggc cgc acc acc tac tgg acc tgc ggc tcg 939 Thr Ala Ile Cys Lys Ser Gly Arg Thr Thr Tyr Trp Thr Cys Gly Ser 100 105 110 gtc cag gcc aag aac gtc acc gtg aac tac gac aac ggt gac ggc acg 987 Val Gln Ala Lys Asn Val Thr Val Asn Tyr Asp Asn Gly Asp Gly Thr 115 120 125 acg agc tcg gtc tcg ggc ctc acg aag tcc aac acc tgc acc gag ggc 1035 Thr Ser Ser Val Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr Glu Gly 130 135 140 145 ggc gac tcc ggc ggc tcc tgg atg gcg ggc aac ctt gcc cag ggc gtg 1083 Gly Asp Ser Gly Gly Ser Trp Met Ala Gly Asn Leu Ala Gln Gly Val 150 155 160 acg agc ggc ggc gcg ggc tac ggc tcc agc gga gtg tgc ggc gag aag 1131 Thr Ser Gly Gly Ala Gly Tyr Gly Ser Ser Gly Val Cys Gly Glu Lys 165 170 175 gtc ggc cag ccc aac atc gcc tac ttc cag ccg gtc ggc gag atc ctc 1179 Val Gly Gln Pro Asn Ile Ala Tyr Phe Gln Pro Val Gly Glu Ile Leu 180 185 190 tcc gcc tac ggc ctc acc ctc aag acg gcc tga 1212 Ser Ala Tyr Gly Leu Thr Leu Lys Thr Ala 195 200 <210> 2 <211> 403 <212> PRT <213> Janibacter HTCC2649 <400> 2 Met Ser Arg Arg Ser Leu Thr Val Leu Ala Gly Thr Leu Ser Ala ‑200 ‑195 ‑190 Ala Ala Ala Ala Thr Ala Leu Cys Val Ala Pro Ala Ala Asn Ala ‑185 ‑180 ‑175 Ala Asn Pro Gly Pro Ser Gly Pro Ser Ser Pro Ser Thr Gly Pro ‑170 ‑165 ‑160 Leu Ala Val Asp Ser Gly Asp Ser Val Ala Glu Met Ser Ala Gln ‑155 ‑150 ‑145 Trp Leu Ala Thr Glu Glu Gly Leu Ser Leu Glu Thr Ala Arg Asp ‑140 ‑135 ‑130 Arg Val Ala Ala Gln Glu Gly Leu Ser Arg Thr Ala Thr Ser Leu ‑125 ‑120 ‑115 Glu Thr Ser Leu Gly Ala Lys Ala Val Gly Thr Trp Ile Asp Gln ‑110 ‑105 ‑100 Ala Thr Gly Val Leu His Val Asn Val Thr Asp Ala Ala Ala Ala Ser ‑95 ‑90 ‑85 ‑80 Thr Val Arg Ser Ala Gly Ala Ser Ala Arg Val Val Ser Ala Asp Lys ‑75 ‑70 ‑65 Ser Arg Leu Ala Ala Ser Glu Lys Ala Ala Thr Thr Val Ala Gly Lys ‑60 ‑55 ‑50 Asp Thr Ile Ala Ser Tyr Val Asp Pro Val Thr Asn Lys Val Ile Leu ‑45 ‑40 ‑35 Thr Val Pro Ala Asp Arg Val Glu Ala Thr Arg Ala Lys Ile Ala Asp ‑30 ‑25 ‑20 Pro Ser Val Thr Val Glu Gly Thr Gln Ala Lys Val Ser Thr Gln Ala ‑15 ‑10 ‑5 ‑1 1 Asn Val Tyr Gly Gly Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys Ser 5 10 15 Leu Gly Phe Asn Ala Thr Lys Ala Gly Ala Pro Val Phe Ile Thr Ala 20 25 30 Gly His Cys Gly Glu Gly Tyr Gln Thr Phe Ser Lys Asn Gly Thr Thr 35 40 45 Leu Gly Lys Thr Gln Ala Phe Ser Phe Pro Gly Asn Asp Tyr Ala Tyr 50 55 60 65 Ser Thr Leu Ala Ser Ser Trp Thr Gly Ile Gly Ala Val Asp Leu Trp 70 75 80 Thr Gly Ser Ala Arg Ala Val Thr Gly Ser Ser Asn Ala Ala Val Gly 85 90 95 Thr Ala Ile Cys Lys Ser Gly Arg Thr Thr Tyr Trp Thr Cys Gly Ser 100 105 110 Val Gln Ala Lys Asn Val Thr Val Asn Tyr Asp Asn Gly Asp Gly Thr 115 120 125 Thr Ser Ser Val Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr Glu Gly 130 135 140 145 Gly Asp Ser Gly Gly Ser Trp Met Ala Gly Asn Leu Ala Gln Gly Val 150 155 160 Thr Ser Gly Gly Ala Gly Tyr Gly Ser Ser Gly Val Cys Gly Glu Lys 165 170 175 Val Gly Gln Pro Asn Ile Ala Tyr Phe Gln Pro Val Gly Glu Ile Leu 180 185 190 Ser Ala Tyr Gly Leu Thr Leu Lys Thr Ala 195 200 <210> 3 <211> 1203 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> CDS <222> (1)..(1200) <220> <221> signal peptide <222> (1)..(81) <220> <221> Mature peptides <222> (592)..(1200) <400> 3 atg aag aaa ccg ttg ggg aaa att gtc gca agc acc gca cta ctc 45 Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu -195 -190 -185 att tct gtt gct ttt agt tca tcg ata gca tcg gct gca aat ccg 90 Ile Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala Ala Asn Pro -180 -175 -170 gga ccg agc gga ccg tca tca ccg tca aca gga ccg ctg gca gtt 135 Gly Pro Ser Gly Pro Ser Ser Pro Ser Thr Gly Pro Leu Ala Val -165 -160 -155 gat tca ggc gat tca gtt gca gaa atg tca gca caa tgg ctg gca 180 Asp Ser Gly Asp Ser Val Ala Glu Met Ser Ala Gln Trp Leu Ala -150 -145 -140 aca gaa gaa ggc ctg tca ctg gaa aca gca aga gat aga gtt gca 225 Thr Glu Glu Gly Leu Ser Leu Glu Thr Ala Arg Asp Arg Val Ala -135 -130 -125 gca caa gaa gga ctt tca aga aca gca aca tca ctt gaa aca agc 270 Ala Gln Glu Gly Leu Ser Arg Thr Ala Thr Ser Leu Glu Thr Ser -120 -115 -110 ctt ggc gca aaa gca gtt ggc aca tgg att gat caa gca aca ggc gtt 318 Leu Gly Ala Lys Ala Val Gly Thr Trp Ile Asp Gln Ala Thr Gly Val -105 -100 -95 ctg cat gtt aat gtt aca gat gca gca gca gca tca aca gtt aga tca 366 Leu His Val Asn Val Thr Asp Ala Ala Ala Ala Ser Thr Val Arg Ser -90 -85 -80 gca ggc gca tca gca aga gtt gtt tca gca gat aaa tca aga ctg gca 414 Ala Gly Ala Ser Ala Arg Val Val Ser Ala Asp Lys Ser Arg Leu Ala -75 -70 -65 -60 gca tca gaa aaa gca gcg aca aca gtt gca ggc aaa gat aca att gca 462 Ala Ser Glu Lys Ala Ala Thr Thr Val Ala Gly Lys Asp Thr Ile Ala -55 -50 -45 tca tat gtt gat ccg gtc acg aac aaa gtt att ctg aca gtt ccg gca 510 Ser Tyr Val Asp Pro Val Thr Asn Lys Val Ile Leu Thr Val Pro Ala -40 -35 -30 gat aga gtc gaa gca aca cgc gca aaa att gca gat ccg tca gtt aca 558 Asp Arg Val Glu Ala Thr Arg Ala Lys Ile Ala Asp Pro Ser Val Thr -25 -20 -15 gtt gaa ggc aca caa gca aaa gtt tca aca caa gcg aat gtt tat ggc 606 Val Glu Gly Thr Gln Ala Lys Val Ser Thr Gln Ala Asn Val Tyr Gly -10 -5 -1 1 5 gga cag caa att gaa ttt agc ggc tat gtt tgc tca ctg ggc ttt aat 654 Gly Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys Ser Leu Gly Phe Asn 10 15 20 gca aca aaa gca ggc gct ccg gtc ttt att aca gca ggc cat tgc gga 702 Ala Thr Lys Ala Gly Ala Pro Val Phe Ile Thr Ala Gly His Cys Gly 25 30 35 gaa ggc tat caa aca ttt tca aaa aat ggc aca aca ctg ggc aaa aca 750 Glu Gly Tyr Gln Thr Phe Ser Lys Asn Gly Thr Thr Leu Gly Lys Thr 40 45 50 cag gca ttt tca ttt ccg gga aac gat tat gca tat tca aca ctt gca 798 Gln Ala Phe Ser Phe Pro Gly Asn Asp Tyr Ala Tyr Ser Thr Leu Ala 55 60 65 tca agc tgg aca ggc att ggc gca gtt gat ctg tgg aca ggc tca gcg 846 Ser Ser Trp Thr Gly Ile Gly Ala Val Asp Leu Trp Thr Gly Ser Ala 70 75 80 85 aga gca gtt aca ggc tca tca aat gca gca gtt gga aca gca att tgc 894 Arg Ala Val Thr Gly Ser Ser Asn Ala Ala Val Gly Thr Ala Ile Cys 90 95 100 aaa agc ggc aga aca aca tat tgg aca tgc ggc tca gtt caa gca aaa 942 Lys Ser Gly Arg Thr Thr Tyr Trp Thr Cys Gly Ser Val Gln Ala Lys 105 110 115 aat gtg aca gtc aac tat gat aat ggc gac ggc aca aca tca tca gtt 990 Asn Val Thr Val Asn Tyr Asp Asn Gly Asp Gly Thr Thr Ser Ser Val 120 125 130 tca ggc ctt aca aaa agc aac aca tgc aca gaa ggc gga gat agc gga 1038 Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr Glu Gly