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CN-107072251-B - Polypeptides having serine protease activity and polynucleotides encoding same and their use in animal feed

CN107072251BCN 107072251 BCN107072251 BCN 107072251BCN-107072251-B

Abstract

The present invention relates to animal feed or animal feed additives comprising polypeptides having protease activity and uses thereof. Specifically, the protease is serine S1 protease from genus Proteus, genus Agrobacterium (Terracoccus) and genus Nocardia (Knoellia), all belonging to the family Mortierella of the order Micrococcus (Micrococineae). These proteases have high activity over a broad pH range (pH 3-7) and are therefore highly active during the complete passage through the digestive tract. It also relates to methods for producing these proteases and using the proteases to improve animal performance and animal feed nutritional value.

Inventors

  • T. Hoff
  • P.R. Eastergao
  • K.F. Pontopidam

Assignees

  • 诺维信公司
  • 诺维信公司

Dates

Publication Date
20260421
Application Date
20151103
Priority Date
20141104

Claims (18)

  1. 1. An animal feed or animal feed additive comprising one or more polypeptides having protease activity, wherein the polypeptides are selected from the group consisting of: (a) A polypeptide, the sequence of which is SEQ ID NO. 5; (b) A polypeptide encoded by a polynucleotide that is: (i) The mature polypeptide coding sequence of SEQ ID NO. 1; And (C) A variant of SEQ ID No. 5, wherein the variant has protease activity and the variant consists of the amino acid substitution S68N or T71N of SEQ ID No. 5; wherein: The animal feed or animal feed additive further comprises one or more components selected from the list consisting 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.
  2. 2. The animal feed or animal feed additive of claim 1, wherein the polypeptide is obtained or obtainable from the order micrococcus.
  3. 3. The animal feed or animal feed additive of any one of claims 1 to 2, wherein the polypeptide is obtained or obtainable from the family mesotheceneae.
  4. 4. The animal feed or animal feed additive of any one of claims 1 to 2, wherein: (i) The polypeptide has at least 2 times higher activity on soybean-corn meal than the activity of protease 10R of SEQ ID NO. 8 at the same pH at pH 4; (ii) The polypeptide has at least 2-fold higher activity on soybean-corn meal than the activity of protease 10R of SEQ ID NO. 8 at the same pH at pH 5, and (Iii) The polypeptide has at least 50% activity at pH 7 on soybean-corn meal compared to protease 10R of SEQ ID NO. 8 at the same pH.
  5. 5. The animal feed or animal feed additive of any one of claims 1 to 2, wherein: (i) The polypeptide has at least 25% activity at pH 4 on soybean-corn meal as compared to activity at pH 7; (ii) The polypeptide has at least 45% activity at pH 5 on soybean-corn meal as compared to activity at pH 7, and (Iii) The polypeptide has at least 50% activity at pH 7 on soybean-corn meal compared to protease 10R of SEQ ID NO. 8 at the same pH.
  6. 6. An animal feed or animal feed additive according to any one of claims 1 to 2 wherein the polypeptide consists of the mature polypeptide of SEQ ID No.5 or amino acids 1 to 203 of SEQ ID No.5 or the polypeptide consists of variant S68N, T N of SEQ ID No. 5.
  7. 7. The animal feed or animal feed additive of any one of claims 1 to 2, wherein the polypeptide comprises one or more of the SEQ ID No. 15 motifs VCG [ E/Q ] KVGQP.
  8. 8. An animal feed or animal feed additive as claimed in any one of claims 1 to 2 having a crude protein content of 50 to 800 g/kg.
  9. 9. The animal feed or animal feed additive of claim 1, wherein the additional enzymes are selected from any one of the group consisting of phytase, xylanase, galactanase, alpha-galactosidase, additional proteases, phospholipase A1, phospholipase A2, lysophospholipase, phospholipase C, phospholipase D, amylase, lysozyme, arabinofuranosidase, beta-xylosidase, acetylxylan esterase, feruloyl esterase, cellulase, cellobiohydrolase, beta-glucosidase, and beta-glucanase, or any combination thereof.
  10. 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 bacillus (Carnobacterium sp.), clostridium, enterococcus, lactobacillus, lactococcus, leuconostoc, megacoccus (Megasphaera sp.), pediococcus, propionibacterium, and streptococcus or any combination thereof.
  11. 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 circulans, bifidobacterium bifidum, bifidobacterium animalis, clostridium butyricum, enterococcus faecium, lactobacillus acidophilus, lactobacillus sausage (Lactobacillus farciminus), lactobacillus rhamnosus, lactobacillus reuteri, lactobacillus salivarius, lactococcus lactis, giant (MEGASPHAERA ELSDENII), pediococcus acidilactici (Pediococcus acidilactici), propionibacterium termitis (Propionibacterium thoenii), or any combination thereof.
  12. 12. Use of an animal feed or an animal feed additive according to any one of claims 1 to 11: in the preparation of a composition for use in animal feed; For improving the nutritional value of animal feeds, but not to methods of treatment of diseases; for increasing digestible and/or soluble proteins in animal feed, but not to methods of treatment of diseases; for increasing the degree of hydrolysis of proteins in animal diets, but not to methods of treatment of diseases; For improving one or more performance parameters in an animal, but not related to a method of treatment of a disease, and/or For treating proteins, but not related to methods of disease treatment.
  13. 13. A method for preparing an animal feed comprising mixing an animal feed additive according to any one of claims 1 to 11 with at least one protein or protein source.
  14. 14. A method for improving the nutritional value of an animal feed, wherein the animal feed or animal feed additive of any one of claims 1 to 11 is added to the feed.
  15. 15. A method for treating proteins comprising the step of adding an animal feed or animal feed additive according to any one of claims 1 to 11 to at least one protein or protein source, wherein the method does not involve a method of treatment of a disease.
  16. 16. A method for increasing the digestibility and/or solubility of a protein comprising mixing an animal feed additive according to any one of claims 1 to 11 with at least one protein or protein source, wherein the method does not involve a method of treatment of a disease.
  17. 17. A method for improving one or more performance parameters in an animal comprising administering the animal feed or animal feed additive of any one of claims 1 to 11 to one or more animals, wherein the method does not involve a method of treatment of a disease.
  18. 18. The use according to claim 12 or the method according to claim 17, wherein the performance parameter is selected from the group consisting of weight gain (BWG), european Production Efficiency Factor (EPEF) or Feed Conversion Rate (FCR).

