阅读说明：本技术 胰蛋白酶样丝氨酸蛋白酶及其用途 (Trypsin-like serine protease and uses thereof ) 是由 S·希波夫斯科夫 Z·邹 S·于 X·顾 于 2018-03-09 设计创作，主要内容包括：本文描述了新颖胰蛋白酶样丝氨酸蛋白酶及其用途。(Described herein are novel trypsin-like serine proteases and uses thereof.)

1. An isolated polypeptide having serine protease activity, selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence having at least 91% identity to the amino acid sequence of SEQ ID NO. 22;

b) a polypeptide comprising an amino acid sequence having at least 94% identity to the amino acid sequence of SEQ ID NO. 23;

c) a polypeptide comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO. 24; and

d) a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO. 25.

2. An isolated polypeptide having serine protease activity and comprising a predicted precursor amino acid sequence selected from the group consisting of: 3, SEQ ID NO; 6, SEQ ID NO; 9, SEQ ID NO; and SEQ ID NO 12.

3. An isolated polypeptide having serine protease activity and comprising a protease catalytic region selected from the group consisting of:

a) an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO. 18;

b) an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO. 19;

c) 20, the amino acid sequence of SEQ ID NO; and

d) an amino acid sequence having at least 91% identity to the amino acid sequence of SEQ ID NO. 21.

4. A recombinant construct comprising a control sequence functional in a production host operably linked to a nucleotide sequence encoding at least one polypeptide of any one of claims 1-3.

5. The recombinant construct of claim 4, wherein said host is selected from the group consisting of: fungi, bacteria and algae.

6. A method for producing at least one polypeptide, the method comprising:

(a) transforming a production host with the recombinant construct of claim 4; and

(b) cultivating the production host of step (a) under conditions to produce at least one polypeptide.

7. The method of claim 6, wherein the polypeptide is optionally recovered from the production host.

8. A culture supernatant containing a serine protease obtained by the method of claim 6 or 7.

9. A recombinant microbial production host for expressing at least one polypeptide, comprising the recombinant construct of claim 4.

10. The production host of claim 9, wherein the host is selected from the group consisting of: bacteria, fungi and algae.

11. An animal feed comprising at least one polypeptide according to any one of claims 1-3, wherein the polypeptide is present in an amount of 1-20g per ton of feed.

12. The animal feed of claim 11, further comprising: a) at least one direct fed microbial, or b) at least one other enzyme, or c) at least one direct fed microbial and at least one other enzyme.

13. A feed, feed additive composition or premix comprising at least one polypeptide having serine protease activity according to any of claims 1-3.

14. The feed, feed additive composition or premix of claim 13 further comprising:

a) at least one direct fed microbial, or b) at least one other enzyme, or c) at least one direct fed microbial and at least one other enzyme.

15. A feed additive composition according to any one of claims 13-14 wherein the composition further comprises at least one component selected from the group consisting of: proteins, peptides, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methyl paraben, and propyl paraben.

16. A granulated feed additive composition for use in animal feed, the granulated feed additive composition comprising a serine protease polypeptide of any of claims 1-3, wherein the granulated feed additive composition comprises granules produced by a method selected from the group consisting of: high shear granulation, drum granulation, extrusion, spheronization, fluidized bed agglomeration, fluidized bed spraying, spray drying, freeze drying, granulation, spray cooling, rotary disk atomization, agglomeration, tableting, or any combination of the foregoing.

17. A granulated feed additive composition as claimed in claim 16 wherein the mean diameter of the granules is greater than 50 microns and less than 2000 microns.

18. A feed additive composition according to claim 17 wherein the composition is in liquid form.

19. A feed additive composition according to claim 18 wherein the composition is in liquid form suitable for spray drying on feed pellets.

Technical Field

The art relates to novel trypsin-like serine proteases and uses thereof.

Background

A protease (also known as peptidase or prion) is an enzyme that is capable of cleaving peptide bonds. Proteases have evolved many times and different classes of proteases can carry out the same reaction by completely different catalytic mechanisms. Proteases can be found in animals, plants, fungi, bacteria, archaea and viruses.

Proteolysis can be achieved by enzymes currently classified into the following six major groups: aspartyl proteases, cysteine proteases, serine proteases (such as, for example, subtilisins or trypsin-like proteases), threonine proteases, glutamine proteases and metalloproteases.

Serine proteases belong to the carbonyl hydrolase subgroup, which comprises different classes of enzymes with a wide range of specificities and biological functions. Despite this functional diversity, the catalytic mechanism of serine proteases has approached at least two genetically distinct enzyme families: 1) a subtilisin; and 2) trypsin-like serine proteases (also known as chymotrypsin-related serine proteases). The two families of serine proteases or serine endopeptidases have very similar catalytic mechanisms. The tertiary structure of these two enzyme families brings together a conserved catalytic triad of amino acids consisting of serine, histidine and aspartic acid.

A great deal of research has been carried out on serine proteases, in particular subtilisins, mainly due to their usefulness in industrial applications. Additional work has focused on adverse environmental conditions (e.g., exposure to oxidizing agents, chelating agents, extremes of temperature and/or pH) that may adversely affect the function of these enzymes in various applications.

Accordingly, there is a continuing need to find new serine proteases (e.g., trypsin-like proteases of prokaryotic origin) that can be used under adverse conditions and that retain or have improved proteolytic activity and/or stability.

Disclosure of Invention

In a first embodiment, an isolated polypeptide having serine protease activity is described, the polypeptide being selected from the group consisting of:

a) a polypeptide having an amino acid sequence with at least 91% identity to the amino acid sequence of SEQ ID NO. 22;

b) a polypeptide having an amino acid sequence with at least 94% identity to the amino acid sequence of SEQ ID NO. 23;

c) a polypeptide having an amino acid sequence with at least 98% identity to the amino acid sequence of SEQ ID NO. 24;

d) a polypeptide having an amino acid sequence with at least 80% identity to the amino acid sequence of SEQ ID NO. 25.

In a second embodiment, an isolated polypeptide having serine protease activity and comprising a predicted precursor amino acid sequence selected from the group consisting of: 3, SEQ ID NO; 6, SEQ ID NO; 9, SEQ ID NO; and SEQ ID NO 12.

In a third embodiment, an isolated polypeptide having serine protease activity and comprising a protease catalytic region selected from the group consisting of:

a) an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO. 18;

b) an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO. 19;

c) 20, the amino acid sequence of SEQ ID NO;

d) an amino acid sequence having at least 91% identity to the amino acid sequence of SEQ ID NO. 21;

in a fourth embodiment, a recombinant construct is described comprising a control sequence functional in a production host operably linked to a nucleotide sequence encoding at least one polypeptide having serine protease activity selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence having at least 91% identity to the amino acid sequence of SEQ ID NO. 22;

b) a polypeptide comprising an amino acid sequence having at least 94% identity to the amino acid sequence of SEQ ID NO. 23;

c) a polypeptide comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO. 24;

d) a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO. 25.

The production host may be selected from the group consisting of: fungi, bacteria, and algae.

In a fifth embodiment, a method for producing at least one serine protease is described, the method comprising:

(a) transforming a production host with a recombinant construct as described herein; and

(b) cultivating the production host of step (a) under conditions to produce at least one polypeptide having serine protease activity.

According to the method, optionally at least one polypeptide having a serine protease is recovered from said production host.

In another aspect, a culture supernatant containing a serine protease can be obtained using any of the methods described herein.

In yet another aspect, a recombinant microbial production host for expressing at least one polypeptide having serine protease activity comprises a recombinant construct as described herein.

Further, the production host is selected from the group consisting of: bacteria, fungi and algae.

In a sixth embodiment, an animal feed is described comprising at least one polypeptide having serine protease activity as described herein, wherein the serine protease is present in an amount of 1-20g per ton of feed.

In addition, such animal feed can comprise (a) at least one direct fed microbial, or (b) at least one other enzyme, or (c) at least one direct fed microbial and at least one other enzyme.

In a seventh embodiment, a feed, a feedstuff additive composition, or a premix comprising at least one polypeptide having serine protease activity as described herein is described. Further, the feed, feedstuff, feed additive composition or premix described herein comprises (a) at least one direct-fed microorganism, or (b) at least one other enzyme, or (c) at least one direct-fed microorganism and at least one other enzyme.

In a seventh embodiment, there is a feed additive composition as described herein, wherein the composition further comprises at least one component selected from the group consisting of: proteins, peptides, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methyl paraben, and propyl paraben.

In an eighth embodiment, a granulated feed additive composition for use in animal feed is described, the granulated feed additive composition comprising at least one polypeptide having serine protease activity described herein, wherein the granulated feed additive composition comprises granules produced by a method selected from the group consisting of: high shear granulation, drum granulation, extrusion, spheronization, fluidized bed agglomeration, fluidized bed spraying, spray drying, freeze drying, granulation, spray cooling, rotary disk atomization, agglomeration, tableting, or any combination of the foregoing.

In another embodiment, the particles of the granulated feed additive composition may have an average diameter of more than 50 microns and less than 2000 microns.

In another aspect, the feed additive composition may be in liquid form, and further, in liquid form suitable for spray drying on feed pellets.

Drawings

FIG. 1. plasmid AprE-SspcPrPro 29 for expression of AprE-SspcPPro 29 protease.

FIG. 2 dose-response of the enzymatic activities of the serine proteases SsppCPro 29, SsppCPro 33, and ProAct to AAPF-pNA substrate.

FIG. 3 pH profiles of the serine proteases SspcPro23, SspPro 29, SspPro 33, and SspPro 59.

FIG. 4 temperature profiles for the serine proteases SspcPro23, SspPro 29, SspPro 33, and SspPro 59.

Figure 5a hydrolysis of corn soybean meal by the serine proteases SspCPro29 and SspCPro33 detected by OPA at pH 6.

Figure 5b hydrolysis of corn soybean meal by serine proteases SspCPro29 and SspCPro33 detected by BCA at pH 6.

FIG. 6. cleaning Performance of SspcPro29 and SspPro 33 proteases in GSM-B ADW detergents at pH 10.3.

FIG. 7 cleaning performance of SspcPro29, SspPro 33, and BPN' Y217L protease in liquid laundry detergent at 16 ℃.

FIG. 8 cleaning performance of SspcPro29, SspcPro33, and BPN' Y217L protease in liquid laundry detergent at 32 ℃.

FIG. 9 cleaning performance of SspcPro29, SspPro 33, and GG36 proteases in powder laundry detergents at 16 ℃.

FIG. 10 cleaning performance of SspcPro29, SspPro 33, and GG36 proteases in powder laundry detergents at 32 ℃.

11A-11D multiple sequence alignments of full-length sequences of trypsin-like serine proteases of various Streptomyces species (Streptomyces sp).

FIGS. 12A-12B. multiplex sequence alignment of predicted catalytic core sequences of trypsin-like serine proteases of Streptomyces species.

The following Sequences comply with 37c.f.r. § 1.821-1.825 ("Requirements for Patent Applications relating to Nucleotide Sequences and/or Amino Acid Sequence disorders-the Sequence Rules [ Requirements-Sequence Rules of Patent Applications Containing Nucleotide and/or Amino Acid Sequence publications ]") and comply with the World Intellectual Property Organization (WIPO) standard st.25(2009), and the European Patent Convention (EPC) and Patent cooperation convention (PCT) regulations clauses 5.2 and 49.5(a-bis), and the Requirements of the administrative clause clauses 208 and annex C on the Sequence listing. The symbols and formats used for nucleotide and amino acid sequence data follow the regulations as described in 37c.f.r. § 1.822.

SEQ ID NO 1 lists the nucleotide sequence of the SspcPro29 gene isolated from Streptomyces species C009.

SEQ ID NO 2 lists the predicted signal sequence for the SsppCPro 29 precursor protein.

SEQ ID NO 3 lists the amino acid sequence of the SspcPro29 precursor protein.

SEQ ID NO 4 lists the nucleotide sequence of the SspCPPro 33 gene isolated from Streptomyces species C001.

SEQ ID NO 5 lists the predicted signal sequence for the SsppCPro 33 precursor protein.

SEQ ID NO 6 sets forth the amino acid sequence of the SspcPro33 precursor protein.

SEQ ID NO 7 lists the nucleotide sequence of the SspcPro23 gene isolated from Streptomyces species C003.

SEQ ID NO 8 lists the predicted signal sequence for the SspCPI 23 precursor protein.

SEQ ID NO 9 sets forth the amino acid sequence of the SsppCPro 23 precursor protein.

SEQ ID NO 10 lists the nucleotide sequence of the SspCPPro 59 gene isolated from Streptomyces species C055.

SEQ ID NO 11 lists the predicted signal sequence for the SspCPI 59 precursor protein.

SEQ ID NO. 12 sets forth the amino acid sequence of the SspcPro59 precursor protein.