Gly Asp Ser Gly 135 140 145 ggc tca tgg atg gca ggc aat ctg gca caa ggc gtt aca tca ggc gga 1086 Gly Ser Trp Met Ala Gly Asn Leu Ala Gln Gly Val Thr Ser Gly Gly 150 155 160 165 gca gga tat ggc tca tca ggc gtc tgc gga gaa aaa gtt gga caa ccg 1134 Ala Gly Tyr Gly Ser Ser Gly Val Cys Gly Glu Lys Val Gly Gln Pro 170 175 180 aat att gca tat ttt caa ccg gtc ggc gaa att ctg tca gca tat ggc 1182 Asn Ile Ala Tyr Phe Gln Pro Val Gly Glu Ile Leu Ser Ala Tyr Gly 185 190 195 ctg aca ctt aaa aca gca taa 1203 Leu Thr Leu Lys Thr Ala 200 <210> 4 <211> 400 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 4 Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu -195 -190 -185 Ile Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala Ala Asn Pro -180 -175 -170 Gly Pro Ser Gly Pro Ser Ser Pro Ser Thr Gly Pro Leu Ala Val -165 -160 -155 Asp Ser Gly Asp Ser Val Ala Glu Met Ser Ala Gln Trp Leu Ala -150 -145 -140 Thr Glu Glu Gly Leu Ser Leu Glu Thr Ala Arg Asp Arg Val Ala -135 -130 -125 Ala Gln Glu Gly Leu Ser Arg Thr Ala Thr Ser Leu Glu Thr Ser -120 -115 -110 Leu Gly Ala Lys Ala Val Gly Thr Trp Ile Asp Gln Ala Thr Gly Val -105 -100 -95 Leu His Val Asn Val Thr Asp Ala Ala Ala Ala Ser Thr Val Arg Ser -90 -85 -80 Ala Gly Ala Ser Ala Arg Val Val Ser Ala Asp Lys Ser Arg Leu Ala -75 -70 -65 -60 Ala Ser Glu Lys Ala Ala Thr Thr Val Ala Gly Lys Asp Thr Ile Ala ‑55 ‑50 ‑45 Ser Tyr Val Asp Pro Val Thr Asn Lys Val Ile Leu Thr Val Pro Ala ‑40 ‑35 ‑30 Asp Arg Val Glu Ala Thr Arg Ala Lys Ile Ala Asp Pro Ser Val Thr ‑25 ‑20 ‑15 Val Glu Gly Thr Gln Ala Lys Val Ser Thr Gln Ala Asn Val Tyr Gly ‑10 ‑5 ‑1 1 5 Gly Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys Ser Leu Gly Phe Asn 10 15 20 Ala Thr Lys Ala Gly Ala Pro Val Phe Ile Thr Ala Gly His Cys Gly 25 30 35 Glu Gly Tyr Gln Thr Phe Ser Lys Asn Gly Thr Thr Leu Gly Lys Thr 40 45 50 Gln Ala Phe Ser Phe Pro Gly Asn Asp Tyr Ala Tyr Ser Thr Leu Ala 55 60 65 Ser Ser Trp Thr Gly Ile Gly Ala Val Asp Leu Trp Thr Gly Ser Ala 70 75 80 85 Arg Ala Val Thr Gly Ser Ser Asn Ala Ala Val Gly Thr Ala Ile Cys 90 95 100 Lys Ser Gly Arg Thr Thr Tyr Trp Thr Cys Gly Ser Val Gln Ala Lys 105 110 115 Asn Val Thr Val Asn Tyr Asp Asn Gly Asp Gly Thr Thr Ser Ser Val 120 125 130 Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr Glu Gly Gly Asp Ser Gly 135 140 145 Gly Ser Trp Met Ala Gly Asn Leu Ala Gln Gly Val Thr Ser Gly Gly 150 155 160 165 Ala Gly Tyr Gly Ser Ser Gly Val Cys Gly Glu Lys Val Gly Gln Pro 170 175 180 Asn Ile Ala Tyr Phe Gln Pro Val Gly Glu Ile Leu Ser Ala Tyr Gly 185 190 195 Leu Thr Leu Lys Thr Ala 200 <210> 5 <211> 203 <212> PRT <213> Janibacter HTCC2649 <220> <221> Mature polypeptide <222> (1)..(203) <400> 5 Ala Asn Val Tyr Gly Gly Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys 1 5 10 15 Ser Leu Gly Phe Asn Ala Thr Lys Ala Gly Ala Pro Val Phe Ile Thr 20 25 30 Ala Gly His Cys Gly Glu Gly Tyr Gln Thr Phe Ser Lys Asn Gly Thr 35 40 45 Thr Leu Gly Lys Thr Gln Ala Phe Ser Phe Pro Gly Asn Asp Tyr Ala 50 55 60 Tyr Ser Thr Leu Ala Ser Ser Trp Thr Gly Ile Gly Ala Val Asp Leu 65 70 75 80 Trp Thr Gly Ser Ala Arg Ala Val Thr Gly Ser Ser Asn Ala Ala Val 85 90 95 Gly Thr Ala Ile Cys Lys Ser Gly Arg Thr Thr Tyr Trp Thr Cys Gly 100 105 110 Ser Val Gln Ala Lys Asn Val Thr Val Asn Tyr Asp Asn Gly Asp Gly 115 120 125 Thr Thr Ser Ser Val Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr Glu 130 135 140 Gly Gly Asp Ser Gly Gly Ser Trp Met Ala Gly Asn Leu Ala Gln Gly 145 150 155 160 Val Thr Ser Gly Gly Ala Gly Tyr Gly Ser Ser Gly Val Cys Gly Glu 165 170 175 Lys Val Gly Gln Pro Asn Ile Ala Tyr Phe Gln Pro Val Gly Glu Ile 180 185 190 Leu Ser Ala Tyr Gly Leu Thr Leu Lys Thr Ala 195 200 <210> 6 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Bacillus clausti secretes signals <400> 6 Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu Ile 1 5 10 15 Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala 20 25 <210> 7 <211> 1596 <212> DNA <213> Nocardiopsis sp. <220> <221> CDS <222> (318) (1463) <220> <221> signal peptide <222> (318) (404) <220> <221> Mature peptides <222> (900)...(1463) <400> 7 acgtttggta cgggtaccgg tgtccgcatg tggccagaat gcccccttgc gacagggaac 60 ggattcggtc ggtagcgcat cgactccgac aaccgcgagg tggccgttcg cgtcgccacg 120 ttctgcgacc gtcatgcgac ccatcatcgg gtgaccccac cgagctctga atggtccacc 180 gttctgacgg tctttccctc accaaaacgt gcacctatgg ttaggacgtt gtttaccgaa 240 tgtctcggtg aacgacaggg gccggacggt attcggcccc gatcccccgt tgatcccccc 300 aggagagtag ggacccc atg cga ccc tcc ccc gtt gtc tcc gcc atc ggt 350 Met Arg Pro Ser Pro Val Val Ser Ala Ile Gly 185-190 acg gga gcg ctg gcc ttc ggt ctg gcg ctg tcc ggt acc ccg ggt 395 Thr Gly Ala Leu Ala Phe Gly Leu Ala Leu Ser Gly Thr Pro Gly 180 - 175 - 170 gcc ctc gcg gcc acc gga gcg ctc ccc cag tca ccc acc ccg gag 440 Ala Leu Ala Ala Thr Gly Ala Leu Pro Gln Ser Pro Thr Pro Glu 165 - 160 - 155 gcc gac gcg gtc tcc atg cag gag gcg ctc cag cgc gac ctc gac 485 Ala Asp Ala Val Ser Met Gln Glu Ala Leu Gln Arg Asp Leu Asp 150 - 145 - 140 ctg acc tcc gcc gag gcc gag gag ctg ctg gcc gcc cag gac acc 530 Leu Thr Ser Ala Glu Ala Glu Glu Leu Leu Ala Ala Gln Asp Thr ‑135 ‑130 ‑125 gcc ttc gag gtc gac gag gcc gcg gcc gag gcc gcc ggg gac gcc 575 Ala Phe Glu Val Asp Glu Ala Ala Ala Glu Ala Ala Gly Asp Ala ‑120 ‑115 ‑110 tac ggc ggc tcc gtc ttc gac acc gag agc ctg gaa ctg acc gtc ctg 623 Tyr Gly Gly Ser Val Phe Asp Thr Glu Ser Leu Glu Leu Thr Val Leu ‑105 ‑100 ‑95 gtc acc gat gcc gcc gcg gtc gag gcc gtg gag gcc acc ggc gcc ggg 671 Val Thr Asp Ala Ala Ala Val Glu Ala Val Glu Ala Thr Gly Ala Gly ‑90 ‑85 ‑80 acc gag ctg gtc tcc tac ggc atc gac ggt ctc gac gag atc gtc cag 719 Thr Glu Leu Val Ser Tyr Gly Ile Asp Gly Leu Asp Glu Ile Val Gln ‑75 ‑70 ‑65 gag ctc aac gcc gcc gac gcc gtt ccc ggt gtg gtc ggc tgg tac ccg 767 Glu Leu Asn Ala Ala Asp Ala Val Pro Gly Val Val Gly Trp Tyr Pro ‑60 ‑55 ‑50 ‑45 gac gtg gcg ggt gac acc gtc gtc ctg gag gtc ctg gag ggt tcc gga 815 Asp Val Ala Gly Asp Thr Val Val Leu Glu Val Leu Glu Gly Ser Gly ‑40 ‑35 ‑30 gcc gac gtc agc ggc ctg ctc gcg gac gcc ggc gtg gac gcc tcg gcc 863 Ala Asp Val Ser Gly Leu Leu Ala Asp Ala Gly Val Asp Ala Ser Ala ‑25 ‑20 ‑15 gtc gag gtg acc acg agc gac cag ccc gag ctc tac gcc gac atc atc 911 Val Glu Val Thr Thr Ser Asp Gln Pro Glu Leu Tyr Ala Asp Ile Ile ‑10 ‑5 ‑1 1 ggt ggt ctg gcc tac acc atg ggc ggc cgc tgt tcg gtc ggc ttc gcg 959 