Description

Polypeptides having serine protease activity and polynucleotides encoding same and their use in animal feed Reference to sequence Listing The present application comprises a sequence listing in computer readable form, which is incorporated herein by reference. Background Technical Field The present invention relates to animal feed or animal feed additives comprising polypeptides having protease activity and uses thereof. It also relates to methods for producing these proteases and using the proteases to improve animal performance and animal feed nutritional value. Background Proteases are used in animal feed (in vivo), and/or the use of such proteases for the treatment of vegetable proteins (in vitro), it being noted that proteins are essential nutritional factors for animals and humans. Most domestic animals and many humans acquire these essential proteins from vegetable protein sources. Important vegetable protein sources are, for example, oilseed crops, legumes and cereals. When, for example, soy flour is included in the feed of monogastric animals such as pigs and poultry, a substantial proportion of the soy flour is not digested effectively (the apparent ileal protein digestibility in piglets, growing pigs and poultry such as broilers, laying hens and roosters is only around 80%). The gastrointestinal tract of animals is composed of a series of segments each presenting a different pH environment. In monogastric animals such as pigs and poultry and many types of fish, the stomach is strongly acidic with a pH potentially as low as 1-2, while the intestines have a more neutral pH of around 6-7.5. In addition to the stomach and intestines, poultry has a crop in front of the stomach. The pH in the crop is mainly determined by the digested feed and is therefore typically in the range of pH 4-6. Digestion of a protein by a protease may occur throughout the digestive tract, provided that the protease is active and is active in conditions of the digestive tract. Thus, proteases that are highly acid stable and therefore survive in the gastric environment and at the same time are effectively active at a wide range of physiological pH of the digestive tract of the target animal are particularly desirable. Since animal feeds are typically formulated in granular form, wherein steam is applied during pelleting, it is also desirable that the proteases used in the animal feed remain active after exposure to the steam treatment. In order to produce proteases for industrial use, it is important to produce proteases in high yields so that a sufficient amount of the product is available to be able to provide the protease at a favourable price. Description of related Art Proteases from the S1 family are known in the art and are used in animal feed. For example, WO 01/58275 discloses the use of acid stable proteases of the subtilisin family in animal feed. WO 01/58276 discloses the use of an acid stable protease derived from nocardia sp NRRL 18262 (10R protease) and a protease derived from nocardia albicans 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 subtilisin/keratinase, PWD-1 from Bacillus licheniformis, in poultry feed. Soybeans and corn are two highly used protein sources in agriculture, and it is therefore important that proteases have good activity on such substrates. There are many disclosures showing the activity of proteases from different bacterial sources on soybean-corn flour, such as korean genus (Kribbella) species (WO 2013/026796), rhodosporidium (WO 2013/110766 and WO 2014/122161), monospora viridis (WO 2013/189972), mycelial australis (WO 2013/041689) and dactylotheca species (WO 2014/096259). In addition, proteases isolated from other bacterial species (e.g., genus bifidogenic (Janibacter sp.)) are known in the art. Sirash (Thrash) et al performed the whole genome bird gun method of the bacterial genus Shelter HTCC2649 as described in "offshore Apis strain HTCC2649 (Genome sequence of THE MARINE Janibacter Sp. Stress HTCC 2649)", 2011, journal of bacteriology (J. Bacterio.) 193:584-585, which strain has been submitted to EMBL/GenBank under accession number AAMN 01000001. From this genome, a polypeptide having Uniprot number A3TJ83 (SEQ ID NO:2 herein) was annotated as serine protease. Jitian (Yoshida) et al performed the genome-wide bird gun method of bacteria Austwickia chelonae NBRC 105200, which were submitted to the EMBL/GenBank/DDBJ databases, and the peptidases (Uniprot: K6VM97, SEQ ID NO: 9) from these databases were annotated with 62.1% sequence identity to SEQ ID NO:5 (corresponding to the mature polypeptides of SEQ ID NO:2 and 4 herein). Commercial products comprising a protease and marketed for use in animal feed includeProAct (DSM NP/Norwev letter Co., ltd.),(DuPont Co.,