SEQ ID NO 13 lists the nucleotide sequence of the synthetic AprE-SspCPI 23,

SEQ ID NO. 14 lists the nucleotide sequence of AprE-SspCPI 29,

SEQ ID NO. 15 lists the nucleotide sequence of AprE-SspCPI 33,

the nucleotide sequence of the AprE-SspCPI 59 gene is shown in SEQ ID NO. 16.

SEQ ID NO 17 lists the AprE signal sequence used to direct secretion of recombinant proteins in B.subtilis.

SEQ ID NO 18 lists the predicted catalytic domains for SspCPPro 29.

SEQ ID NO 19 lists the predicted catalytic domains for SspCPPro 23.

SEQ ID NO 20 lists the predicted catalytic domains for SspCPPro 33.

SEQ ID NO 21 lists the predicted catalytic domains for SspCPPro 59.

SEQ ID NO. 22 lists the predicted full-length amino acid sequence for SspCPPro 29.

SEQ ID NO 23 sets forth the predicted full-length amino acid sequence for SspCPPro 33.

SEQ ID NO 24 lists the predicted full-length amino acid sequence for SspCPPro 23.

SEQ ID NO. 25 lists the predicted full-length amino acid sequence for SspCPPro 59.

SEQ ID NO 26 lists the sequence of the serine protease WP _064069271 of the Streptomyces species.

SEQ ID NO 27 lists the sequence of the serine protease WP _043225562 of the Streptomyces species.

SEQ ID NO 28 lists the sequence of the serine protease WP _024756173 of the Streptomyces species.

SEQ ID NO 29 lists the sequence of the serine protease WP _030548298 of the Streptomyces species.

SEQ ID NO 30 lists the sequence of the serine protease WP _005320871 of the Streptomyces species.

SEQ ID NO 31 lists the sequence of the serine protease WP _055639793 of the Streptomyces species.

SEQ ID NO 32 lists the sequences of the Streptomyces species serine protease WO 2015048332-44360.

SEQ ID NO 33 lists the sequences of the Streptomyces species serine protease WO 2015048332-44127.

SEQ ID NO 34 lists the sequence of the serine protease WP _030313004 of the Streptomyces species.

SEQ ID NO 35 lists the sequence of the serine protease WP _030212164 of the Streptomyces species.

SEQ ID NO 36 lists the sequence of the serine protease WP _030749137 of the Streptomyces species.

SEQ ID NO 37 lists the sequence of the serine protease WP _031004112 of the Streptomyces species.

SEQ ID NO 38 lists the sequence of the serine protease WP _026277977 of the Streptomyces species.

SEQ ID NO:39 sets forth the amino acid sequence of the catalytic domain of Streptomyces protease (Streptgrisin) C.

SEQ ID NO 40 lists the amino acid sequence of the BPN' -Y217L protein.

SEQ ID NO 41 lists the amino acid sequence of the GG36 protein.

42 lists the amino acid sequence of residues 204-394 of S.albugus (S _ albulus) WP 064069271.

43 lists the amino acid sequence of residue 204-394 of the S _ sp _ NRRL _ F-5193_ WP _043225562 protein.

The amino acid sequence of residues 201-391 of S.defoliatus (S _ exfoliatus) _ WP _024756173 is shown in SEQ ID NO: 44.

SEQ ID NO:45 lists the amino acid sequence at residue 207-397 of S.albus (S _ albus) _ WP _ 030548298.

SEQ ID NO 46 lists the amino acid sequence of residues 204-394 of Streptomyces pristinaespiralis WP 005320871.

SEQ ID NO:47 lists the amino acid sequence of residue 138-328 of S.lexuwenhoekii (S _ leewenhoekii) _ WP _ 029386953.

SEQ ID NO 48 lists the amino acid sequence at residue 207-397 of Streptomyces species CNT372_ WP _ 026277977.

SEQ ID NO:49 lists the amino acid sequence of residue 208-398 of Streptomyces cyaneogriseus P _ 044383230.

SEQ ID NO:50 lists the amino acid sequence of residue 193-383 of Streptomyces niveus WP _ 069630550.

SEQ ID NO:51 lists the amino acid sequence of residue 201-391 of Streptomyces venezuelae (WP _ 055639793).

SEQ ID NO 52 lists the amino acid sequence of residue 211-401 of Streptomyces species NRRL F-5755_ WP _ 053699044.

SEQ ID NO:53 lists the amino acid sequence of residues 205-395 of Streptomyces fradiae WP 031135572.

SEQ ID NO 54 lists the predicted catalytic domain consensus sequence from figure 12.

Detailed Description

All patents, patent applications, and publications cited are incorporated by reference herein in their entirety.

In this disclosure, a number of terms and abbreviations are used. The following definitions apply unless otherwise specifically indicated.

The articles "a/an" and "the" preceding an element or component are intended to be non-limiting with respect to the number of instances (i.e., occurrences) of the element or component. Thus, "a" and "the" are to be understood as including one or at least one and the singular forms of an element or component also include the plural unless the number clearly dictates otherwise.

The term "comprising" means the presence of the stated features, integers, steps or components as referred to in the claims, but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. The term "comprising" is intended to include embodiments encompassed by the terms "consisting essentially of … …" and "consisting of … …". Similarly, the term "consisting essentially of … …" is intended to include embodiments encompassed by the term "consisting of … …".

Where present, all ranges are inclusive and combinable. For example, when a range of "1 to 5" is recited, the recited range should be interpreted to include ranges of "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like.

As used herein in connection with numerical values, the term "about" refers to a range of +/-0.5 of the numerical value unless the term is otherwise specifically defined in context. For example, the phrase "a pH of about 6" means a pH of from 5.5 to 6.5 unless the pH is otherwise specifically defined.

Every maximum numerical limitation given throughout this specification is intended to include every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

The term "protease" means a protein or polypeptide domain derived from a microorganism (e.g., fungi, bacteria) or derived from a plant or animal, and which has the ability to catalyze the cleavage of peptide bonds at one or more different positions of the protein backbone (e.g., e.c. 3.4). The terms "protease", "peptidase" and "protease" are used interchangeably. Proteases can be found in animals, plants, fungi, bacteria, archaea and viruses. Proteolysis can be achieved by enzymes currently classified into the following six major groups based on their catalytic mechanism: aspartyl proteases, cysteine proteases, trypsin-like serine proteases, threonine proteases, glutamine proteases and metalloproteases.

The term "serine protease" refers to an enzyme that cleaves peptide bonds in proteins, where serine serves as a nucleophilic amino acid at the active site of the enzyme. Serine proteases are classified into two major classes based on their structure: chymotrypsin-like (trypsin-like) and subtilisin. In the MEROPS protease classification system, proteases are distributed among 16 superfamilies and numerous families. Family S8 includes subtilisins and family S1 includes chymotrypsin-like (trypsin-like) enzymes. Subfamily S1E includes trypsin-like serine proteases from Streptomyces organisms, such as Streptomyces proteases (Streptogricin) A, B and C. The terms "serine protease", "trypsin-like serine protease" and "chymotrypsin-like protease" are used interchangeably herein.

The terms "animal" and "subject" are used interchangeably herein. "animal" includes all non-ruminants (including humans) and ruminants. In particular embodiments, the animal is a non-ruminant animal, such as horses and monogastric animals. Examples of monogastric animals include, but are not limited to, pigs (pigs and swine), such as piglets, growing pigs, sows; poultry, such as turkeys, ducks, chickens, broiler chicks, laying hens; fish, such as salmon, trout, tilapia, catfish, and carp; and crustaceans such as shrimp and prawn. In further embodiments, the animal is a ruminant animal including, but not limited to, cattle, calves, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, antelope, pronghorn, and deer antelope.

By "feed" and "food" is meant any natural or artificial diet, meal, etc., or component of such meal, respectively, which is intended or suitable for consumption, ingestion, digestion by non-human animals and humans, respectively.

As used herein, the term "food" is used in a broad sense and encompasses food and food products for humans as well as food (i.e., feed) for non-human animals.

The term "feed" is used in relation to products which are fed to animals when raised in livestock. The terms "feed" and "animal feed" are used interchangeably.

As used herein, the term "direct fed microbial" ("DFM") is a source of live (viable) naturally occurring microorganisms. DFMs may comprise one or more such naturally occurring microorganisms, such as bacterial strains. The categories of DFM include Bacillus (Bacillus), lactic acid bacteria and yeast. Thus, the term DFM encompasses one or more of: direct fed bacteria, direct fed yeast and combinations thereof.

Bacilli (Bacilli) are unique, spore-forming, gram-positive Bacilli. These spores are very stable and can withstand environmental conditions such as heat, moisture and a range of pH. These spores germinate into viable vegetative cells when ingested by the animal and can be used in diets that are kibbled and pressed into pellets. Lactic acid bacteria are gram-positive cocci and produce lactic acid which is antagonistic to pathogens. Since lactic acid bacteria seem to be somewhat heat sensitive, they cannot be used in a diet pressed into pellets. The species of lactic acid bacteria include Bifidobacterium (Bifidobacterium), Lactobacillus (Lactobacillus), and Streptococcus (Streptococcus).

The term "probiotic" means an indigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of beneficial bacteria.

As used herein, the term "probiotic culture" defines a live microorganism (including, for example, bacteria or yeast) that beneficially affects a host organism (i.e., by imparting one or more demonstrable health benefits to the host organism) when, for example, ingested in sufficient quantities or applied topically. The probiotic may improve the microbial balance of one or more mucosal surfaces. For example, the mucosal surface may be the intestine, urinary tract, respiratory tract or skin. As used herein, the term "probiotic" also encompasses live microorganisms that can stimulate the beneficial branches of the immune system while reducing the inflammatory response in mucosal surfaces (e.g., the intestine). Although there is no lower or upper limit for probiotic intake, it has been shown that at least 10⁶-10¹²Preferably toLess than 10⁶-10¹⁰Preferably 10⁸-10⁹cfu as a daily dose will be effective to achieve a beneficial health effect in a subject.

As used herein, the term "CFU" means "colony forming unit" and is a measure of viable cells in a colony representing the aggregation of cells derived from a single progenitor cell.

The term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance, including but not limited to any host cell, enzyme, variant, nucleic acid, protein, peptide, or cofactor, which is at least partially removed from one or more or all of the naturally occurring components with which it is naturally associated; (3) any substance modified by the human hand (relative to substances found in nature); or (4) any substance that is modified by increasing the amount of the substance relative to other ingredients with which it is naturally associated. The terms "isolated nucleic acid molecule," "isolated polynucleotide," and "isolated nucleic acid fragment" will be used interchangeably and refer to a polymer of RNA or DNA that is single-or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, or synthetic DNA.

The term "purified," as applied to a nucleic acid or polypeptide, generally refers to a nucleic acid or polypeptide that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., the purified polypeptide or polynucleotide forms discrete bands in an electrophoretic gel, chromatographic eluate, and/or media that is subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that produces a substantial band in an electrophoretic gel is "purified". The purified nucleic acid or polypeptide is at least about 50% pure, typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or more pure (e.g., percent by weight on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" means that a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component is present in a composition at a relative or absolute concentration that is higher than the starting composition.

As used herein, the term "functional assay" refers to an assay that provides an indication of protein activity. In some embodiments, the term refers to an assay system in which a protein is analyzed for its ability to function in its usual capacity. For example, in the case of proteases, functional assays involve determining the effectiveness of the protease to hydrolyze a protein substrate.

The terms "peptide," "protein," and "polypeptide" are used interchangeably herein and refer to a polymer of amino acids linked together by peptide bonds. A "protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. The single letter and 3-letter codes for amino acids as defined by the Joint Commission on biochemical nomenclature, JCBN, for IUPAC-IUB biochemical terminology are used throughout this disclosure. The single letter X refers to any of the twenty amino acids. It is also understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence. Mutations may be named by the single letter code of the parent amino acid, followed by a position number, and then the single letter code of the variant amino acid. For example, the mutation of glycine (G) to serine (S) at position 87 is denoted as "G087S" or "G87S". When describing modifications, the amino acids listed in parentheses after a position indicate the list of substitutions at that position by any of the listed amino acids. For example, 6(L, I) means that position 6 can be substituted with leucine or isoleucine. Sometimes, in the sequence, a slash (/) is used to define a substitution, e.g., F/V indicates a particular position at which there may be a phenylalanine or valine.

"pro sequence" or "propeptide sequence" refers to an amino acid sequence between the signal peptide sequence and the mature protease sequence that is necessary for proper folding and secretion of the protease; they are sometimes referred to as intramolecular chaperones. Cleavage of the pro sequence or pro peptide sequence yields the mature active protease. Proteases are typically expressed as proenzymes.

The terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that can be involved in the secretion or targeted transport of the mature or precursor form of a protein. Typically, the signal sequence is located at the N-terminus of the precursor or mature protein sequence. The signal sequence may be endogenous or exogenous. The signal sequence is generally absent from the mature protein. Typically, after protein transport, the signal sequence is cleaved from the protein by a signal peptidase.