Gly Gly Leu Ala Tyr Thr Met Gly Gly Arg Cys Ser Val Gly Phe Ala 5 10 15 20 gcc acc aac gcc gcc ggt cag ccc ggg ttc gtc acc gcc ggt cac tgc 1007 Ala Thr Asn Ala Ala Gly Gln Pro Gly Phe Val Thr Ala Gly His Cys 25 30 35 ggc cgc gtg ggc acc cag gtg acc atc ggc aac ggc agg ggc gtc ttc 1055 Gly Arg Val Gly Thr Gln Val Thr Ile Gly Asn Gly Arg Gly Val Phe 40 45 50 gag cag tcc gtc ttc ccc ggc aac gac gcg gcc ttc gtc cgc ggt acg 1103 Glu Gln Ser Val Phe Pro Gly Asn Asp Ala Ala Phe Val Arg Gly Thr 55 60 65 tcc aac ttc acg ctg acc aac ctg gtc agc cgc tac aac acc ggc ggg 1151 Ser Asn Phe Thr Leu Thr Asn Leu Val Ser Arg Tyr Asn Thr Gly Gly 70 75 80 tac gcc acg gtc gcc ggt cac aac cag gcc ccc atc ggc tcc tcc gtc 1199 Tyr Ala Thr Val Ala Gly His Asn Gln Ala Pro Ile Gly Ser Ser Val 85 90 95 100 tgc cgc tcc ggc tcc acc acc ggt tgg cac tgc ggc acc atc cag gcc 1247 Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly Thr Ile Gln Ala 105 110 115 cgc ggc cag tcg gtg agc tac ccc gag ggc acc gtc acc aac atg acc 1295 Arg Gly Gln Ser Val Ser Tyr Pro Glu Gly Thr Val Thr Asn Met Thr 120 125 130 cgg acc acc gtg tgc gcc gag ccc ggc gac tcc ggc ggc tcc tac atc 1343 Arg Thr Thr Val Cys Ala Glu Pro Gly Asp Ser Gly Gly Ser Tyr Ile 135 140 145 tcc ggc acc cag gcc cag ggc gtg acc tcc ggc ggc tcc ggc aac tgc 1391 Ser Gly Thr Gln Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn Cys 150 155 160 cgc acc ggc ggg acc acc ttc tac cag gag gtc acc ccc atg gtg aac 1439 Arg Thr Gly Gly Thr Thr Phe Tyr Gln Glu Val Thr Pro Met Val Asn 165 170 175 180 tcc tgg ggc gtc cgt ctc cgg acc tgatccccgc ggttccaggc ggaccgacgg 1493 Ser Trp Gly Val Arg Leu Arg Thr 185 tcgtgacctg agtaccaggc gtccccgccg cttccagcgg cgtccgcacc ggggtgggac 1553 cgggcgtggc cacggcccca cccgtgaccg gaccgcccgg cta 1596 <210> 8 <211> 382 <212> PRT <213> Nocardiopsis <400> 8 Met Arg Pro Ser Pro Val Val Ser Ala Ile Gly Thr Gly Ala Leu ‑190 ‑185 ‑180 Ala Phe Gly Leu Ala Leu Ser Gly Thr Pro Gly Ala Leu Ala Ala ‑175 ‑170 ‑165 Thr Gly Ala Leu Pro Gln Ser Pro Thr Pro Glu Ala Asp Ala Val ‑160 ‑155 ‑150 Ser Met Gln Glu Ala Leu Gln Arg Asp Leu Asp Leu Thr Ser Ala ‑145 ‑140 ‑135 Glu Ala Glu Glu Leu Leu Ala Ala Gln Asp Thr Ala Phe Glu Val ‑130 ‑125 ‑120 Asp Glu Ala Ala Ala Glu Ala Ala Gly Asp Ala Tyr Gly Gly Ser ‑115 ‑110 ‑105 Val Phe Asp Thr Glu Ser Leu Glu Leu Thr Val Leu Val Thr Asp Ala ‑100 ‑95 ‑90 Ala Ala Val Glu Ala Val Glu Ala Thr Gly Ala Gly Thr Glu Leu Val ‑85 ‑80 ‑75 Ser Tyr Gly Ile Asp Gly Leu Asp Glu Ile Val Gln Glu Leu Asn Ala ‑70 ‑65 ‑60 Ala Asp Ala Val Pro Gly Val Val Gly Trp Tyr Pro Asp Val Ala Gly ‑55 ‑50 ‑45 Asp Thr Val Val Leu Glu Val Leu Glu Gly Ser Gly Ala Asp Val Ser ‑40 ‑35 ‑30 ‑25 Gly Leu Leu Ala Asp Ala Gly Val Asp Ala Ser Ala Val Glu Val Thr ‑20 ‑15 ‑10 Thr Ser Asp Gln Pro Glu Leu Tyr Ala Asp Ile Ile Gly Gly Leu Ala ‑5 ‑1 1 5 Tyr Thr Met Gly Gly Arg Cys Ser Val Gly Phe Ala Ala Thr Asn Ala 10 15 20 Ala Gly Gln Pro Gly Phe Val Thr Ala Gly His Cys Gly Arg Val Gly 25 30 35 40 Thr Gln Val Thr Ile Gly Asn Gly Arg Gly Val Phe Glu Gln Ser Val 45 50 55 Phe Pro Gly Asn Asp Ala Ala Phe Val Arg Gly Thr Ser Asn Phe Thr 60 65 70 Leu Thr Asn Leu Val Ser Arg Tyr Asn Thr Gly Gly Tyr Ala Thr Val 75 80 85 Ala Gly His Asn Gln Ala Pro Ile Gly Ser Ser Val Cys Arg Ser Gly 90 95 100 Ser Thr Thr Gly Trp His Cys Gly Thr Ile Gln Ala Arg Gly Gln Ser 105 110 115 120 Val Ser Tyr Pro Glu Gly Thr Val Thr Asn Met Thr Arg Thr Thr Val 125 130 135 Cys Ala Glu Pro Gly Asp Ser Gly Gly Ser Tyr Ile Ser Gly Thr Gln 140 145 150 Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asn Cys Arg Thr Gly Gly 155 160 165 Thr Thr Phe Tyr Gln Glu Val Thr Pro Met Val Asn Ser Trp Gly Val 170 175 180 Arg Leu Arg Thr 185 <210> 9 <211> 415 <212> PRT <213> Austwickia chelonae NBRC 105200 <400> 9 Met Asn Lys Arg Ile Gly Ala Thr Leu Ala Leu Leu Ser Ser Ala Ala 1 5 10 15 Leu Leu Pro Ala Ala Ala Pro Leu Thr Ala Gln Ala Ala Asp Glu Lys 20 25 30 Lys Ser Glu Glu Leu Ser Thr Ile Gln Glu Met Ser Ala Glu Trp Leu 35 40 45 Ser Lys Ser Tyr Gly Leu Asp Gly Glu Glu Ala Lys Arg Arg Val Lys 50 55 60 Asp Gln Asp Gly His Ala Gln Lys Ala Lys Asp Ile Glu Gly Asp Leu 65 70 75 80 Gly Asp Lys Thr Ala Gly Ser Phe Ile Asp Gln Lys Arg Gly Lys Leu 85 90 95 Val Val Thr Val Thr Asp Glu Lys Ala Val Asp Lys Val Lys Glu Lys 100 105 110 Asp Lys Asn Ala Asp Val Arg Val Val Lys Asn Ser Ala Ser Lys Leu 115 120 125 Asn Asp Ser Lys Lys Asn Ala Glu Asp Lys Val Gly Asp Lys Met Ala 130 135 140 Ala Ser Tyr Val Asp Val Glu Arg Asn Val Val Val Leu Thr Val Pro 145 150 155 160 Lys Asp Lys Ala Glu Glu Ala Lys Lys Gln Val Lys Asp Val Gln Gly 165 170 175 Val Glu Val Gln Glu Val Glu Ala Thr Ile Glu Ala Gln Ala Asn Leu 180 185 190 Tyr Gly Gly Gln Glu Ile Gln Phe Gly Arg Ser Val Cys Ser Val Gly 195 200 205 Phe Pro Ala Thr Lys Asp Gly Lys Asn Val Phe Ile Thr Ala Gly His 210 215 220 Cys Ala Ala Gly Gly Gln Ala Phe Arg Arg Asn Gly Gln Asn Leu Gly 225 230 235 240 Lys Pro Val Lys Tyr Asn Phe Pro Gly Pro Asp Met Ala Tyr Ser Thr 245 250 255 Met Glu Asn Gly Trp Asn Gly Val Gly Ala Val Asp His Trp Asn Gly 260 265 270 Lys Ala Val Ala Val Ala Gly Ser Gln Glu Ala Pro Val Gly Ala Thr 275 280 285 Val Cys Lys Ser Gly Arg Thr Thr Arg Trp Thr Cys Gly Val Ile Gln 290 295 300 Ala Lys Asn Val Thr Val Arg Tyr Gly Lys Pro Gly Gly Gly Gln Asp 305 310 315 320 Ile Val Arg Gly Met Thr Gln Ala Asn Val Cys Ser Glu Gly Gly Asp 325 330 335 Ser Gly Gly Ser Trp Ile Ala Gly Asn Gln Ala Gln Gly Val Thr Ser 340 345 350 Gly Gly Ala Gly Tyr Gly Pro Asn Lys Ser Cys Gly Glu Lys Val Gly 355 360 365 Arg Pro Asn Val Ala Tyr Phe Gln Pro Leu Asn Pro Ile Leu Lys Asp 370 375 380 Tyr Gly Leu Lys Leu Thr Thr His Asn Gly Gly Lys Gly Gly Gly Asp 385 390 395 400 Asn Gly Gly Asp Arg Asn Arg Asp Asn Arg Lys Gly Gly Arg Tyr 405 410 415 <210> 10 <211> 1203 <212> DNA <213> Terracoccus sp.