The term "mature" form of a protein, polypeptide or peptide refers to a functional form of the protein, polypeptide or enzyme that is free of signal peptide sequences and propeptide sequences.

The term "pro" form of a protein or peptide refers to the mature form of the protein having a pre-sequence operatively linked to the amino-or carboxy-terminus of the protein. The precursor may also have a "signal" sequence operatively linked to the amino terminus of the pro sequence. The precursor may also have additional polypeptides involved in post-translational activity (e.g., polypeptides from which cleavage leaves the protein or peptide in a mature form).

With respect to amino acid sequences or nucleic acid sequences, the term "wild-type" indicates that the amino acid sequence or nucleic acid sequence is a native or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance (e.g., protein, amino acid, or nucleic acid sequence) found in nature. In contrast, the term "non-naturally occurring" refers to anything not found in nature (e.g., recombinant nucleic acid and protein sequences produced in the laboratory, or modifications of wild-type sequences).

As used herein, with respect to amino acid residue positions, "corresponding to" (or corresponds to) or "corresponding to" refers to an amino acid residue at a position listed in a protein or peptide, or an amino acid residue that is similar, homologous, or identical to a residue listed in a protein or peptide. As used herein, "corresponding region" generally refers to a similar position in a related protein or a reference protein.

The terms "derived from" and "obtained from" refer not only to proteins produced by or producible by the strain of the organism in question, but also to proteins encoded by DNA sequences isolated from such strains and produced in host organisms containing such DNA sequences. In addition, the term refers to proteins encoded by DNA sequences of synthetic and/or cDNA origin and having the identifying characteristics of the protein in question.

With respect to the polypeptides described herein, the term "reference" refers to a naturally occurring polypeptide that does not include artificial substitutions, insertions, or deletions at one or more amino acid positions, as well as a naturally occurring or synthetic polypeptide that does include one or more artificial substitutions, insertions, or deletions at one or more amino acid positions. Similarly, with respect to polynucleotides, the term "reference" refers to a naturally occurring polynucleotide that does not include artificial substitutions, insertions, or deletions at one or more nucleosides, as well as a naturally occurring or synthetic polynucleotide that does include one or more artificial substitutions, insertions, or deletions at one or more nucleosides. For example, a polynucleotide encoding a wild-type or parent polypeptide is not limited to a naturally occurring polynucleotide, and encompasses any polynucleotide encoding a wild-type or parent polypeptide.

The term "amino acid" refers to the basic chemical building block of a protein or polypeptide. Abbreviations used herein may be found in table 2 to identify particular amino acids.

TABLE 2 Single letter and three letter amino acid abbreviations

One skilled in the art will recognize that modifications can be made to the amino acid sequences disclosed herein while retaining the functionality associated with the disclosed amino acid sequences. For example, it is common for a gene alteration to result in the production of a chemically equivalent amino acid at a given site without affecting the functional properties of the encoded protein, as is well known in the art. For example, any particular amino acid in an amino acid sequence disclosed herein can be substituted for another functionally equivalent amino acid. For the purposes of this disclosure, substitution is defined as an exchange in one of the following five groups:

1. small aliphatic, non-polar or slightly polar residues: ala, Ser, Thr (Pro, Gly);

2. polar, negatively charged residues and their amides: asp, Asn, Glu, Gln;

3. polar, positively charged residues: his, Arg, Lys;

4. large aliphatic, non-polar residues: met, Leu, Ile, Val (Cys); and

5. large aromatic residues: phe, Tyr, and Trp.

Thus, the codon for the amino acid alanine (a hydrophobic amino acid) may be replaced by a codon encoding another less hydrophobic residue (e.g., glycine) or a more hydrophobic residue (e.g., valine, leucine, or isoleucine). Similarly, changes that result in the substitution of one negatively charged residue for another (e.g., thermostable serine for glutamic acid) or one positively charged residue for another (e.g., lysine for arginine) can also be expected to yield functionally equivalent products. In many cases, nucleotide changes that result in changes in the N-terminal and C-terminal portions of a protein molecule will also not be expected to alter the activity of the protein. Each of the proposed modifications is well within the routine skill in the art, such as determining the retention of biological activity of the encoded product.

The term "codon optimized", as it refers to genes or coding regions of nucleic acid molecules used to transform various hosts, refers to codon changes in the genes or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA.

The term "gene" refers to a nucleic acid molecule that expresses a particular protein, including regulatory sequences preceding (5 'non-coding sequences) and following (3' non-coding sequences) the coding sequence. "native gene" refers to a gene found in nature with its own regulatory sequences. "chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Thus, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different than that which occurs in nature. "endogenous gene" refers to a native gene that is located in a native location in the genome of an organism. A "foreign" gene refers to a gene that is not normally found in the host organism, but is introduced into the host organism by gene transfer. The foreign gene may comprise a native gene or a chimeric gene inserted into a non-native organism. A "transgene" is a gene that is introduced into the genome by a transformation procedure.

The term "coding sequence" refers to a nucleotide sequence that encodes a specific amino acid sequence. "suitable regulatory sequences" refer to nucleotide sequences located upstream (5 'non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which affect transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, RNA processing sites, effector binding sites, and stem-loop structures.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid molecule such that the function of one nucleic acid fragment is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). The coding sequence may be operably linked to regulatory sequences in sense or antisense orientation.

The terms "regulatory sequence" or "control sequence" are used interchangeably herein and refer to a segment of a nucleotide sequence that is capable of increasing or decreasing the expression of a particular gene in an organism. Examples of regulatory sequences include, but are not limited to, promoters, signal sequences, operators, and the like. As noted above, the regulatory sequences may be operably linked to the coding sequence/gene of interest in either sense or antisense orientation.

"promoter" or "promoter sequence" refers to a DNA sequence that defines where RNA polymerase begins gene transcription. Promoter sequences are usually located directly upstream or at the 5' end of the transcription start site. Promoters may be derived in their entirety from a natural or naturally occurring sequence, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It will be appreciated by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions ("inducible promoters").

The "3' non-coding sequence" refers to a DNA sequence located downstream of a coding sequence and includes sequences that encode regulatory signals capable of affecting mRNA processing or gene expression (e.g., transcription termination).

As used herein, the term "transformation" refers to the transfer or introduction of a nucleic acid molecule into a host organism. The nucleic acid molecule may be introduced as a linear or circular form of DNA. The nucleic acid molecule may be an autonomously replicating plasmid, or it may be integrated into the genome of the production host. A production host containing a transformed nucleic acid is referred to as a "transformed" or "recombinant" or "transgenic" organism or "transformant".

As used herein, the term "recombinant" refers to the artificial combination of two otherwise isolated nucleic acid sequence segments, e.g., by chemical synthesis or by manipulating the isolated nucleic acid segments via genetic engineering techniques. For example, DNA in which one or more segments or genes have been inserted, either naturally or by laboratory manipulation, from a different molecule, another part of the same molecule or an artificial sequence, results in the introduction of a new sequence in the gene and subsequently in the organism. The terms "recombinant," "transgenic," "transformed," "engineered," or "exogenous gene expression modification" are used interchangeably herein.

The terms "recombinant construct", "expression construct", "recombinant expression construct" and "expression cassette" are used interchangeably herein. Recombinant constructs comprise nucleic acid fragments, such as artificial combinations of regulatory and coding sequences not all found together in nature. For example, a construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different than that which occurs in nature. Such a construct may be used alone or may be used in combination with a vector. If a vector is used, the choice of vector will depend on the method to be used to transform the host cell, as is well known to those skilled in the art. For example, plasmid vectors can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate the host cell. The skilled artisan will also recognize that different independent transformation events may result in different levels and patterns of expression (Jones et al, (1985) EMBO J [ J. European society of molecular biology ]4: 2411-2418; De Almeida et al, (1989) Mol Gen Genetics [ molecular and general Genetics ]218:78-86), and that multiple events are therefore typically screened to obtain lines exhibiting the desired levels and patterns of expression. Such screening may be accomplished by standard molecular biology assays, biochemical assays, and other assays including blot analysis of DNA, Northern analysis of mRNA expression, PCR, real-time quantitative PCR (qpcr), reverse transcription PCR (RT-PCR), immunoblot analysis of protein expression, enzymatic or activity assays, and/or phenotypic analysis.

The terms "production host", "host" and "host cell" are used interchangeably herein and refer to any organism or cell thereof, whether human or non-human, into which a recombinant construct may be stably or transiently introduced to express a gene. The term encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during propagation.

The term "percent identity" is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences as determined by comparing these sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the number of matching nucleotides or amino acids between strings of such sequences. "identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in the following documents: computational Molecular Biology [ Computational Molecular Biology ] (Lesk, A.M. ed.) Oxford University Press [ Oxford University Press ], New York, State (1988); biocontrol information and Genome Projects [ biological: informatics and genomic projects ] (Smith, d.w. eds.), Academic Press [ Academic Press ], new york, 1993; computer Analysis of Sequence Data, section I, (Griffin, A.M. and Griffin, edited by H.G.) Humana Press, Humata Press, New Jersey (1994); sequence Analysis in Molecular Biology [ Sequence Analysis in Molecular Biology ] (von Heinje, g. eds.), Academic Press [ Academic Press ] (1987); sequence Analysis Primer (Gribskov, M. and Devereux, J. eds.) Stockton Press [ Stockton Press ], N.Y. (1991). Methods of determining identity and similarity are programmed into publicly available computer programs.

As used herein, "% identity" or "percent identity" or "PID" refers to protein sequence identity. Percent identity can be determined using standard techniques known in the art. Useful algorithms include the BLAST algorithm (see Altschul et al, J Mol Biol [ J. Mol. Biol., 215:403 + 410, 1990; and Karlin and Altschul, Proc Natl Acad Sci USA [ Proc. Natl. Acad. Sci., USA ], 90:5873 + 5787, 1993). The BLAST program uses several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most relevant sequences in terms of biological similarity, but is not recommended for query sequences of less than 20 residues (Altschul et al, Nucleic Acids SRs [ Nucleic Acids Res., 25: 3389. sup. 3402, 1997; and Schafer et al, Nucleic Acids Res. [ Nucleic Acids Res. ]29: 2994. sup. 3005, 2001). Exemplary default BLAST parameters for nucleic acid sequence searches include: the adjacent word length threshold is 11; e-value cutoff is 10; score matrix-nuc.3.1 (match-1, mismatch-3); vacancy opening is 5; and a vacancy extension of 2. Exemplary default BLAST parameters for amino acid sequence searches include: the word length is 3; e-value cutoff is 10; score matrix BLOSUM 62; vacancy opening is 11; and a vacancy extension of 1. Percent (%) amino acid sequence identity values are determined by dividing the number of matching identical residues by the total number of residues in the "reference" sequence, including any gaps created by the program for optimal/maximum alignment. The BLAST algorithm refers to "reference" sequences as "query" sequences.

As used herein, "homologous protein" or "homologous protease" refers to proteins having different similarities in primary, secondary and/or tertiary structure. Protein homology may refer to the similarity of linear amino acid sequences when aligning proteins. Homology searches for protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST using a threshold (E-value cut-off) of 0.001. (Altschul SF, Madde TL, Shaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. gapped BLAST and PSI BLAST a new generation of protein database programs) [ gap BLAST and PSI BLAST: New Generation protein database search program ] Nucleic Acids Res 1997 group 1; 25(17): 3389-. Using this information, protein sequences can be grouped. The amino acid sequence can be used to construct phylogenetic trees.

Sequence alignments and percent identity calculations can be performed using the Megalign program of the LASERGENE bioinformation calculation package (DNASTAR, Madison, Wis.), the AlignX program of Vector NTI v.7.0 (Informatx, Besserda, Md.), or the EMBOSS open software package (EMBL-EBI; Rice et al, Trends in Genetics 16, (6): 276-. Multiple alignments of sequences can be performed using CLUSTAL alignment methods with default parameters (e.g., CLUSTALW; e.g., version 1.83) (Higgins and Sharp, CABIOS, 5: 151-. Suitable parameters for CLUSTALW protein alignments include a gap existence penalty of 15, a gap extension of 0.2, a matrix of Gonnet (e.g., Gonnet250), a protein end gap (ENDGAP) -1, a protein gap distance (gapist) -4, and KTUPLE-1. In one embodiment, in the case of slow alignment, fast or slow alignment is used, along with default settings. Alternatively, parameters using the CLUSTALW method (e.g., version 1.83) may be modified to also use KTUPLE ═ 1, gap penalty ═ 10, gap extension ═ 1, matrix ═ BLOSUM (e.g., BLOSUM64), WINDOW (WINDOW) ═ 5, and TOP stored diagonal (TOP DIAGONALS SAVED) ═ 5.