[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​tcc gac ccg acc ccc gcg ccg acg cca gcg gcg tcc acc gac tca 135 Ser Asp Pro Thr Pro Ala Pro Thr Pro Ala Ala Ser Thr Asp Ser ‑165 ‑160 ‑155 ggc cag tcc gtc gcc gag atg tcg gcg cgc tgg ctc gcg aag gac 180 Gly Gln Ser Val Ala Glu Met Ser Ala Arg Trp Leu Ala Lys Asp ‑150 ‑145 ‑140 cgt gcc atc agc ctc gcc acg gct cgc cag cgc gtc gcg gcc cag 225 Arg Ala Ile Ser Leu Ala Thr Ala Arg Gln Arg Val Ala Ala Gln ‑135 ‑130 ‑125 gac gga cag acc cgc acg gcg gcc tcg ctc gag cgc gcg ctc ggc 270 Asp Gly Gln Thr Arg Thr Ala Ala Ser Leu Glu Arg Ala Leu Gly ‑120 ‑115 ‑110 gcc cgg gcc gca ggg tcc tac atc gac gcc acc tcc ggc gcg ctc gtc 318 Ala Arg Ala Ala Gly Ser Tyr Ile Asp Ala Thr Ser Gly Ala Leu Val ‑105 ‑100 ‑95 gtc aac gtc gtc gac acc gcg tcc gtc gcc agg gtg ctg tct gcc ggt 366 Val Asn Val Val Asp Thr Ala Ser Val Ala Arg Val Leu Ser Ala Gly ‑90 ‑85 ‑80 ‑75 gcc gtc gcc aag gtc gtc gac cgc tcg acg agc gag ctg tcc gcg acc 414 Ala Val Ala Lys Val Val Asp Arg Ser Thr Ser Glu Leu Ser Ala Thr ‑70 ‑65 ‑60 gag cgc gcg gca cgc gca cgt gcc ggg tcg gcc gtc gtg tcc tcc tac 462 Glu Arg Ala Ala Arg Ala Arg Ala Gly Ser Ala Val Val Ser Ser Tyr ‑55 ‑50 ‑45 acc gac ccc gtc acc aac ggc gtc gtc ctg acc gtc ccc agc gcg cgg 510 Thr Asp Pro Val Thr Asn Gly Val Val Leu Thr Val Pro Ser Ala Arg ‑40 ‑35 ‑30 gtc tcg gag gtc cgc agc gag gtc gtt ggt ctc gac ggg gtg acc gtc 558 Val Ser Glu Val Arg Ser Glu Val Val Gly Leu Asp Gly Val Thr Val ‑25 ‑20 ‑15 gca ggc acc gac gca cgg acg acg acg cag gcc aac gtc tac ggc ggc 606 Ala Gly Thr Asp Ala Arg Thr Thr Thr Gln Ala Asn Val Tyr Gly Gly ‑10 ‑5 ‑1 1 5 cag cag atc gag ttc agc ggc tac gtc tgc tcg ctc ggc ttc aac gcc 654 Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys Ser Leu Gly Phe Asn Ala 10 15 20 acc cgc ggc ggt gct ccg gtg ttc gtc acc gcc ggc cac tgt ggt gag 702 Thr Arg Gly Gly Ala Pro Val Phe Val Thr Ala Gly His Cys Gly Glu 25 30 35 ggt tac cag acc ttc agc aag gga ggc acg acg ctg ggg tcg acg cag 750 Gly Tyr Gln Thr Phe Ser Lys Gly Gly Thr Thr Leu Gly Ser Thr Gln 40 45 50 gcg tac tcc ttc ccg ggc aac gac tac gcc tac tcg acc ctg acg tcg 798 Ala Tyr Ser Phe Pro Gly Asn Asp Tyr Ala Tyr Ser Thr Leu Thr Ser 55 60 65 70 agc tgg acc ggg gtc ggt gcc gtc gac ctc tac gac ggc gtc aac gcc 846 Ser Trp Thr Gly Val Gly Ala Val Asp Leu Tyr Asp Gly Val Asn Ala 75 80 85 cgc cgg gtc tcg ggc tac tcg aac gcc ccg gtc ggg acc gcg atc tgc 894 Arg Arg Val Ser Gly Tyr Ser Asn Ala Pro Val Gly Thr Ala Ile Cys 90 95 100 aag tcg ggc cgc acg acc ggc tgg acc tgc ggc tcg gtg cag gcc aag 942 Lys Ser Gly Arg Thr Thr Gly Trp Thr Cys Gly Ser Val Gln Ala Lys 105 110 115 aac gtc acc gtc aac tac agc aac gcc gac ggc tcg acg agc acc gtg 990 Asn Val Thr Val Asn Tyr Ser Asn Ala Asp Gly Ser Thr Ser Thr Val 120 125 130 agc ggc ctg acg aag agc aac acc tgc acc gag ggt ggc gac tcg ggc 1038 Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr Glu Gly Gly Asp Ser Gly 135 140 145 150 ggc tcg tgg atg gcg agc acg tcg gca cag ggc gtg acg agc ggt ggt 1086 Gly Ser Trp Met Ala Ser Thr Ser Ala Gln Gly Val Thr Ser Gly Gly 155 160 165 gcc ggc tac ggc gcc aac agc gtc tgc ggc cag aag gtc ggc cag ccc 1134 Ala Gly Tyr Gly Ala Asn Ser Val Cys Gly Gln Lys Val Gly Gln Pro 170 175 180 aac atc gcc tac ttc cag ccc gtc gac gag atc gtg tcg gcc tac ggc 1182 Asn Ile Ala Tyr Phe Gln Pro Val Asp Glu Ile Val Ser Ala Tyr Gly 185 190 195 ctg acg ctc aag acc tcc tga 1203 Leu Thr Leu Lys Thr Ser 200 <210> 11 <211> 400 <212> PRT <213> Terracoccus sp. <400> 11 Met Ile Arg Thr Ser Leu Thr Thr Leu Ala Ala Thr Ala Ala Ile -195 -190 -185 Ala Thr Ala Ile Thr Val Met Pro Ala Gln Ala Ser Thr Leu Ala -180 -175 -170 Ser Asp Pro Thr Pro Ala Pro Thr Pro Ala Ala Ser Thr Asp Ser -165 -160 -155 Gly Gln Ser Val Ala Glu Met Ser Ala Arg Trp Leu Ala Lys Asp -150 -145 -140 Arg Ala Ile Ser Leu Ala Thr Ala Arg Gln Arg Val Ala Ala Gln -135 -130 -125 Asp Gly Gln Thr Arg Thr Ala Ala Ser Leu Glu Arg Ala Leu Gly -120 -115 -110 Ala Arg Ala Ala Gly Ser Tyr Ile Asp Ala Thr Ser Gly Ala Leu Val ‑105 ‑100 ‑95 Val Asn Val Val Asp Thr Ala Ser Val Ala Arg Val Leu Ser Ala Gly ‑90 ‑85 ‑80 ‑75 Ala Val Ala Lys Val Val Asp Arg Ser Thr Ser Glu Leu Ser Ala Thr ‑70 ‑65 ‑60 Glu Arg Ala Ala Arg Ala Arg Ala Gly Ser Ala Val Val Ser Ser Tyr ‑55 ‑50 ‑45 Thr Asp Pro Val Thr Asn Gly Val Val Leu Thr Val Pro Ser Ala Arg ‑40 ‑35 ‑30 Val Ser Glu Val Arg Ser Glu Val Val Gly Leu Asp Gly Val Thr Val ‑25 ‑20 ‑15 Ala Gly Thr Asp Ala Arg Thr Thr Thr Gln Ala Asn Val Tyr Gly Gly ‑10 ‑5 ‑1 1 5 Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys Ser Leu Gly Phe Asn Ala 10 15 20 Thr Arg Gly Gly Ala Pro Val Phe Val Thr Ala Gly His Cys Gly Glu 25 30 35 Gly Tyr Gln Thr Phe Ser Lys Gly Gly Thr Thr Leu Gly Ser Thr Gln 40 45 50 Ala Tyr Ser Phe Pro Gly Asn Asp Tyr Ala Tyr Ser Thr Leu Thr Ser 55 60 65 70 Ser Trp Thr Gly Val Gly Ala Val Asp Leu Tyr Asp Gly Val Asn Ala 75 80 85 Arg Arg Val Ser Gly Tyr Ser Asn Ala Pro Val Gly Thr Ala Ile Cys 90 95 100 Lys Ser Gly Arg Thr Thr Gly Trp Thr Cys Gly Ser Val Gln Ala Lys 105 110 115 Asn Val Thr Val Asn Tyr Ser Asn Ala Asp Gly Ser Thr Ser Thr Val 120 125 130 Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr Glu Gly Gly Asp Ser Gly 135 140 145 150 Gly Ser Trp Met Ala Ser Thr Ser Ala Gln Gly Val Thr Ser Gly Gly 155 160 165 Ala Gly Tyr Gly Ala Asn Ser Val Cys Gly Gln Lys Val Gly Gln Pro 170 175 180 Asn Ile Ala Tyr Phe Gln Pro Val Asp Glu Ile Val Ser Ala Tyr Gly 185 190 195 Leu Thr Leu Lys Thr Ser 200 <210> 12 <211> 1206 <212> DNA <213> Artificial sequence <220> <223> Expression builder <220> <221> CDS <222> (1)..(1203) <220> <221> signal peptide <222> (1)..(81) <220> <221> Mature peptides <222> (592)..(1203) <400> 12 atg aag aaa ccg ttg ggg aaa att gtc gca agc acc gca cta ctc 45 Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu -195 -190 -185 att tct gtt gct ttt agt tca tcg atc gca tcg gct tca aca ctg 90 Ile Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala Ser Thr Leu -180 -175 -170 gca tca gat ccg aca ccg gca cct aca ccg gca gca agc aca gat 135 Ala Ser Asp Pro Thr Pro Ala Pro Thr Pro Ala Ala Ser Thr Asp -165 -160 -155 tca ggc caa tca gtt gca gaa atg tca gca aga tgg ctg gca aaa 180 Ser Gly Gln Ser Val Ala Glu Met Ser Ala Arg Trp Leu Ala Lys -150 -145 -140 gat aga gca att tca ctg gca aca gca aga cag cgc gtt gca gca 225 Asp Arg Ala Ile Ser Leu Ala Thr Ala Arg Gln Arg Val Ala Ala -135 -130 -125 caa gat ggc caa aca aga aca gca gca tca ctg gaa aga gca ctt 270 Gln Asp Gly Gln Thr Arg Thr Ala Ala Ser Leu Glu Arg Ala Leu -120 -115 -110 ggc gca aga gca gca ggc tca tat att gat gca aca tca ggc gca ctg 318 Gly Ala Arg Ala Ala Gly Ser Tyr Ile Asp Ala Thr Ser Gly Ala Leu -105 -100 -95 gtt gtt aat gtt gtt gat aca gca agc gtt gca aga gtt ctg tca gca 366 Val Val Asn Val Val Asp Thr Ala Ser Val Ala Arg Val Leu Ser Ala -90 -85 -80 ggc gca gtt gca aaa gtt gtc gat aga tca aca tca gaa ctg agc gca 414 Gly Ala Val Ala Lys Val Val Asp Arg Ser Thr Ser Glu Leu Ser Ala -75 -70 -65 -60 aca gaa aga gcg gca aga gcg aga gca ggc agc gca gtt gtt tca tca 462 Thr Glu Arg Ala Ala Arg Ala Arg Ala Gly Ser Ala Val Val Ser Ser -55 -50 -45 tat aca gat ccg gtt aca aat ggc gtt gtt ctg aca gtt ccg agc gca 510 Tyr Thr Asp Pro Val Thr Asn Gly Val Val Leu Thr Val Pro Ser Ala -40 -35 -30 aga gtt tca gat gat gat agc agc gat gtt ggc ctg gat ggc gtt aca 558 Arg Val Ser Glu Val Arg Ser Glu Val Val Gly Leu Asp Gly Val Thr -25 -20 -15 gtt gca ggc aca gat gca aga aca aca aca caa gca aat gtt tat ggc 606 Val Ala Gly Thr Asp Ala Arg Thr Thr Thr Gln Ala Asn Val Tyr Gly -10 -5 -1 1 5 gga cag cag att gaa ttt tca ggc tat gtt tgc tca ctg ggc ttt aat 654 Gly Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys Ser Leu Gly Phe Asn 10 15 20 gca aca aga ggc gga gca ccg gtt ttt gtt aca gca ggc cat tgc gga 702 Ala Thr Arg Gly Gly Ala Pro Val Phe Val Thr Ala Gly His Cys Gly 25 30 35 gaa ggc tat caa aca ttt tca aaa ggc gga aca aca ctg ggc agc aca 750 Glu Gly Tyr Gln Thr Phe Ser Lys Gly Gly Thr Thr Leu Gly Ser Thr 40 45 50 caa gca tat tca ttt ccg gga aac gat tat gca tat agc aca ctg aca 798 Gln Ala Tyr Ser Phe Pro Gly Asn Asp Tyr Ala Tyr Ser Thr Leu Thr 55 60 65 tca tca tgg aca ggc gtt gga gca gtt gat ctg tat gat ggc gtc aat 846 Ser Ser Trp Thr Gly Val Gly Ala Val Asp Leu Tyr Asp Gly Val Asn 70 75 80 85 gca aga aga gtt agc ggc tat tca aat gca ccg gtt ggc aca gca att 894 Ala Arg Arg Val Ser Gly Tyr Ser Asn Ala Pro Val Gly Thr Ala Ile 90 95 100 tgc aaa agc ggc aga aca aca ggc tgg aca tgc ggc tca gtt caa gca 942 Cys Lys Ser Gly Arg Thr Thr Gly Trp Thr Cys Gly Ser Val Gln Ala 105 110 115 aaa aat gtc aca gtc aat tat agc aat gca gat ggc tca aca tca aca 990 Lys Asn Val Thr Val Asn Tyr Ser Asn Ala Asp Gly Ser Thr Ser Thr 120 125 130 gtt tca ggc ctt aca aaa agc aac aca tgc aca gaa ggc gga gat agc 1038 Val Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr Glu Gly Gly Asp Ser 135 140 145 gga ggc tca tgg atg gca tca aca agc gca caa ggc gtt aca agc gga 1086 Gly Gly Ser Trp Met Ala Ser Thr Ser Ala Gln Gly Val Thr Ser Gly 150 155 160 165 ggc gca ggc tat ggc gca aat tca gtt tgc gga caa aaa gtt gga caa 1134 Gly Ala Gly Tyr Gly Ala Asn Ser Val Cys Gly Gln Lys Val Gly Gln 170 175 180 ccg aac att gca tat ttt caa ccg gtc gat gaa att gtt agc gca tat 1182 Pro Asn Ile Ala Tyr Phe Gln Pro Val Asp Glu Ile Val Ser Ala Tyr 185 190 195 ggc ctg aca ctg aaa aca tca taa 1206 Gly Leu Thr Leu Lys Thr Ser 200 <210> 13 <211> 401 <212> PRT <213> artificial sequence <220> <223> Synthesize construction <400> 13 Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu ‑195 ‑190 ‑185 Ile Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala Ser Thr Leu ‑180 ‑175 ‑170 Ala Ser Asp Pro Thr Pro Ala Pro Thr Pro Ala Ala Ser Thr Asp ‑165 ‑160 ‑155 Ser Gly Gln Ser Val Ala Glu Met Ser Ala Arg Trp Leu Ala Lys ‑150 ‑145 ‑140 Asp Arg Ala Ile Ser Leu Ala Thr Ala Arg Gln Arg Val Ala Ala ‑135 ‑130 ‑125 Gln Asp Gly Gln Thr Arg Thr Ala Ala Ser Leu Glu Arg Ala Leu ‑120 ‑115 ‑110 Gly Ala Arg Ala Ala Gly Ser Tyr Ile Asp Ala Thr Ser Gly Ala Leu ‑105 ‑100 ‑95 Val Val Asn Val Val Asp Thr Ala Ser Val Ala Arg Val Leu Ser Ala ‑90 ‑85 ‑80 Gly Ala Val Ala Lys Val Val Asp Arg Ser Thr Ser Glu Leu Ser Ala ‑75 ‑70 ‑65 ‑60 Thr Glu Arg Ala Ala Arg Ala Arg Ala Gly Ser Ala Val Val Ser Ser ‑55 ‑50 ‑45 Tyr Thr Asp Pro Val Thr Asn Gly Val Val Leu Thr Val Pro Ser Ala ‑40 ‑35 ‑30 Arg Val Ser Glu Val Arg Ser Glu Val Val Gly Leu Asp Gly Val Thr ‑25 ‑20 ‑15 Val Ala Gly Thr Asp Ala Arg Thr Thr Thr Gln Ala Asn Val Tyr Gly ‑10 ‑5 ‑1 1 5 Gly Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys Ser Leu Gly Phe Asn 10 15 20 Ala Thr Arg Gly Gly Ala Pro Val Phe Val Thr Ala Gly His Cys Gly 25 30 35 Glu Gly Tyr Gln Thr Phe Ser Lys Gly Gly Thr Thr Leu Gly Ser Thr 40 45 50 Gln Ala Tyr Ser Phe Pro Gly Asn Asp Tyr Ala Tyr Ser Thr Leu Thr 55 60 65 Ser Ser Trp Thr Gly Val Gly Ala Val Asp Leu Tyr Asp Gly Val Asn 70 75 80 85 Ala Arg Arg Val Ser Gly Tyr Ser Asn Ala Pro Val Gly Thr Ala Ile 90 95 100 Cys Lys Ser Gly Arg Thr Thr Gly Trp Thr Cys Gly Ser Val Gln Ala 105 110 115 Lys Asn Val Thr Val Asn Tyr Ser Asn Ala Asp Gly Ser Thr Ser Thr 120 125 130 Val Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr Glu Gly Gly Asp Ser 135 140 145 Gly Gly Ser Trp Met Ala Ser Thr Ser Ala Gln Gly Val Thr Ser Gly 150 155 160 165 Gly Ala Gly Tyr Gly Ala Asn Ser Val Cys Gly Gln Lys Val Gly Gln 170 175 180 Pro Asn Ile Ala Tyr Phe Gln Pro Val Asp Glu Ile Val Ser Ala Tyr 185 190 195 Gly Leu Thr Leu Lys Thr Ser 200 <210> 14 <211> 204 <212> PRT <213> Terracoccus sp. <220> <221> Mature peptide <222> (1)..(204) <400> 14 [[ID=了44]]Ala Asn Val Tyr Gly Gly Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys 1 5 10 15 It should be noted that there is an error in "了44" in the original text, which should be "44". The above translation is based on the corrected content.Ser Leu Gly Phe Asn Ala Thr Arg Gly Gly Ala Pro Val Phe Val Thr 20 25 30 Ala Gly His Cys Gly Glu Gly Tyr Gln Thr Phe Ser Lys Gly Gly Thr 35 40 45 Thr Leu Gly Ser Thr Gln Ala Tyr Ser Phe Pro Gly Asn Asp Tyr Ala 50 55 60 Tyr Ser Thr Leu Thr Ser Ser Trp Thr Gly Val Gly Ala Val Asp Leu 65 70 75 80 Tyr Asp Gly Val Asn Ala Arg Arg Val Ser Gly Tyr Ser Asn Ala Pro 85 90 95 Val Gly Thr Ala Ile Cys Lys Ser Gly Arg Thr Thr Gly Trp Thr Cys 100 105 110 Gly Ser Val Gln Ala Lys Asn Val Thr Val Asn Tyr Ser Asn Ala Asp 115 120 125 Gly Ser Thr Ser Thr Val Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr 130 135 140 Glu Gly Gly Asp Ser Gly Gly Ser Trp Met Ala Ser Thr Ser Ala Gln 145 150 155 160 Gly Val Thr Ser Gly Gly Ala Gly Tyr Gly Ala Asn Ser Val Cys Gly 165 170 175 Gln Lys Val Gly Gln Pro Asn Ile Ala Tyr Phe Gln Pro Val Asp Glu 180 185 190 Ile Val Ser Ala Tyr Gly Leu Thr Leu Lys Thr Ser 195 200 <210> 15 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Motif VCG[E / Q]KVGQP. <220> <221> Unclassified features <222> (4)..(4) <223> The amino acid at position 4 of this conserved motif is either glutamic acid (Glu, E) or glutamine (Gln, Q). <400> 15 Val Cys Gly Xaa Lys Val Gly Gln Pro 1 5 <210> 16 <211> 1209 <212> DNA <213> Knoellia flava <220> <221> CDS <222> (1)..(1206) <220> <221> signal peptide <222> (1)..(87) <220> <221> Mature peptides <222> (595)..