The MUSCLE program (Robert C.Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput [ MUSCLE: multiple sequence alignment with high accuracy ], nucleic acids Res. [ nucleic acids research ] (2004)32(5): 1792-.

Various polypeptide amino acid sequences and polynucleotide sequences are disclosed herein. Variants of these sequences having at least about 70% -85%, 85% -90%, or 90% -95% identity to the sequences disclosed herein may be used in certain embodiments. Alternatively, a variant polypeptide sequence or polynucleotide sequence in certain embodiments may be at least 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%, or 99% identical to a sequence disclosed herein. The variant amino acid sequence or polynucleotide sequence has the same function as the disclosed sequence, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function of the disclosed sequence.

With respect to polypeptides, the term "variant" refers to a polypeptide that differs from a designated wild-type, parent or reference polypeptide in that it includes one or more naturally occurring or artificial amino acid substitutions, insertions, or deletions. Similarly, with respect to polynucleotides, the term "variant" refers to a polynucleotide that differs in nucleotide sequence from the specified wild-type, parent or reference polynucleotide. The identity of the wild-type, parent or reference polypeptide or polynucleotide will be apparent from the context.

The terms "plasmid", "vector" and "cassette" mean an extrachromosomal element, which typically carries a gene that is not part of the central metabolism of the cell, and is typically in the form of double-stranded DNA. Such elements may be autonomously replicating sequences, genome integrating sequences, phage, or nucleotide sequences in linear or circular form derived from any source, single-or double-stranded DNA or RNA, in which a number of nucleotide sequences have been linked or recombined into a unique configuration capable of introducing a polynucleotide of interest into a cell. "transformation cassette" refers to a particular vector that contains a gene and has elements other than the gene that facilitate transformation of a particular host cell. The terms "expression cassette" and "expression vector" are used interchangeably herein and refer to a specific vector that contains a gene and has elements other than the gene that allow expression of the gene in a host.

As used herein, the term "expression" refers to the production of a functional end product (e.g., mRNA or protein) in either a precursor or mature form. Expression may also refer to translation of mRNA into a polypeptide.

Expression of a gene involves transcription of the gene and translation of the mRNA into a precursor or mature protein. "mature" protein refers to a post-translationally processed polypeptide; i.e. a polypeptide from which any propeptide (propeptide) or propeptide (propeptide) present in the primary translation product has been removed. "precursor" protein refers to the primary product of translation of mRNA; i.e. with the pro and pro peptides still present. The propeptide or propeptide may be, but is not limited to, an intracellular localization signal. "Stable transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, including the genome of the nucleus and organelles, resulting in genetically stable inheritance. In contrast, "transient transformation" refers to the transfer of a nucleic acid fragment into the nucleus of a host organism or into a DNA-containing organelle, resulting in gene expression without integration or stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms.

The expression vector may be one of any number of vectors or cassettes used to transform suitable production hosts known in the art. Typically, the vector or cassette will include sequences that direct the transcription and translation of the gene of interest, a selectable marker, and sequences that permit autonomous replication or chromosomal integration. Suitable vectors typically include a 5 'region containing the gene for transcriptional initiation control and a 3' region containing the DNA segment for transcriptional termination control. Both control regions may be derived from genes homologous to genes of the transformed production host cell and/or genes native to the production host, although such control regions need not be so derived.

The possible initiation control regions or promoters that may be included in an expression vector are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable, including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (for expression in saccharomyces); AOX1 (for expression in Pichia (Pichia)); and lac, araB, tet, trp, lP_L、lP_RT7, tac, and trc (for expression in Escherichia coli), as well as the amy, apr, npr promoters and various phage promoters for expression in bacillus. In some embodiments, the promoter is a constitutive or inducible promoter. A "constitutive promoter" is a promoter that is active under most environmental and developmental conditions. An "inducible" or "repressible" promoter refers to a promoter that is active under environmental or developmental regulation. In some embodiments, the promoter is inducible or repressible due to a change in environmental factors including, but not limited to, carbon, nitrogen or other available nutrients, temperature, pH, osmotic pressure, presence of one or more heavy metals, concentration of inhibitors, stress, or a combination thereof, as is known in the art. In some embodiments, the inducible or repressible promoter is induced or inhibited by a metabolic factor, e.g., the level of a certain carbon source, the level of a certain energy source, the level of a certain catabolite, or a combination thereofCombinations, as known in the art. In one embodiment, the promoter is native to the host cell. For example, when trichoderma reesei (t. reesei) is the host, the promoter is a native trichoderma reesei promoter, such as the cbh1 promoter, deposited under accession number D86235 in GenBank.

Non-limiting examples of suitable promoters include cbh1, cbh2, egl1, egl2, egl3, egl4, egl5, xyn1, and xyn2, the repressible acid phosphatase Gene (phoA) promoter of Penicillium chrysogenum (P.chrysogenum) (see, e.g., Graessle et al, (1997) appl. environ. Microbiol. [ application and environmental microbiology ],63: 753-. Examples of other useful promoters include the promoters from the genes for Aspergillus awamori (A.awamori) and Aspergillus niger (A.niger) glucoamylase (see Nunberg et al, (1984) mol.cell Biol. [ molecular and cellular biology ] 154: 2306-2315 and Boel et al, (1984) EMBO J. [ J. European society of molecular biology ]3: 1581-1585). Furthermore, the promoter of the trichoderma reesei xln1 gene may be useful (see, e.g., EPA 137280 Al).

The DNA segment controlling the termination of transcription may also be derived from various genes native to the preferred production host cell. In certain embodiments, the inclusion of a termination control region is optional. In certain embodiments, the expression vector includes a termination control region derived from a preferred host cell.

The expression vector may be comprised in the production host, in particular in a cell of a microbial production host. The production host cell may be a microbial host found in a fungal or bacterial family and which grows under a wide range of temperatures, pH values and solvent tolerance. For example, it is contemplated that any of bacteria, algae, and fungi (e.g., filamentous fungi and yeast) may suitably contain the expression vector.

The inclusion of the expression vector in the production host cell may be used to express the protein of interest such that it may be present intracellularly, extracellularly, or a combination of intracellularly and extracellularly. Extracellular expression makes it easier to recover the desired protein from the fermentation product than the method used to recover the protein produced by intracellular expression.

Certain embodiments of the present disclosure relate to isolated polypeptides having serine protease activity selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence having at least 91% identity to the amino acid sequence of SEQ ID NO. 22;

b) a polypeptide comprising an amino acid sequence having at least 94% identity to the amino acid sequence of SEQ ID NO. 23;

c) a polypeptide comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO. 24;

d) a polypeptide having an amino acid sequence with at least 80% identity to the amino acid sequence of SEQ ID NO. 25.

In another embodiment, an isolated polypeptide having serine protease activity and comprising a predicted precursor amino acid sequence selected from the group consisting of: 3, SEQ ID NO; 6, SEQ ID NO; 9 is SEQ ID NO; and SEQ ID NO 12.

In yet another embodiment, an isolated polypeptide having serine protease activity and comprising a protease catalytic region selected from the group consisting of:

a) an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO. 18;

b) an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO. 19;

c) 20, the amino acid sequence of SEQ ID NO;

d) amino acid sequence having at least 91% identity to the amino acid sequence of SEQ ID NO 21

Other embodiments include a recombinant construct comprising a control sequence functional in a production host operably linked to a nucleotide sequence encoding at least one polypeptide having serine protease activity selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence having at least 91% identity to the amino acid sequence of SEQ ID NO. 22;

b) a polypeptide comprising an amino acid sequence having at least 94% identity to the amino acid sequence of SEQ ID NO. 23;

c) a polypeptide comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO. 24;

d) a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO. 25.

The production host is selected from the group consisting of: fungi, bacteria and algae. The production host may be used in a method for producing at least one polypeptide having serine protease activity, the method comprising:

(a) transforming a production host with a recombinant construct as described herein; and

(b) cultivating the production host of step (a) under conditions to produce at least one polypeptide having serine protease activity.

According to the method, optionally from the production host recovery of at least one polypeptide. In another aspect, the culture supernatant containing the serine protease is obtained by using any of the methods described herein.

Also described herein are recombinant microbial production hosts for expressing at least one polypeptide described herein, comprising a recombinant construct described herein. In another embodiment, the recombinant microbial production host is selected from the group consisting of: bacteria, fungi and algae.

Expression will be understood to include any step of producing at least one polypeptide described herein, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Techniques for modifying nucleic acid sequences using cloning methods are well known in the art.

Polynucleotides encoding trypsin-like serine proteases can be manipulated in a variety of ways to provide for expression of the polynucleotides in a bacillus host cell. Manipulation of the polynucleotide sequence prior to insertion into a nucleic acid construct or vector may be desirable or necessary depending on the nucleic acid construct or vector or the Bacillus host cell. Techniques for modifying nucleotide sequences using cloning methods are well known in the art.

Regulatory sequences are defined above. They include all components necessary or advantageous for the expression of trypsin-like serine proteases. Each control sequence may be native or foreign to the nucleic acid sequence encoding the trypsin-like serine protease. Such regulatory sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal sequence, and transcription terminator. Control sequences with linkers may be provided to introduce specific restriction sites to facilitate ligation of the control sequences with the coding region of the nucleotide sequence encoding a trypsin-like serine protease.

A nucleic acid construct comprising a polynucleotide encoding a trypsin-like serine protease may be operably linked to one or more control sequences capable of directing the expression of the coding sequence in a bacillus host cell under conditions compatible with the control sequences.

Each control sequence may be native or foreign to the polynucleic acid encoding the trypsin-like serine protease. Such control sequences include, but are not limited to, a leader sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. Control sequences with linkers may be provided to introduce specific restriction sites to facilitate ligation of the control sequences with the coding region of the polynucleotide encoding a trypsin-like serine protease.

The control sequence may be an appropriate promoter region, a nucleotide sequence recognized by a Bacillus host cell for expression of a polynucleotide encoding a trypsin-like serine protease. The promoter region contains transcriptional control sequences that mediate the expression of trypsin-like serine proteases. The promoter region may be any nucleotide sequence that shows transcriptional activity in the Bacillus host cell of choice and may be obtained from genes homologous or heterologous to the Bacillus host cell that direct the synthesis of extracellular or intracellular polypeptides having biological activity.

The promoter region may comprise a single promoter or a combination of promoters. When the promoter regions comprise a combination of promoters, the promoters are preferably linked in series. The promoter of the promoter region may be any promoter that can initiate transcription of a polynucleotide encoding a polypeptide having biological activity in a bacillus host cell of interest. The promoter may be native, foreign, or a combination thereof, relative to the nucleotide sequence encoding the polypeptide having biological activity. Such promoters may be obtained from genes homologous or heterologous to the Bacillus host cell that direct the synthesis of biologically active extracellular or intracellular polypeptides.

Thus, in certain embodiments, the promoter region comprises a promoter obtained from a bacterial source. In other embodiments, the promoter region comprises a promoter obtained from a gram-positive or gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Streptococcus, Streptomyces, Staphylococcus (Staphylococcus), Enterococcus (Enterococcus), Lactobacillus, Lactococcus (Lactococcus), Clostridium (Clostridium), Geobacillus (Geobacillus), and Bacillus marinus (Oceanobacillus). Gram-negative bacteria include, but are not limited to, Escherichia coli (E.coli), Pseudomonas (Pseudomonas), Salmonella (Salmonella), Campylobacter (Campylobacter), Helicobacter (Helicobacter), Flavobacterium (Flavobacterium), Clostridium (Fusobacterium), Corynebacterium (Ilyobacter), Neisseria (Neisseria), and Ureabasma (Ureabasma).

The promoter region may comprise a promoter obtained from a Bacillus strain (e.g., Bacillus mucilaginosus (Bacillus agaradherens), Bacillus alkalophilus (Bacillus alkalophilus), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus brevis (Bacillus brevis), Bacillus circulans (Bacillus circulans), Bacillus clausii (Bacillus clausii), Bacillus coagulans (Bacillus coemululans), Bacillus firmus (Bacillus firmus), Bacillus lautus (Bacillus lautus), Bacillus lentus (Bacillus lentus), Bacillus licheniformis (Bacillus licheniformis), Bacillus megaterium (Bacillus megaterium), Bacillus pumilus (Bacillus thermophilus), Bacillus stearothermophilus (Bacillus thermophilus), Bacillus subtilis (Bacillus subtilis), or Bacillus thuringiensis (Bacillus thuringiensis)); or from a Streptomyces strain (e.g., Streptomyces lividans or Streptomyces murinus).