(1206) <400> 16 atg tct cgc aga cgt ctc acc gtc ctc gcc ggg ggc ctc tcg gcc 45 Met Ser Arg Arg Arg Leu Thr Val Leu Ala Gly Gly Leu Ser Ala ‑195 ‑190 ‑185 gcg gcg gca gcc acc gcc ctc tgc gtc gcg ccc gcg tcc gcc gcc 90 Ala Ala Ala Ala Thr Ala Leu Cys Val Ala Pro Ala Ser Ala Ala ‑180 ‑175 ‑170 acc tcc gcg gcg ggc ggt ccg gag ccg agc acc ggc cct ctc gcc 135 Thr Ser Ala Ala Gly Gly Pro Glu Pro Ser Thr Gly Pro Leu Ala ‑165 ‑160 ‑155 acc gac tcg ggc gcg tcc gtc gcc gag atg tcg gcc cgg tgg ctc 180 Thr Asp Ser Gly Ala Ser Val Ala Glu Met Ser Ala Arg Trp Leu ‑150 ‑145 ‑140 gcc aag gag cac gac ctg agc atc gag acc gct cgt gag cgg atc 225 Ala Lys Glu His Asp Leu Ser Ile Glu Thr Ala Arg Glu Arg Ile ‑135 ‑130 ‑125 gcg tcc cag gag gac aag agt cgc aag gcc gag gct ctc gaa cgg 270 Ala Ser Gln Glu Asp Lys Ser Arg Lys Ala Glu Ala Leu Glu Arg ‑120 ‑115 ‑110 tcg ctc ggc gcg cga gcc gtg ggc tcg ttc atc gac cag acc ggc ggc 318 Ser Leu Gly Ala Arg Ala Val Gly Ser Phe Ile Asp Gln Thr Gly Gly ‑105 ‑100 ‑95 gtg ctc gtc gtc aac gtc acc gac gcc gac gcc gct gcc cgc gtg cag 366 Val Leu Val Val Asn Val Thr Asp Ala Asp Ala Ala Ala Arg Val Gln ‑90 ‑85 ‑80 aag gcg ggc gcg acc gcc cgc gtc gtc acc gag gac aag gcg gag ctc 414 Lys Ala Gly Ala Thr Ala Arg Val Val Thr Glu Asp Lys Ala Glu Leu ‑75 ‑70 ‑65 ggc gcg tcg cag gcg agg gcg gtc aag gcg ctc ggc gcc acg gtc atc 462 Gly Ala Ser Gln Ala Arg Ala Val Lys Ala Leu Gly Ala Thr Val Ile ‑60 ‑55 ‑50 ‑45 gac agc tcc gtc gac ccg gtg acc aac aag gtc gtc gtc acc gtg ccc 510 Asp Ser Ser Val Asp Pro Val Thr Asn Lys Val Val Val Thr Val Pro ‑40 ‑35 ‑30 acc gcc gac gtc gcc gcc gcg cgg gcg cgc acg agc gac ccg tcg gtg 558 Thr Ala Asp Val Ala Ala Ala Arg Ala Arg Thr Ser Asp Pro Ser Val ‑25 ‑20 ‑15 acg atc cag ggc acc gac gcg acg gtg tcg acg cag gcc aac gtc tat 606 Thr Ile Gln Gly Thr Asp Ala Thr Val Ser Thr Gln Ala Asn Val Tyr ‑10 ‑5 ‑1 1 ggc ggg cag cag atc gag ttc agc ggc tac gtc tgc tcg ctg ggc ttc 654 Gly Gly Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys Ser Leu Gly Phe 5 10 15 20 aac gcg acg aag tcc ggc acc ccg gtc ttc atc acc gcc ggc cac tgc 702 Asn Ala Thr Lys Ser Gly Thr Pro Val Phe Ile Thr Ala Gly His Cys 25 30 35 gcc gag ggg aac cag acc ttc acg cgc aac ggc acg acc ctc ggc acg 750 Ala Glu Gly Asn Gln Thr Phe Thr Arg Asn Gly Thr Thr Leu Gly Thr 40 45 50 acc cgc ggc tgg tcc ttc ccg ggc aac gac tac gcc tac tcg agc ctc 798 Thr Arg Gly Trp Ser Phe Pro Gly Asn Asp Tyr Ala Tyr Ser Ser Leu 55 60 65 acc tcg agc tgg acc ggc atc ggc gcc gtc gac ctg tgg aac ggc acg 846 Thr Ser Ser Trp Thr Gly Ile Gly Ala Val Asp Leu Trp Asn Gly Thr 70 75 80 agc gcg cgc tcc gtc acg ggc tcg agc aac gcc gcc gtc ggc acc gcg 894 Ser Ala Arg Ser Val Thr Gly Ser Ser Asn Ala Ala Val Gly Thr Ala 85 90 95 100 atc tgc aag tcg ggc cgc acg acc ggc tgg acc tgt ggc tcg gtc cag 942 Ile Cys Lys Ser Gly Arg Thr Thr Gly Trp Thr Cys Gly Ser Val Gln 105 110 115 acc aag aac gtc acc gtc aac tac aac aac ggc gac ggc acc tac tcg 990 Thr Lys Asn Val Thr Val Asn Tyr Asn Asn Gly Asp Gly Thr Tyr Ser 120 125 130 acc gtg agc ggc ctg acg aag tcc aac acc tgc acc gag ggt ggc gac 1038 Thr Val Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr Glu Gly Gly Asp 135 140 145 tcc ggc ggc tcg tgg atg gcg ggc aac ctc gcc cag ggc gtg acg agc 1086 Ser Gly Gly Ser Trp Met Ala Gly Asn Leu Ala Gln Gly Val Thr Ser 150 155 160 ggc ggc gcc ggc tac ggc tcc aac ggc gtc tgc ggc cag aag gtc ggc 1134 Gly Gly Ala Gly Tyr Gly Ser Asn Gly Val Cys Gly Gln Lys Val Gly 165 170 175 180 cag ccc aac atc gcc tac ttc cag ccg atc ggc gag atc ctc tcc gtc 1182 Gln Pro Asn Ile Ala Tyr Phe Gln Pro Ile Gly Glu Ile Leu Ser Val 185 190 195 tac ggc ctc acc ctc aag acc gcc tga 1209 Tyr Gly Leu Thr Leu Lys Thr Ala 200 <210> 17 <211> 402 <212> PRT <213> Nocardia flavescens <400> 17 Met Ser Arg Arg Arg Leu Thr Val Leu Ala Gly Gly Leu Ser Ala ‑195 ‑190 ‑185 Ala Ala Ala Ala Thr Ala Leu Cys Val Ala Pro Ala Ser Ala Ala ‑180 ‑175 ‑170 Thr Ser Ala Ala Gly Gly Pro Glu Pro Ser Thr Gly Pro Leu Ala ‑165 ‑160 ‑155 Thr Asp Ser Gly Ala Ser Val Ala Glu Met Ser Ala Arg Trp Leu ‑150 ‑145 ‑140 Ala Lys Glu His Asp Leu Ser Ile Glu Thr Ala Arg Glu Arg Ile ‑135 ‑130 ‑125 Ala Ser Gln Glu Asp Lys Ser Arg Lys Ala Glu Ala Leu Glu Arg ‑120 ‑115 ‑110 Ser Leu Gly Ala Arg Ala Val Gly Ser Phe Ile Asp Gln Thr Gly Gly ‑105 ‑100 ‑95 Val Leu Val Val Asn Val Thr Asp Ala Asp Ala Ala Ala Arg Val Gln ‑90 ‑85 ‑80 Lys Ala Gly Ala Thr Ala Arg Val Val Thr Glu Asp Lys Ala Glu Leu ‑75 ‑70 ‑65 Gly Ala Ser Gln Ala Arg Ala Val Lys Ala Leu Gly Ala Thr Val Ile ‑60 ‑55 ‑50 ‑45 Asp Ser Ser Val Asp Pro Val Thr Asn Lys Val Val Val Thr Val Pro ‑40 ‑35 ‑30 Thr Ala Asp Val Ala Ala Ala Arg Ala Arg Thr Ser Asp Pro Ser Val ‑25 ‑20 ‑15 Thr Ile Gln Gly Thr Asp Ala Thr Val Ser Thr Gln Ala Asn Val Tyr ‑10 ‑5 ‑1 1 Gly Gly Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys Ser Leu Gly Phe 5 10 15 20 Asn Ala Thr Lys Ser Gly Thr Pro Val Phe Ile Thr Ala Gly His Cys 25 30 35 Ala Glu Gly Asn Gln Thr Phe Thr Arg Asn Gly Thr Thr Leu Gly Thr 40 45 50 Thr Arg Gly Trp Ser Phe Pro Gly Asn Asp Tyr Ala Tyr Ser Ser Leu 55 60 65 Thr Ser Ser Trp Thr Gly Ile Gly Ala Val Asp Leu Trp Asn Gly Thr 70 75 80 Ser Ala Arg Ser Val Thr Gly Ser Ser Asn Ala Ala Val Gly Thr Ala 85 90 95 100 Ile Cys Lys Ser Gly Arg Thr Thr Gly Trp Thr Cys Gly Ser Val Gln 105 110 115 Thr Lys Asn Val Thr Val Asn Tyr Asn Asn Gly Asp Gly Thr Tyr Ser 120 125 130 Thr Val Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr Glu Gly Gly Asp 135 140 145 Ser Gly Gly Ser Trp Met Ala Gly Asn Leu Ala Gln Gly Val Thr Ser 150 155 160 Gly Gly Ala Gly Tyr Gly Ser Asn Gly Val Cys Gly Gln Lys Val Gly 165 170 175 180 Gln Pro Asn Ile Ala Tyr Phe Gln Pro Ile Gly Glu Ile Leu Ser Val 185 190 195 Tyr Gly Leu Thr Leu Lys Thr Ala 200 <210> 18 <211> 1200 <212> DNA <213> Artificial sequence <220> <223> Codon-optimized synthetic genes <220> <221> CDS <222> (1)..(1200) <220> <221> signal peptide <222> (1)..(81) <220> <221> Mature peptides <222> (589)...(1200) <400> 18 atg aag aaa ccg ttg ggg aaa att gtc gca agc acc gca cta ctc 45 Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu -195 -190 -185 att tct gtt gct ttt agt tca tcg atc gca tcg gct gct aca tct 90 Ile Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala Ala Thr Ser -180 -175 -170 gca gct ggt ggc cca gaa cca tca act ggc cca ctt gct aca gat 135 Ala Ala Gly Gly Pro Glu Pro Ser Thr Gly Pro Leu Ala Thr Asp ‑165 ‑160 ‑155 tct ggc gct tca gtt gcc gag atg tca gca cgc tgg ctt gcg aaa 180 Ser Gly Ala Ser Val Ala Glu Met Ser Ala Arg Trp Leu Ala Lys ‑150 ‑145 ‑140 gaa cac gat ctt tca att gag act gca cgt gag cgc atc gct agc 225 Glu His Asp Leu Ser Ile Glu Thr Ala Arg Glu Arg Ile Ala Ser ‑135 ‑130 ‑125 cag gaa gac aaa tct cgc aag gct gaa gct ctt gaa cgc tca ctt 270 Gln Glu Asp Lys Ser Arg Lys Ala Glu Ala Leu Glu Arg Ser Leu ‑120 ‑115 ‑110 ggc gct cgt gct gtt ggc agc ttt atc gac caa act ggc ggt gta ttg 318 Gly Ala Arg Ala Val Gly Ser Phe Ile Asp Gln Thr Gly Gly Val Leu ‑105 ‑100 ‑95 gta gta aac gtt act gat gcg gat gca gca gct cgc gtt caa aag gca 366 Val Val Asn Val Thr Asp Ala Asp Ala Ala Ala Arg Val Gln Lys Ala ‑90 ‑85 ‑80 ‑75 gga gct aca gct cgt gtt gtt act gag gat aag gct gaa ctt ggc gct 414 Gly Ala Thr Ala Arg Val Val Thr Glu Asp Lys Ala Glu Leu Gly Ala ‑70 ‑65 ‑60 tct caa gct cgt gct gtt aag gct ctt ggt gcc act gta att gat agc 462 Ser Gln Ala Arg Ala Val Lys Ala Leu Gly Ala Thr Val Ile Asp Ser ‑55 ‑50 ‑45 tca gtt gac cct gta acg aac aaa gtt gta gtt aca gta cct act gct 510 Ser Val Asp Pro Val Thr Asn Lys Val Val Val Thr Val Pro Thr Ala ‑40 ‑35 ‑30 gat gtt gcg gca gca cgt gca cgt aca agc gac cca tct gta act att 558 Asp Val Ala Ala Ala Arg Ala Arg Thr Ser Asp Pro Ser Val Thr Ile ‑25 ‑20 ‑15 caa gga aca gac gca acg gtt tct aca cag gct aac gtt tat ggt ggc 606 Gln Gly Thr Asp Ala Thr Val Ser Thr Gln Ala Asn Val Tyr Gly Gly ‑10 ‑5 ‑1 1 5 cag cag atc gag ttc tct gga tac gta tgt tca tta ggt ttc aac gca 654 Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys Ser Leu Gly Phe Asn Ala 10 15 20 act aaa tct gga act cct gtt ttc atc aca gct ggc cat tgt gcg gaa 702 Thr Lys Ser Gly Thr Pro Val Phe Ile Thr Ala Gly His Cys Ala Glu 25 30 35 ggt aac cag act ttc act cgt aat ggt aca aca ttg ggt aca aca cgc 750 Gly Asn Gln Thr Phe Thr Arg Asn Gly Thr Thr Leu Gly Thr Thr Arg 40 45 50 ggt tgg tct ttt cca ggt aac gat tat gcg tac tca tct ctt act tct 798 Gly Trp Ser Phe Pro Gly Asn Asp Tyr Ala Tyr Ser Ser Leu Thr Ser 55 60 65 70 tct tgg act ggt att gga gct gtt gac tta tgg aat gga aca tca gct 846 Ser Trp Thr Gly Ile Gly Ala Val Asp Leu Trp Asn Gly Thr Ser Ala 75 80 85 cgc tct gta act ggc tca tca aac gct gct gtt gga act gca att tgc 894 Arg Ser Val Thr Gly Ser Ser Asn Ala Ala Val Gly Thr Ala Ile Cys 90 95 100 aaa tct ggt cgt aca acg gga tgg aca tgt ggt tct gta caa acg aaa 942 Lys Ser Gly Arg Thr Thr Gly Trp Thr Cys Gly Ser Val Gln Thr Lys 105 110 115 aac gta act gta aac tat aac aac gga gat ggt aca tat tct act gta 990 Asn Val Thr Val Asn Tyr Asn Asn Gly Asp Gly Thr Tyr Ser Thr Val 120 125 130 tct ggt ctt aca aaa agc aat act tgc act gaa ggt gga gat tca ggc 1038 Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr Glu Gly Gly Asp Ser Gly 135 140 145 150 ggt tct tgg atg gct ggc aac tta gca caa ggt gta act agc ggt ggt 1086 Gly Ser Trp Met Ala Gly Asn Leu Ala Gln Gly Val Thr Ser Gly Gly 155 160 165 gct ggc tat ggt agc aat gga gta tgt ggc cag aaa gta ggt caa ccg 1134 Ala Gly Tyr Gly Ser Asn Gly Val Cys Gly Gln Lys Val Gly Gln Pro 170 175 180 aac att gct tac ttt cag cct atc ggt gaa atc ​​ttg tct gtt tat ggt 1182 Asn Ile Ala Tyr Phe Gln Pro Ile Gly Glu Ile Leu Ser Val Tyr Gly 185 190 195 ctt aca ttg aaa aca gct 1200 Leu Thr Leu Lys Thr Ala 200 <210> 19 <211> 400 <212> PRT <213> artificial sequence <220> <223> Synthesis structure <400> 19 Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu -195 -190 -185 Ile Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala Ala Thr Ser -180 -175 -170 Ala Ala Gly Gly Pro Glu Pro Ser Thr Gly Pro Leu Ala Thr Asp -165 -160 -155 Ser Gly Ala Ser Val Ala Glu Met Ser Ala Arg Trp Leu Ala Lys -150 -145 -140 Glu His Asp Leu Ser Ile Glu Thr Ala Arg Glu Arg Ile Ala Ser -135 -130 -125 Gln Glu Asp Lys Ser Arg Lys Ala Glu Ala Leu Glu Arg Ser Leu ‑120 ‑115 ‑110 Gly Ala Arg Ala Val Gly Ser Phe Ile Asp Gln Thr Gly Gly Val Leu ‑105 ‑100 ‑95 Val Val Asn Val Thr Asp Ala Asp Ala Ala Ala Arg Val Gln Lys Ala ‑90 ‑85 ‑80 ‑75 Gly Ala Thr Ala Arg Val Val Thr Glu Asp Lys Ala Glu Leu Gly Ala ‑70 ‑65 ‑60 Ser Gln Ala Arg Ala Val Lys Ala Leu Gly Ala Thr Val Ile Asp Ser ‑55 ‑50 ‑45 Ser Val Asp Pro Val Thr Asn Lys Val Val Val Thr Val Pro Thr Ala ‑40 ‑35 ‑30 Asp Val Ala Ala Ala Arg Ala Arg Thr Ser Asp Pro Ser Val Thr Ile ‑25 ‑20 ‑15 Gln Gly Thr Asp Ala Thr Val Ser Thr Gln Ala Asn Val Tyr Gly Gly ‑10 ‑5 ‑1 1 5 Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys Ser Leu Gly Phe Asn Ala 10 15 20 Thr Lys Ser Gly Thr Pro Val Phe Ile Thr Ala Gly His Cys Ala Glu 25 30 35 Gly Asn Gln Thr Phe Thr Arg Asn Gly Thr Thr Leu Gly Thr Thr Arg 40 45 50 Gly Trp Ser Phe Pro Gly Asn Asp Tyr Ala Tyr Ser Ser Leu Thr Ser 55 60 65 70 Ser Trp Thr Gly Ile Gly Ala Val Asp Leu Trp Asn Gly Thr Ser Ala 75 80 85 Arg Ser Val Thr Gly Ser Ser Asn Ala Ala Val Gly Thr Ala Ile Cys 90 95 100 Lys Ser Gly Arg Thr Thr Gly Trp Thr Cys Gly Ser Val Gln Thr Lys 105 110 115 Asn Val Thr Val Asn Tyr Asn Asn Gly Asp Gly Thr Tyr Ser Thr Val 120 125 130 Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr Glu Gly Gly Asp Ser Gly 135 140 145 150 Gly Ser Trp Met Ala Gly Asn Leu Ala Gln Gly Val Thr Ser Gly Gly 155 160 165 Ala Gly Tyr Gly Ser Asn Gly Val Cys Gly Gln Lys Val Gly Gln Pro 170 175 180 Asn Ile Ala Tyr Phe Gln Pro Ile Gly Glu Ile Leu Ser Val Tyr Gly 185 190 195 Leu Thr Leu Lys Thr Ala 200 <210> 20 <211> 204 <212> PRT <213> Nocardia flava <220> <221> mature peptide <222> (1)..(204) <400> 20 Ala Asn Val Tyr Gly Gly Gln Gln Ile Glu Phe Ser Gly Tyr Val Cys 1 5 10 15 Ser Leu Gly Phe Asn Ala Thr Lys Ser Gly Thr Pro Val Phe Ile Thr 20 25 30 Ala Gly His Cys Ala Glu Gly Asn Gln Thr Phe Thr Arg Asn Gly Thr 35 40 45 Thr Leu Gly Thr Thr Arg Gly Trp Ser Phe Pro Gly Asn Asp Tyr Ala 50 55 60 Tyr Ser Ser Leu Thr Ser Ser Trp Thr Gly Ile Gly Ala Val Asp Leu 65 70 75 80 Trp Asn Gly Thr Ser Ala Arg Ser Val Thr Gly Ser Ser Asn Ala Ala 85 90 95 Val Gly Thr Ala Ile Cys Lys Ser Gly Arg Thr Thr Gly Trp Thr Cys 100 105 110 Gly Ser Val Gln Thr Lys Asn Val Thr Val Asn Tyr Asn Asn Gly Asp 115 120 125 Gly Thr Tyr Ser Thr Val Ser Gly Leu Thr Lys Ser Asn Thr Cys Thr 130 135 140 Glu Gly Gly Asp Ser Gly Gly Ser Trp Met Ala Gly Asn Leu Ala Gln 145 150 155 160 Gly Val Thr Ser Gly Gly Ala Gly Tyr Gly Ser Asn Gly Val Cys Gly 165 170 175 Gln Lys Val Gly Gln Pro Asn Ile Ala Tyr Phe Gln Pro Ile Gly Glu 180 185 190 Ile Leu Ser Val Tyr Gly Leu Thr Leu Lys Thr Ala 195 200