Examples of suitable promoters for directing transcription of polynucleotides encoding polypeptides having biological activity in the methods of the present disclosure are promoters obtained from: coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus lentus or Bacillus clausii alkaline protease gene (aprH), Bacillus licheniformis alkaline protease gene (subtilisin Carlsberg), Bacillus subtilis levan sucrase gene (sacB), Bacillus subtilis alpha-amylase gene (amyE), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis Walkmeria CryIIIA gene (cryIIIA) or parts thereof, prokaryotic beta-lactamase gene (Villa-Kamaroff et al, 1978, Proceedings of the Natal Acemency of the American Academy of Sciences USA 3727, USA 31, And the Bacillus megaterium xylA Gene (Rygus and Hillen,1992, J.Bacteriol. [ J.Bacteriol ]174: 3049-3055; Kim et al, 1996, Gene [ Gene ]181: 71-76). Other examples are the promoter of the spo1 bacteriophage and the promoter of the tac promoter (DeBoer et al, 1983, Proceedings of the national academy of Sciences USA [ Proc. Natl. Acad. Sci. USA ]80: 21-25). Additional promoters are described in "Useful proteins from recombinant bacteria" in Scientific American [ Scientific Americans ], 1980,242: 74-94; and Sambrook, Fritsch and Maniatis,1989, Molecular Cloning, A Laboratory Manual, 2 nd edition, Cold spring harbor, N.Y..

The promoter region may comprise a promoter that is a "consensus" promoter having the sequence TTGACA for the "-35" region and TATAAT for the "-10" region. The consensus promoter may be obtained from any promoter that can function in a Bacillus host cell. Construction of a "consensus" promoter can be accomplished by site-directed mutagenesis using methods well known in the art to generate a promoter that more perfectly conforms to the established consensus sequence for the "-10" and "-35" regions of the vegetative "σ A-type" promoter of B.subtilis (Voskuil et al, 1995, Molecular Microbiology [ Molecular Microbiology ]17: 271-279).

The control sequence may also be a suitable transcription terminator sequence, such as a sequence recognized by a Bacillus host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the trypsin-like serine protease. Any terminator which is functional in a Bacillus host cell may be used.

The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the Bacillus host cell. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence and directs the synthesis of a biologically active polypeptide. Any leader sequence which is functional in the Bacillus host cell of choice may be used in the present invention.

The control sequence may also be an mRNA stabilizing sequence. The term "mRNA stabilizing sequence" is defined herein as a sequence located downstream of a promoter region operably linked to allow processing of all mRNA synthesized from the promoter region to produce an mRNA transcript having a stabilizing sequence at the 5' end of the transcript and upstream of the polynucleotide coding sequence encoding the trypsin-like serine protease. For example, the presence of such stabilizing sequences at the 5' end of mRNA transcripts increases their half-life (Agaisse and Lereclus,1994, supra, Hue et al, 1995, Journal of Bacteriology 177: 3465-. The mRNA processing/stabilizing sequence is complementary to the 3' end of bacterial 16S ribosomal RNA. In certain embodiments, the mRNA processing/stabilizing sequence produces a transcript of substantially a single size with the stabilizing sequence at the 5' end of the transcript. The mRNA processing/stabilizing sequence is preferably complementary to the 3' end of the bacterial 16S ribosomal RNA. See, U.S. Pat. No. 6,255,076 and U.S. Pat. No. 5,955,310.

The nucleic acid construct can then be introduced into a bacillus host cell for introduction and expression of the trypsin-like serine protease using methods known in the art or those described herein.

Nucleic acid constructs comprising a DNA of interest encoding a protein of interest can also be similarly constructed as described above.

To achieve secretion of the protein of interest from the introduced DNA, the control sequence may also include a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of the polypeptide that can direct the expressed polypeptide into the cell's secretory pathway. The signal peptide coding region may be native to the polypeptide or may be obtained from a foreign source. The 5' end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region which is foreign to the portion of the coding sequence which encodes the secreted polypeptide. In the case where the coding sequence does not normally contain a signal peptide coding region, a foreign signal peptide coding region may be required. Alternatively, the foreign signal peptide coding region may simply replace the native signal peptide coding region in order to obtain enhanced secretion of the polypeptide relative to the native signal peptide coding region normally associated with the coding sequence. The signal peptide coding region may be obtained from an amylase or protease gene of a Bacillus species. However, any signal peptide coding region capable of directing the expressed polypeptide into the secretory pathway of a Bacillus host cell of choice may be used in the present invention.

An effective signal peptide coding region for use in a Bacillus host cell is the signal peptide coding region obtained from: the maltogenic amylase gene from Bacillus NCIB 11837, the Bacillus stearothermophilus alpha-amylase gene, the Bacillus licheniformis subtilisin gene, the Bacillus licheniformis beta-lactamase gene, the Bacillus stearothermophilus neutral proteases genes (nprT, nprS, nprM), and the Bacillus subtilis prsA gene.

Thus, a polynucleotide construct comprising a nucleic acid encoding a trypsin-like serine protease construct comprising a nucleic acid encoding a polypeptide of interest (POI) can be constructed such that the host cell expresses the polypeptide of interest. Due to the known degeneracy in the genetic code, different polynucleotides encoding the same amino acid sequence can be designed and prepared using techniques that are routine in the art. For example, codon optimization may be applied to optimize production in a particular host cell.

The nucleic acid encoding the protein of interest can be incorporated into a vector, which can be transferred to a host cell using well-known transformation techniques, such as those disclosed herein.

The vector may be any vector that can be transformed into a host cell and replicated in the host cell. For example, a vector comprising a nucleic acid encoding a POI can be transformed and replicated in a bacterial host cell as a means of propagating and amplifying the vector. The vector may also be transformed into a bacillus expression host of the present disclosure such that the nucleic acid encoding the protein (e.g., ORF) may be expressed as a functional protein.

A representative vector that can be modified by conventional techniques to contain and express a nucleic acid encoding a POI is the vector p2JM103 BBI.

The polynucleotide encoding the trypsin-like serine protease or POI may be operably linked to a suitable promoter that allows transcription in the host cell. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Means for assessing promoter activity/strength are routine to those skilled in the art.

Examples of suitable promoters for directing transcription of the polynucleotide sequences encoding the comS1 polypeptides or POIs of the present disclosure (particularly in bacterial host cells) include the promoter of the lactose operon of E.coli, the Streptomyces coelicolor agarase gene dagA or celA promoter, the promoter of the Bacillus licheniformis alpha-amylase gene (amyL), the promoter of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoter of the Bacillus amyloliquefaciens alpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes, and the like.

The promoter used to direct transcription of the polynucleotide sequence encoding the POI can be the wild-type aprE promoter, a mutant aprE promoter, or a consensus aprE promoter as set forth in PCT International publication WO 2001/51643. In certain other embodiments, the promoter used to direct transcription of the polynucleotide sequence encoding the POI is a wild-type spoVG promoter, a mutant spoVG promoter, or a consensus spoVG promoter (Frisby and Zuber, 1991).

The promoter used to direct transcription of the polynucleotide sequence encoding the trypsin-like serine protease or POI is a ribosomal promoter, such as a ribosomal RNA promoter or a ribosomal protein promoter. The ribosomal RNA promoter may be an rrn promoter derived from bacillus subtilis, more specifically, the rrn promoter may be an rrnB, rrnI or rrnE ribosomal promoter from bacillus subtilis. In certain embodiments, the ribosomal RNA promoter is the P2 rrnI promoter from bacillus subtilis as described in PCT international publication No. WO 2013/086219.

Suitable vectors may further comprise nucleic acid sequences enabling the vector to replicate in a host cell. Examples of such energized sequences include the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1, pIJ702, and the like.

Suitable vectors may also comprise a selectable marker, e.g., a gene whose product complements a defect in the isolated host cell, such as the dal genes from B.subtilis or B.licheniformis, or a gene that confers antibiotic resistance (e.g., ampicillin resistance, kanamycin resistance, chloramphenicol resistance, tetracycline resistance, etc.).

Suitable expression vectors typically include components of a cloning vector, such as, for example, elements that permit autonomous replication of the vector in a selected host organism and one or more phenotypically detectable markers for selection purposes. Expression vectors typically also contain control nucleotide sequences such as, for example, promoters, operators, ribosome binding sites, translation initiation signals, and optionally repressor genes, one or more activator gene sequences, or the like.

In addition, suitable expression vectors may further comprise sequences encoding amino acid sequences capable of targeting the protein of interest to a host cell organelle (e.g., peroxisome) or to a particular host cell compartment. Such targeting sequences may be, for example, the amino acid sequence "SKL". For expression under the direction of a control sequence, the nucleic acid sequence of the protein of interest is operatively linked to the control sequence in a suitable manner such that expression occurs.

Protocols for ligating DNA constructs, promoters, terminators and/or other elements encoding a protein of interest as described herein and inserting them into suitable vectors containing the information necessary for replication are well known to those skilled in the art.

An isolated cell comprising the polynucleotide construct or expression vector is advantageously used as a host cell for recombinant production of the POI. The cell may be transformed with the DNA construct encoding the POI, conveniently by integrating the construct (in one or more copies) into the host chromosome. Integration is generally considered to be advantageous because the DNA sequence so introduced is more likely to be stably maintained in the cell. Integration of the DNA construct into the host chromosome may be carried out using conventional methods, for example by homologous or heterologous recombination. For example, PCT International publication No. WO 2002/14490 describes methods for transformation of Bacillus, transformants thereof, and libraries thereof. Alternatively, the cells may be transformed with expression vectors as described above in connection with different types of host cells.

In other embodiments, it may be advantageous to delete a gene from an expression host, wherein the gene defect can be cured by the expression vector. Known methods can be used to obtain bacterial host cells having one or more inactivated genes. Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation, or by any other means that renders the gene inoperative for its intended purpose such that the gene is prevented from expressing a functional protein.

Techniques for transforming bacteria and culturing bacteria are standard and well known in the art. They may be used to transform the improved hosts of the present invention to produce recombinant proteins of interest. Introduction of a DNA construct or vector into a host cell includes techniques such as: transformation; electroporation; nuclear microinjection; transduction; transfection, such as lipid-mediated transfection and DEAE-dextrin-mediated transfection; incubation with calcium phosphate DNA pellet; bombarding with DNA coated particles at high speed; gene gun (gene gun) or biolistic transformation and protoplast fusion, etc. Brigidi et al also disclose methods for transformation and expression of bacteria (1990). Ferrari et al (U.S. Pat. No. 5,264,366) describe a general transformation and expression scheme for protease deficient Bacillus strains.

Methods for converting nucleic acids into filamentous fungi (e.g., Aspergillus spp), such as Aspergillus oryzae (a. oryzae) or Aspergillus niger (a. niger), humicola grisea (h.grisea), humicola insolens (h.insolens), and trichoderma reesei, are well known in the art. Suitable procedures for transforming an aspergillus host cell are described in, for example, EP 238023. Suitable procedures for transforming Trichoderma host cells are described, for example, in Steiger et al, 2011, appl.environ.Microbiol. [ applied and environmental microbiology ]77: 114-.

The choice of production host may be any suitable microorganism, such as bacteria, fungi and algae.

Typically, the choice will vary depending on the gene encoding the trypsin-like serine protease and its source.

Introduction of the DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection (e.g., lipofection-mediated and DEAE-dextran-mediated transfection); incubating with calcium phosphate DNA precipitate; bombarding with DNA coated particles at high speed; and protoplast fusion. Basic documents disclosing general methods that may be used include Sambrook et al, Molecular Cloning, a Laboratory Manual [ Molecular Cloning: a laboratory manual ] (2 nd edition, 1989); a Laboratory Manual [ Gene Transfer and Expression: a laboratory manual ] (1990); and Ausubel et al, Current Protocols in Molecular Biology (modern methods in Molecular Biology) (1994)). The methods of transformation of the present invention may result in the stable integration of all or a portion of the transformation vector into the genome of a host cell (e.g., a filamentous fungal host cell). However, transformation of extrachromosomal transformation vectors that result in maintenance of autonomous replication is also contemplated.

Many standard transfection methods can be used to generate bacterial and filamentous fungal (e.g., Aspergillus or Trichoderma) cell lines that express large amounts of protease. Some disclosed methods for introducing a DNA construct into a cellulase producing strain of trichoderma include: lorito, Hayes, DiPietro and Harman, (1993) Curr. Genet. [ modern genetics ]24: 349-356; goldman, VanMettagu and Herrera-Estralla, (1990) Curr. Genet. [ modern genetics ]17: 169-; and Penttila, Nevalainen, Ratto, Salminen and Knowles, (1987) Gene [ Gene ]6:155-164, see also USP 6.022,725; USP 6,268,328 and Nevalainen et al, "The Molecular Biology of Trichoderma and applications thereof to The Expression of Both homologus and heterologous Genes [ Molecular Biology of Trichoderma and its use in Homologous and heterologous gene Expression ]", in: molecular Industrial Mycology, eds, Leong and Berka, Markel Dekker Inc. (Marcel Dekker Inc.), N.Y. (1992), p.129-; for Aspergillus including Yelton, Hamer and Timberlake, (1984) Proc. Natl. Acad. Sci. USA [ Proc. Sci. USA ]81: 1470-: a laboratory Manual, [ John Inneski Council ], Novich, UK and Fernandez-Abalos et al, Microbiol [ microbiology ]149: 1623-.