Claims

1. An animal feed or animal feed additive comprising one or more polypeptides having protease activity, wherein the polypeptide is selected from the group consisting of the following: (a) A polypeptide having the sequence SEQ ID NO: 5; (b) A polypeptide encoded by a polynucleotide, wherein the polynucleotide is: (i) The mature polypeptide coding sequence of SEQ ID NO: 1; as well as (c) A variant of SEQ ID NO: 5, wherein the variant has protease activity and the variant is composed of amino acids of SEQ ID NO: 5 substituted with S68N or T71N; in: The animal feed or animal feed additive further comprises one or more components selected from the following list, which consists of the following items: One or more other 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.

2. The animal feed or animal feed additive as described in claim 1, wherein the polypeptide is obtained from or can be obtained from the order Micrococciles.

3. The animal feed or animal feed additive according to any one of claims 1 to 2, wherein the polypeptide is obtained from or can be obtained from the family Mesophyceae.

4. The animal feed or animal feed additive as described in any one of claims 1 to 2, wherein: (i) The polypeptide exhibits at least twice the activity of protease 10R of SEQ ID NO: 8 at the same pH at pH 4; (ii) The polypeptide exhibits at least twice the activity of protease 10R (SEQ ID NO: 8) at the same pH, compared to the activity at pH 5, in soybean-corn flour; and (iii) Compared with protease 10R of SEQ ID NO: 8 at the same pH, this polypeptide has at least 50% activity against soybean-corn flour at pH 7.

5. The animal feed or animal feed additive as described in any one of claims 1 to 2, wherein: (i) The peptide exhibits at least 25% activity against soybean-corn flour at pH 4, compared to its activity at pH 7; (ii) Compared to its activity at pH 7, the peptide exhibits at least 45% activity against soybean-corn flour at pH 5; and (iii) Compared with protease 10R of SEQ ID NO: 8 at the same pH, this polypeptide has at least 50% activity against soybean-corn flour at pH 7.

6. The animal feed or animal feed additive according to any one of claims 1 to 2, wherein the polypeptide is composed of the mature polypeptide of SEQ ID NO: 5, or amino acids 1 to 203 of SEQ ID NO: 5; or the polypeptide is composed of variants S68N and T71N of SEQ ID NO:

5.

7. The animal feed or animal feed additive according to any one of claims 1 to 2, wherein the polypeptide comprises one or more of the SEQ ID NO: 15 motif VCG[E / Q]KVGQP.

8. The animal feed or animal feed additive as described in any one of claims 1 to 2, having a crude protein content of 50 to 800 g / kg.

9. The animal feed or animal feed additive of claim 1, wherein these additional enzymes are selected from any one of the following groups or any combination thereof, the group consisting of the following: Phytase, xylanase, galactanase, α-galactosidase, other proteases, phospholipase A1, phospholipase A2, lysophospholipase, phospholipase C, phospholipase D, amylase, lysozyme, arabinofuranase, β-xylosidase, acetylxylan esterase, ferulic acid esterase, cellulase, cellobiase, β-glucosidase, and β-glucanase.

10. The animal feed or animal feed additive of claim 1, wherein the one or more microorganisms are selected from the group consisting of: Carnivorous bacteria (Carnobacterium sp.) Clostridium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Giant Cocci ( Megasphaera sp. Pediococcus, Propionibacterium, and Streptococcus or any combination thereof.

11. The animal feed or animal feed additive of claim 1, wherein the one or more microorganisms are selected from the group consisting of: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circumflex, Bifidobacterium bifidum, Bifidobacterium animalis, Clostridium butyricum, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus sausageii ( Lactobacillus farciminus Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactococcus lactis, and Giant Pleurotus erythrocytee (Gastroenterococcus) Megasphaera elsdenii ), Pediococcus acidilactici ( Pediococcus acidilactici Propionibacterium tergenti ( Propionibacterium thoenii (or any combination thereof).

12. Use of the animal feed or animal feed additive as described in any one of claims 1 to 11: In the preparation of compositions for use in animal feed; Used to improve the nutritional value of animal feed, but not for the treatment of diseases; Used to increase the digestible and / or soluble protein in animal feed, but not for the treatment of diseases; Used to increase the degree of protein hydrolysis in animal diets, but not for disease treatment; Used to improve one or more performance parameters in animals, but not related to disease treatment methods; and / or It is used for processing proteins, but not for treating diseases.

13. A method for preparing animal feed, comprising mixing an animal feed additive as described in any one of claims 1 to 11 with at least one protein or protein source.

14. A method for improving the nutritional value of animal feed, wherein the animal feed or animal feed additive as described in any one of claims 1 to 11 is added to the feed.

15. A method for processing proteins, comprising the following steps: Adding animal feed or animal feed additives as described in any one of claims 1 to 11 to at least one protein or protein source; wherein the method does not involve a treatment for a disease.

16. A method for increasing the digestibility and / or solubility of protein, comprising mixing an animal feed additive as described in any one of claims 1 to 11 with at least one protein or protein source; wherein the method does not relate to a treatment for a disease.

17. A method for improving one or more performance parameters in an animal, comprising administering an animal feed or animal feed additive as described in any one of claims 1 to 11 to one or more animals; wherein the method does not involve a treatment for a disease.

18. The use as described in claim 12 or the method as described in claim 17, wherein the performance parameter is selected from the following: weight gain (BWG), European Production Efficiency Factor (EPEF), or Feed Conversion Rate (FCR).

Images