However, any well-known procedure for introducing an exogenous nucleotide sequence into a host cell may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistic methods, liposomes, microinjection, protoplast vectors, viral vectors, and any other well known method for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell (see, e.g., Sambrook et al, supra). Also used is the Agrobacterium-mediated transfection method described in U.S. Pat. No. 6,255,115. It is only necessary to use specific genetic engineering procedures that can successfully introduce at least one gene into a host cell capable of expressing said gene.

After introduction of the expression vector into the cell, the transfected or transformed cell is cultured under conditions conducive to expression of the gene under the control of the promoter sequence.

The medium used to culture the cells can be any conventional medium suitable for growing the host cells and obtaining expression of the alpha-glucosidase polypeptide. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).

Thermostable serine polypeptides secreted from the host cell (with minimal post-production processing) can be used as whole broth formulations.

Depending on the host cell used, post-transcriptional and/or post-translational modifications may be made. One non-limiting example of a post-transcriptional and/or post-translational modification is "clipping" or "truncation" of a polypeptide. This may, for example, result in the trypsin-like serine protease being brought from an inactive or substantially inactive state to an active state, as is the case for the propeptide after further post-translational processing to the enzymatically active mature peptide. In another instance, the cleavage can result in obtaining a mature thermostable serine protease polypeptide and further removing the N-terminal or C-terminal amino acid to produce a truncated form of the thermostable serine protease that retains enzymatic activity.

Other examples of post-transcriptional or post-translational modifications include, but are not limited to, myristoylation, glycosylation, truncation, lipidation, and tyrosine, serine, or threonine phosphorylation. One skilled in the art will recognize that the type of post-transcriptional or post-translational modification that a protein may undergo may depend on the host organism in which the protein is expressed.

In some embodiments, the preparation of the spent whole fermentation broth of the recombinant microorganism can be achieved using any culturing method known in the art, resulting in the expression of a trypsin-like serine protease.

Thus, fermentation is understood to include shake flask culture, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the alpha-glucosidase enzyme to be expressed or isolated. The term "spent whole fermentation broth" is defined herein as the unfractionated content of fermentation material that includes culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It is to be understood that the term "spent whole fermentation broth" also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.

The host cell may be cultured under suitable conditions allowing expression of the trypsin-like serine protease. Expression of these enzymes may be constitutive, such that they are produced continuously, or inducible, requiring stimulation to initiate expression. In the case of inducible expression, protein production can be initiated when desired, for example by adding an inducing substance, such as dexamethasone or IPTG or sophorose, to the culture medium.

Any fermentation method known in the art may suitably be used to ferment the transformed or derived fungal strain as described above. In some embodiments, the fungal cell is grown under batch or continuous fermentation conditions.

Classical batch fermentations are closed systems in which the composition of the medium is set at the beginning of the fermentation and does not change during the fermentation. At the beginning of the fermentation, the medium is inoculated with one or more desired organisms. In other words, the entire fermentation process takes place without adding any components to the fermentation system all the way through.

Alternatively, batch fermentation qualifies as "batch" for the addition of carbon sources. In addition, attempts are usually made to control factors such as pH and oxygen concentration throughout the fermentation process. Typically, the metabolite and biomass composition of a batch system is constantly changing until such time as fermentation stops. In batch culture, cells progress through a static lag phase to a high log phase of growth, eventually entering a stationary phase where growth rates are reduced or halted. Without treatment, cells in the stationary phase will eventually die. Generally, cells in log phase are responsible for the bulk production of the product. A suitable variant of a standard batch system is a "fed-batch fermentation" system. In this variation of a typical batch system, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when it is known that catabolite repression will inhibit the metabolism of a cell and/or where it is desirable to have a limited amount of substrate in the fermentation medium. Measuring the actual substrate concentration in a fed-batch system is difficult and therefore it is estimated based on the change in measurable factors such as pH, dissolved oxygen, and partial pressure of exhaust gases such as CO 2. Batch and fed-batch fermentations are well known in the art.

Continuous fermentation is another known fermentation process. It is an open system in which defined fermentation medium is continuously added to the bioreactor while an equal amount of conditioned medium is removed for processing. Continuous fermentation typically maintains the culture at a constant density, wherein the cells are maintained primarily in log phase growth. Continuous fermentation allows for the modulation of one or more factors that affect cell growth and/or product concentration. For example, limiting nutrients (such as carbon or nitrogen sources) can be maintained at a fixed rate and allow all other parameters to be adjusted. In other systems, many factors that affect growth may be constantly changing, while the cell concentration, as measured by media turbidity, remains constant. Continuous systems strive to maintain steady-state growth conditions. Thus, the cell loss due to the transfer of the medium should be balanced with the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes and techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.

Isolation and concentration techniques are known in the art, and conventional methods can be used to prepare a concentrated solution or broth comprising the trypsin-like serine protease polypeptides of the invention.

After fermentation, a fermentation broth is obtained, and the microbial cells and various suspended solids (including remaining crude fermentation material) are removed by conventional separation techniques to obtain a trypsin-like serine protease solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultrafiltration, extraction or chromatography, or the like is typically used.

It may sometimes be desirable to concentrate a solution or broth comprising the alpha-glucosidase polypeptide to optimize recovery. The use of an unconcentrated solution or broth will generally increase the incubation time in order to collect the enriched or purified enzyme precipitate.

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

The solution or broth containing the trypsin-like serine protease can be concentrated until the enzymatic activity of the concentrated solution or broth containing the trypsin-like serine protease polypeptide reaches a desired level.

Concentration may be carried out using, for example, a precipitating agent, such as a metal halide precipitating agent. Metal halide precipitants include, but are not limited to, alkali metal chlorides, alkali metal bromides, and blends of two or more of these metal halides.

Exemplary metal halides include sodium chloride, potassium chloride, sodium bromide, potassium bromide, and blends of two or more of these metal halides. Metal halide precipitants, sodium chloride, may also be used as corrosion inhibitors. For production scale recovery, the trypsin-like serine protease polypeptide can be enriched or partially purified via removal of cells by flocculation with a polymer as generally described above. Alternatively, the enzyme may be enriched or purified by microfiltration and then concentrated by ultrafiltration using available membranes and equipment. However, for some applications, the enzyme need not be enriched or purified, and whole broth cultures can be lysed and used without further processing. The enzyme may then be processed into, for example, granules.

Serine proteases can be isolated or purified in various ways known to those skilled in the art, depending on the other components present in the sample. Standard purification methods include, but are not limited to, chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatofocusing, immunology, and size exclusion), electrophoresis (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), extractive microfiltration, biphasic separation. For example, the protein of interest can be purified using a standard anti-protein of interest antibody column. Ultrafiltration and diafiltration techniques in combination with protein concentration are also useful. For general guidance in suitable Purification techniques, see Scopes, Protein Purification (1982). The degree of purification required will vary depending on the use of the protein of interest. In some cases, purification will not be required.

Assays for detecting and measuring the enzymatic activity of enzymes, such as trypsin-like serine protease polypeptides, are well known. Various assays for detecting and measuring the activity of a protease (e.g., a thermostable serine protease polypeptide) are also known to those of ordinary skill in the art. In particular, the assay can be used to measure protease activity based on the release of acid soluble peptides from casein or hemoglobin as an absorbance measurement at 280nm or as a colorimetric measurement using the Folin method and the hydrolysis of dye-labeled azocasein as an absorbance measurement at 440-450nm

Other exemplary assays involve the dissolution of chromogenic substrates (see, e.g., Ward, "proteases" in Fogarty (eds.), Microbial Enzymes and Biotechnology, Applied Science publishers, London, 1983, p. 251-. A protease detection method using highly labeled Fluorescein Isothiocyanate (FITC) casein as a substrate, and modified versions of the program described in Twining [ Twining, S.S. (1984) "fluorescent Isothiocyanate-Labeled Casein Assay for protease Enzymes" [ Fluorescein Isothiocyanate labeled casein proteolysis Assay ] anal. biochem. [ biochem ]143:30-34] can also be used.

Other exemplary assays include, but are not limited to: cleavage of casein into trichloroacetic acid soluble peptides containing tyrosine and tryptophan residues followed by reaction with the forskol reagent and colorimetric detection of the product at 660nm internally quenches cleavage of the FRET (fluorescence resonance energy transfer) polypeptide substrate, followed by detection of the product using a fluorometer. Fluorescence Resonance Energy Transfer (FRET) is the non-radiative transfer of energy from an excited fluorophore (or donor) to a suitable quencher (or acceptor) molecule. FRET is used in a variety of applications, including protease activity assays involving substrates in which a fluorophore is separated from a quencher by a short peptide sequence containing an enzyme cleavage site. Proteolysis of the peptide produces fluorescence when the fluorophore and quencher are separated. Many additional references known to those skilled in the art provide suitable methods (see, e.g., Wells et al, Nucleic acids SRs [ Nucleic acids research ]11:7911-7925[1983 ]; Christianson et al, anal. biochem. [ analytical biochemistry ]223:119-129[1994], and Hsia et al, anal. biochem. [ analytical biochemistry ]242:221-227[1999 ]).

In yet another aspect, a feed, feed additive composition, premix, food, or cereal product comprising at least one polypeptide having serine protease activity as described herein, alone or in combination (a) with at least one direct-fed microbial, or (b) with at least one other enzyme, or (c) with at least one direct-fed microbial and at least one other enzyme, is disclosed.

The at least one enzyme may be selected from, but is not limited to, enzymes such as, for example, alpha-amylase, amyloglucosidase, phytase, pullulanase, beta-glucanase, cellulase, xylanase, and the like.

Any of these enzymes is used in a range from 0.5 to 500 micrograms/g feed or feed.

Alpha-amylases (alpha-1, 4-glucose-4-glucanohydrolases, EC 3.2.1.1.) hydrolyze the alpha-1, 4-glucosidic linkages in starch, producing primarily smaller molecular weight glucose at random. These polypeptides are mainly used in starch processing and alcohol production. Any alpha-amylase may be used, for example as described in U.S. Pat. Nos. 8,927,250 and 7,354,752.

Amyloglucosidase catalyzes the hydrolysis of terminal 1, 4-linked alpha-D-glucose residues from the non-reducing ends of malto-oligo-and polysaccharides sequentially, with the release of beta-D-glucose. Any amyloglucosidase may be used.

Phytase refers to a protein or polypeptide that is capable of catalyzing the hydrolysis of phytate to (1) inositol, and/or (2) mono-, di-, tri-, tetra-and/or pentaphosphate, and (3) inorganic phosphate. For example, an enzyme having catalytic activity is as defined in the enzyme commission EC number 3.1.3.8 or EC number 3.1.3.26. Any phytase may be used, for example as described in U.S. Pat. nos. 8,144,046, 8,673,609, and 8,053,221.

Pullulanase (EC 3.2.1.41) is a specific glucanase, an amylolytic endo-enzyme that degrades amylopectin, a polysaccharide polymer composed of maltose units, also known as α -1, 4-; α -1, 6-glucan. Thus, this is an example of a debranching enzyme. Pullulanase is also known as pullulanase-6-glucan hydrolase. Pullulanases are typically secreted by bacillus species. For example, Bacillus debranching (Bacillus deramificans) (U.S. Pat. No. 5,817,498; 1998), Bacillus acidophilus (Bacillus acidopulyticus) (European patent No. 0063909) and Bacillus megaterium (Bacillus nanoensis) (U.S. Pat. No. 5,055,403). Production of commercially used enzymes having pullulanase Activity, e.g., from Bacillus species (trade name from DuPont-Jennegaceae)l-100 and from NovixinD2) In that respect Debranching enzyme and process for producing the sameExamples include, but are not limited to, isoamylases from Sulfolobus solfataricus, pseudomonas species, and thermostable pullulanases from scintillation bacilli nodosum (e.g., WO 2010/76113). Isoamylase derived from Pseudomonas species can be used as a purified enzyme from Megazyme International. Any pullulanase may be used.

Glucanase is an enzyme that breaks down glucans, and is a polysaccharide composed of glucose subunits. When they undergo hydrolysis of glycosidic bonds, they are hydrolases.

Beta-glucanase (EC 3.2.1.4) digests fibers. It contributes to the breakdown of plant cell walls (cellulose).

Cellulases are one of several enzymes produced by fungi, bacteria and protozoa that catalyze fibrinolysis, the breakdown of cellulose and some related polysaccharides. This name also applies to any naturally occurring mixture or complex of various such enzymes that act sequentially or synergistically to break down cellulosic material. Any cellulase may be used.

Xylanases (EC 3.2.1.8) are the names given to a class of enzymes that degrade the linear polysaccharide β -1, 4-xylan into xylose, which breaks down hemicellulose, one of the major components of plant cell walls. Any xylanase can be used.

The at least one DFM may comprise at least one live microorganism, such as a live bacterial strain or a live yeast or a live fungus. Preferably, the DFM comprises at least one live bacterium.

DFM may be a spore-forming strain, and thus the term DFM may consist of or comprise spores, such as bacterial spores. Thus, as used herein, a "live microorganism" may include a spore of the microorganism, such as an endospore or a conidium. Alternatively, the DFM in the feed additive compositions described herein may not consist of or comprise microbial spores, such as endospores or conidia.

The microorganism may be a naturally occurring microorganism or a transformed microorganism.

DFMs as described herein may comprise microorganisms from one or more of the following genera: lactobacillus, lactococcus, Streptococcus, Bacillus, Pediococcus (Pediococcus), enterococcus, Leuconostoc (Leuconostoc), Carnobacterium (Carnobacterium), Propionibacterium (Propionibacterium), Bifidobacterium, Clostridium and Macrosphaera (Megasphaera) and combinations thereof.

Preferably, the DFM comprises one or more bacterial strains of bacillus species selected from the group consisting of: bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus pumilus and Bacillus amyloliquefaciens.

As used herein, "bacillus" includes all species within "bacillus" as known to those skilled in the art, including but not limited to: bacillus subtilis, bacillus licheniformis, bacillus lentus, bacillus brevis, bacillus stearothermophilus, bacillus alkalophilus, bacillus amyloliquefaciens, bacillus clausii, bacillus halodurans (b.halodurans), bacillus megaterium, bacillus coagulans, bacillus circulans, bacillus gibsonii (b.gibsonii), bacillus pumilus and bacillus thuringiensis. It is recognized that bacillus continues to undergo taxonomic recombination. Thus, the genus is intended to include species that have been reclassified, including but not limited to such organisms as Bacillus stearothermophilus (now referred to as "geobacillus stearothermophilus") or Bacillus polymyxa (now "Paenibacillus polymyxa"). The production of resistant endospores under stress environmental conditions is considered to be a defining feature of Bacillus, although this feature also applies to the recently named Alicyclobacillus (Alicyclobacillus), Bacillus bisporus (Amphibacillus), Thiamine Bacillus (Aneurinibacillus), anaerobic Bacillus (Anoxybacillus), Brevibacterium (Brevibacillus), linearized Bacillus (Filobacillus), parenchyma Bacillus (Gracilobacillus), Halobacterium (Halobacillus), Paenibacillus (Paenibacillus), Salibacillus (Salibacillus), Thermobacterium (Thermobacillus), Urenibacillus (Ureibacillus) and Mycobacterium (Virgibacillus).

In another aspect, DFM may be further combined with lactobacillus species as follows: streptococcus cremoris (Lactococcus cremoris) and Lactococcus lactis (Lactococcus lactis) and combinations thereof.

DFM can be further combined with lactobacillus species as follows: lactobacillus buchneri (Lactobacillus buchneri), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus casei (Lactobacillus casei), Lactobacillus kefir (Lactobacillus kefir), Lactobacillus bifidus (Lactobacillus bifidus), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus salivarius (Lactobacillus rhamnoides), Lactobacillus curvatus (Lactobacillus curvatus), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus sake (Lactobacillus sakesii), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus crispatus (Lactobacillus), Lactobacillus plantarum (Lactobacillus), Lactobacillus crispatus (Lactobacillus), Lactobacillus plantarum), Lactobacillus (Lactobacillus), Lactobacillus crispatus (Lactobacillus), Lactobacillus plantarum (Lactobacillus), Lactobacillus plantarum), Lactobacillus (Lactobacillus), Lactobacillus crispatus (Lactobacillus), Lactobacillus crispatus, Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus casei, Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus casei, Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus brevis, Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus casei, Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus casei, Lactobacillus), Lactobacillus casei, Lactobacillus (Lactobacillus), Lactobacillus (, Lactobacillus gasseri (Lactobacillus gasseri), Lactobacillus johnsonii (Lactobacillus johnsonii) and Lactobacillus jensenii (Lactobacillus jensenii) and any combination thereof.

In yet another aspect, DFM may be further combined with bifidobacterium species (bifidobacterium spp) as follows: bifidobacterium lactis (Bifidobacterium lactis), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium animalis (Bifidobacterium animalis), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium infantis (Bifidobacterium longum), Bifidobacterium catenulatum (Bifidobacterium catenulatum), Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum), Bifidobacterium adolescentis (Bifidobacterium adolescentis) and Bifidobacterium horn (Bifidobacterium angulus) and any combination thereof.

The following species of bacteria may be mentioned: bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus pumilus, enterococcus species and pediococcus species, lactobacillus species, Bifidobacterium species, lactobacillus acidophilus, pediococcus acidilactici (pediococcus acidilactici), lactococcus lactis, Bifidobacterium bifidum (Bifidobacterium bifidum), bacillus subtilis, propionibacterium thoenii (propionibacterium thoenii), lactobacillus coli, lactobacillus rhamnosus, escherichia coli (Megasphaera elsdenii), Clostridium butyricum (Clostridium butyricum), Bifidobacterium animalis subspecies (Bifidobacterium animalis), lactobacillus reuteri, bacillus cereus, lactobacillus salivarius, propionibacterium species, and combinations thereof.

The direct fed microbial described herein comprises one or more bacterial strains, which may be of the same type (genus, species and strain) or may comprise a mixture of genera, species and/or strains.

Alternatively, DFM may be combined with one or more of the products disclosed in WO 2012110778 or the microorganisms contained in these products, summarized as follows:

bacillus subtilis strain 2084 accession number NRRl B-50013, Bacillus subtilis strain LSSAO1 accession number NRRL B-50104, and Bacillus subtilis strain 15A-P4 ATCC accession number PTA-6507 (from Enviva)(formerly known as) (ii) a Bacillus subtilis strain C3102 (from) (ii) a Bacillus subtilis strain PB6 (from) (ii) a Bacillus pumilus (8G-134); enterococcus NCIMB 10415(SF68) (from) (ii) a Bacillus subtilis strain C3102 (from&) (ii) a Bacillus licheniformis (from Bacillus licheniformis)) (ii) a Enterococcus and Pediococcus (from Poultry)) (ii) a The Lactobacillus, Bifidobacterium and/or enterococcus are derived from) (ii) a Bacillus subtilis strain QST713 (from) (ii) a Bacillus amyloliquefaciens CECT-5940 (from&Plus); enterococcus faecium (Enterococcus faecium) SF68 (from Enterococcus faecium)) (ii) a Bacillus subtilis and Bacillus licheniformis (from Bacillus subtilis) (ii) a Lactobacillus-7 enterococcus faecium (from) (ii) a Bacillus strain (from)) (ii) a Saccharomyces cerevisiae (from Saccharomyces cerevisiae)) (ii) a Enterococcus (from Biomin)) (ii) a Pediococcus acidilactici, enterococcus, Bifidobacterium animalis subspecies, Lactobacillus reuteri, Lactobacillus salivarius subspecies (from Biomin)) (ii) a Lactobacillus Coli (from)) (ii) a Enterococcus (from Oralin)) (ii) a Enterococcus (2 strains), lactococcus lactis DSM 1103 (from Probios-pioneer)) (ii) a Lactobacillus rhamnosus and (from)) (ii) a Bacillus subtilis (from Bacillus subtilis)) (ii) a Enterococcus (from)) (ii) a Saccharomyces cerevisiae (from Levucell SB)) (ii) a Saccharomyces cerevisiae (from Levucell SC 0)&ME); pediococcus acidilactici (from Bactocell); saccharomyces cerevisiae (from)(formerly known as) ); saccharomyces cerevisiae NCYC Sc47 (from Saccharomyces cerevisiaeSC 47); clostridium butyricum (from Miya-) (ii) a Enterococcus (from Fecinor and Fecinor)) (ii) a Saccharomyces cerevisiae NCYC R-625 (from) (ii) a Saccharomyces cerevisiae (from)) (ii) a Enterococcus and Lactobacillus rhamnosus (from) (ii) a Bacillus subtilis and Aspergillus oryzae (from PepSoyGen-) (ii) a Bacillus cereus (from Bacillus cereus)) (ii) a Bacillus cereus variant toyoi NCIMB 40112/CNCM I-1012 (from Bacillus cereus) Or other DFMs, e.g., Bacillus licheniformis and Bacillus subtilis (from Bacillus licheniformis and Bacillus subtilis)YC) and Bacillus subtilis (from)。

DFM may be related toThe combination of the PRO and the protein,PRO is commercially available from Danisco. EnvivaIs a combination of bacillus strain 2084 accession No. NRRl B-50013, bacillus strain LSSAO1 accession No. NRRl B-50104, and bacillus strain 15A-P4 ATCC accession No. PTA-6507 (as taught in US 7,754,469B-incorporated herein by reference).

The DFMs described herein may also be combined with yeast from the genera: pseudomonas species.

Preferably, the DFMs described herein include microorganisms that are Generally Recognized As Safe (GRAS), preferably GRAS-approved microorganisms.

One of ordinary skill in the art will readily know the particular microbial species and/or strains from the genera described herein that are used in the food and/or agricultural industries and that are generally considered suitable for animal consumption.

In certain embodiments, it is important that the DFM have resistance to heat, i.e., heat resistance. This is particularly true when the feed is compressed into pellets. Thus, in another embodiment, the DFM may be a thermotolerant microorganism, such as a thermotolerant bacterium, including, for example, a bacillus species.

In other aspects, it may be desirable for the DFM to comprise a spore-forming bacterium, such as a bacillus species, for example. The bacilli form stable endospores under unfavorable growth conditions and are highly resistant to heat, pH, moisture and disinfectants.

The DFMs described herein can reduce or prevent the establishment of pathogenic microorganisms, such as Clostridium perfringens (Clostridium perfringens) and/or escherichia coli and/or Salmonella spp and/or Campylobacter spp, in the gut. In other words, DFMs may be anti-pathogenic. The term "anti-pathogenic" as used herein refers to the action (negative effect) of DFMs against pathogens.

As described above, the DFM may be any suitable DFM. For example, the following assay "DFM assay" can be used to determine whether a microorganism is suitable for being a DFM. The DFM assay as used herein is described in more detail in US 2009/0280090. For the avoidance of doubt, the DFM selected as an inhibitory strain (or anti-pathogenic DFM), according to the "DFM assay" taught herein, is a DFM suitable for use according to the present disclosure, i.e. for use in a feed additive composition according to the present disclosure.

Each test tube was implanted with a representative pathogen (e.g., bacteria) from a representative cluster.

Supernatants from potential DFMs, cultured aerobically or anaerobically, were added to inoculated tubes (except for controls without added supernatant) and incubated. After incubation, the Optical Density (OD) of the tubes of the control and treated supernatants was determined for each pathogen.

Colonies of strains that produced a lower OD (potential DFM) compared to the control (without any supernatant) could be classified as inhibitory strains (or anti-pathogenic DFM). Thus, the DFM assay as used herein is described in more detail in US 2009/0280090.

Preferably, representative pathogens for use in the present DFM assay may be one (or more) of the following: clostridium, such as Clostridium perfringens and/or Clostridium difficile (Clostridium difficile), and/or Escherichia coli and/or Salmonella species and/or Campylobacter species. In a preferred embodiment, the assay is performed with one or more clostridium perfringens and/or clostridium difficile and/or escherichia coli, preferably clostridium perfringens and/or clostridium difficile, more preferably clostridium perfringens.

Anti-pathogenic DFMs include one or more of the following bacteria and are described in WO 2013029013:

bacillus subtilis strain 3BP5 accession number NRRL B-50510,

Bacillus subtilis strain 918ATCC accession number NRRL B-50508, and

bacillus subtilis strain 1013ATCC accession number NRRL B-50509.

DFMs can be prepared as one or more cultures and one or more carriers (if used) and they can be added to a ribbon or paddle mixer and mixed for about 15 minutes, although time can be increased or decreased. The components are mixed such that a homogeneous mixture of culture and carrier results. The final product is preferably a dry flowable powder. DFMs include those containing one or more strains that can then be added to the animal feed or feed premix, added to the water of the animal, or administered by other routes known in the art (preferably simultaneously with the enzymes described herein).

The proportion of individual strains contained in the DFM mixture may vary from 1% to 99%, preferably from 25% to 75%.

Suitable dosage ranges for DFM in animal feed are about 1x 10³CFU/g feed to about 1x 10¹⁰CFU/g feed, suitably at about 1x 10⁴CFU/g feed to about 1x 10⁸Between CFU/g feed, suitably at about 7.5x 10⁴CFU/g feed to about 1x 10⁷CFU/g feed.

In another aspect, the dose of DFM in the feed exceeds about 1x 10³CFU/g feed, suitably in excess of about 1x 10⁴CFU/g feed, suitably in excess of about 5x 10⁴CFU/g feed, or suitably in excess of about 1x 10⁵CFU/g feed.

The dosage of DFM in the feed additive composition is from about 1x 10³CFU/g composition to about 1x 10¹³CFU/g composition, preferably 1x 10⁵CFU/g composition to about 1x 10¹³CFU/g composition, more preferably at about 1x 10⁶CFU/g composition to about 1x 10¹²CFU/g composition, and most preferably about 3.75x 10⁷CFU/g composition to about 1x 10¹¹CFU/g composition. In another aspect, the dosage of DFM in the feed additive composition is greater than about 1x 10⁵CFU/g composition, preferably greater than about 1x 10⁶CFU/g composition, and most preferably greater than about 3.75x 10⁷CFU/g composition. In one embodiment, the dosage of DFM in the feed additive composition is greater than about 2x10⁵CFU/g composition, suitably greater than about 2x10⁶CFU/g composition, suitably greater than about 3.75x 10⁷CFU/g composition.

A feed additive composition for use in animal feed may comprise at least one polypeptide having serine protease activity as described herein, used alone or in combination with (a) at least one direct fed microbial, or (b) at least one other enzyme, or (c) at least one direct fed microbial and at least one other enzyme, and (d) at least one component selected from the group consisting of: proteins, peptides, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methyl paraben, and propyl paraben.

In yet another aspect, a granulated feed additive composition for use in animal feed is disclosed, the granulated feed additive composition comprising at least one polypeptide having serine protease activity as described herein, alone or in combination with at least one direct fed microbial, or in combination with at least one other enzyme, or in combination with at least one direct fed microbial and at least one other enzyme, wherein the granulated feed additive composition comprises a granulate produced by a process selected from the group consisting of: high shear granulation, drum granulation, extrusion, spheronization, fluidized bed agglomeration, fluidized bed spraying, spray drying, freeze drying, granulation, spray cooling, rotary disk atomization, agglomeration, tableting, or any combination of the foregoing.

Further, the particles of the granulated feed additive composition may have an average diameter of more than 50 microns and less than 2000 microns.

The feed additive composition may be in liquid form, and the liquid form may also be as described as suitable for spray drying on feed pellets.

Animal feed may include plant material such as corn, wheat, sorghum, soybean, canola, sunflower, or mixtures of any of these plant materials or plant protein sources for poultry, swine, ruminants, aquaculture, and pets. It is expected that animal performance parameters such as growth, feed intake and feed efficiency, but at the same time improved uniformity, reduced ammonia concentration in the animal's house and thus improved welfare and health of the animal, will all be improved. More specifically, "animal performance" as used herein may be determined by the feed efficiency and/or weight gain of the animal and/or by the feed conversion rate and/or by the digestibility of nutrients in the feed (e.g. amino acid digestibility) and/or the digestible or metabolic energy in the feed and/or by the nitrogen retention and/or by the ability of the animal to avoid the negative effects of necrotic enteritis and/or by the immune response of the subject.

Preferably, the "animal performance" is determined by feed efficiency and/or animal weight gain and/or feed conversion ratio.

By "improved animal performance" is meant an increased feed efficiency and/or increased weight gain and/or a decreased feed conversion ratio and/or an improved digestibility of nutrients or energy in the feed and/or an improved ability to nitrogen retention and/or to avoid negative effects of necrotic enteritis and/or an improved immune response in a subject as a result of the use of the feed additive composition compared to a feed not comprising the feed additive composition of the invention.

Preferably, "improved animal performance" means the presence of increased feed efficiency and/or increased weight gain and/or decreased feed conversion ratio. As used herein, the term "feed efficiency" refers to the amount of animal weight gain that occurs when an animal is fed ad libitum or a prescribed amount of food over a period of time.

By "increased feed efficiency" is meant that the use of the feed additive composition according to the invention in a feed results in an increased weight gain per unit feed intake compared to an animal fed in the absence of the feed additive composition according to the invention.

As used herein, the term "feed conversion ratio" refers to a specified amount of feed that is fed to an animal to increase the weight of the animal.

Improved feed conversion ratio means lower feed conversion ratio.

By "lower feed conversion ratio" or "improved feed conversion ratio" is meant the amount of feed required to use the feed additive composition in a feed to result in an animal gaining a weight by a given amount to feed the animal that is lower than the amount of feed required to gain the animal by the same amount in the case of the feed additive composition.

Nutrient digestibility as used herein refers to the rate of nutrients that disappear from the gastrointestinal tract or a particular segment of the gastrointestinal tract (e.g., the small intestine). Nutrient digestibility may be measured as the difference between the nutrient administered to the subject and the nutrient excreted in the stool of the subject, or the difference between the nutrient administered to the subject and the nutrient retained in the digest over a specified segment of the gastrointestinal tract (e.g., the ileum).

As used herein, nutrient digestibility may be determined by collecting total excreta over a period of time, measuring the difference between the nutrient ingested and the nutrient excreted; or by an inert marker that is not absorbed by the animal and allows the researcher to calculate the amount of nutrients lost throughout the gastrointestinal tract or sections of the gastrointestinal tract. Such inert markers may be titanium dioxide, chromium oxide or acid insoluble ash. Digestibility may be expressed as a percentage of the nutrients in the feed or as a mass unit of digestible nutrients/mass unit of nutrients in the feed.

Nutrient digestibility as used herein encompasses starch digestibility, fat digestibility, protein digestibility and amino acid digestibility.

Energy digestibility as used herein means the total energy of feed consumed minus the total energy of feces, or the total energy of feed consumed minus the total energy of remaining digesta in a given segment of the animal's gastrointestinal tract (e.g., ileum). As used herein, metabolic energy refers to the apparent metabolic energy and means the total energy of the feed consumed minus the total energy contained in the feces, urine and digested gas products. The energy digestibility and metabolic energy can be measured by the difference between the intake of total energy and the total energy of faecal output, or the total energy of digesta present in a particular segment of the gastrointestinal tract (e.g. the ileum), using the same method as for determining nutrient digestibility, with appropriate correction for nitrogen excretion to calculate the metabolic energy of the feed.

In some embodiments, the compositions described herein can increase the digestibility or availability of dietary hemicellulose or fiber by a subject. In some embodiments, the subject is a pig.

Nitrogen retention as used herein means the ability of a subject to retain nitrogen in the diet as body weight. When the nitrogen excretion exceeds daily intake, negative nitrogen balance occurs, which is commonly observed when muscles are reduced. Positive nitrogen balance is often associated with muscle growth, especially for growing animals.

Nitrogen retention can be measured as the difference between the intake of nitrogen over a period of time and the output of nitrogen by the complete collection of feces and urine. It is understood that excreted nitrogen includes undigested protein in the feed, secretion of endogenous proteins, microbial proteins and urinary nitrogen.

The term survival rate, as used herein, refers to the number of surviving subjects. The term "improved survival" is another statement of "reduced mortality".

The term carcass yield as used herein refers to the amount of carcass that is part of the live weight after a commercial or experimental slaughter process. The term carcass refers to the body of an animal that has been slaughtered for consumption and has the head, internal organs, limbs, and feathers or skin removed. As used herein, the term meat yield refers to the amount of edible meat as part of the weight of a living body, or the amount of a particular piece of meat as part of the weight of a living body.

By "increased weight gain" is meant an animal that gains weight when fed a feed comprising the feed additive composition as compared to an animal fed a feed that does not contain the feed additive composition.

The term "animal" as used herein includes all non-ruminants and ruminants. In particular embodiments, the animal is a non-ruminant animal, such as horses and monogastric animals. Examples of monogastric animals include, but are not limited to, pigs (pigs and swine), such as piglets, growing pigs, sows; poultry, such as turkeys, ducks, chickens, broiler chicks, laying hens; fish, such as salmon, trout, tilapia, catfish, and carp; and crustaceans such as shrimp and prawn. In further embodiments, the animal is a ruminant animal including, but not limited to, cattle, calves, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, antelope, pronghorn, and deer antelope.

In the context of the present invention, the term "pet food" is intended to be understood as meaning a food for: domestic animals such as, but not limited to, dogs, cats, gerbils, hamsters, chinchillas, brown rats, guinea pigs; avian pets such as canaries, parakeets and parrots; reptile pets such as turtles, lizards, and snakes; and aquatic pets such as tropical fish and frogs.

The terms "animal feed composition", "feed", and "fodder" (interchangeably) are used interchangeably and comprise one or more feed stocks selected from the group comprising: a) cereals, such as small grain cereals (e.g. wheat, barley, rye, oats and combinations thereof) and/or large grain cereals, such as corn or sorghum; b) by-products from cereals, such as corn gluten meal, Distillers Dried Grains with Solubles (DDGS) (in particular corn-based Distillers Dried Grains with Solubles (cdddgs)), wheat bran, wheat middlings, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) proteins obtained from the following sources: such as soybean, sunflower, peanut, lupin, pea, broad bean, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from plant and animal sources; and/or e) minerals and vitamins.

The trypsin-like serine protease or feed additive composition described herein may be used as a feed or in the preparation of a feed. The terms "feed additive composition" and "enzyme composition" are used interchangeably herein.

Depending on the use and/or mode of application and/or mode of administration, the feed can be in the form of a solution or in the form of a solid or in the form of a semi-solid.

When used as or in the preparation of a feed (e.g., a functional feed), the enzymes or feed additive compositions described herein may be used in combination with one or more of the following: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient. For example, mention is made of at least one component selected from the group consisting of: proteins, peptides, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methyl paraben, and propyl paraben.

In a preferred embodiment, the enzyme or feed additive composition of the invention is mixed with feed components to form a feed. As used herein, "feed component" means all or part of a feed. Part of a feed may mean one ingredient of the feed or more than one (e.g., 2 or 3 or 4 or more) ingredients of the feed. In one embodiment, the term "feed component" encompasses a premix or premix ingredients. Preferably, the feed may be a silage or a premix thereof, a compound feed or a premix thereof. The feed additive composition may be mixed with or to a premix of the composite feed or to a premix of the fodder, fodder component or fodder.

Any feed material described herein may comprise one or more feed materials selected from the group comprising: a) cereals, such as small grain cereals (e.g., wheat, barley, rye, oats, triticale, and combinations thereof) and/or large grain cereals such as maize or sorghum; b) by-products from cereals, such as corn gluten meal, wet cake (in particular corn-based wet cake), Distillers Dried Grains (DDG) (in particular corn-based distillers dried grains (cDDG)), distillers dried grains with Solubles (DDGs) (in particular corn-based distillers dried grains with solubles (cDDGS)), wheat bran, wheat middlings, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) proteins obtained from the following sources: such as soybean, sunflower, peanut, lupin, pea, broad bean, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from plant and animal sources; e) minerals and vitamins.

As used herein, the term "fodder" means any food provided to the animal (rather than the animal having to forage for it itself). The fodder covers the plants that have been cut off. Furthermore, the fodder material comprises silage, compressed and pelleted fodder, oil and mixed ration, also sprouted grain and beans.

The fodder may be obtained from one or more of the plants selected from: corn (maize), alfalfa (alfalfa), barley, lotus roots, brassica, huma cabbage (Chau moellier), kale, rapeseed (canola), rutabaga (swedish), radish, clover, hybrid clover, red clover, subterranean clover, white clover, fescue, bromeline, millet, oat, sorghum, soybean, trees (used as pruned tree shoots for hay), wheat, and legumes.

The term "compound feed" means a commercial feed in the form of meal, pellets (nut), cake or crumbles. The compound feed can be blended by various raw materials and additives. These blends are formulated according to the specific requirements of the target animal.

The compound feed may be a complete feed providing all the daily required nutrients, a concentrate providing a part of the ration (protein, energy) or a supplement providing only additional micronutrients like minerals and vitamins.

The main ingredient used in the compound feed is feed grain, which includes corn, wheat, canola meal, rapeseed meal, lupins, soybean, sorghum, oats and barley.

Suitably, the premixes mentioned herein may be compositions consisting of micro-ingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products and other essential ingredients. Premixes are generally compositions suitable for blending into commercial rations.

In one embodiment, the feed comprises or consists of: corn, DDGS (e.g., cDDGS), wheat bran, or any combination thereof.

In one embodiment, the feed component can be corn, DDGS (e.g., cdddgs), wheat bran, or a combination thereof. In one embodiment, the feed comprises or consists of: corn, DDGS (e.g., cDDGS), or a combination thereof.

The feeds described herein may contain at least 30%, at least 40%, at least 50%, or at least 60% by weight of corn and soy flour or corn and full fat soy, or wheat flour or sunflower flour.

For example, the feed may contain about 5% to about 40% corn DDGS. For poultry, the feedstuffs may contain on average about 7% to 15% corn DDGS. For swine (swine or pig), the feed may contain an average of 5% to 40% corn DDGS. It may also contain corn as the single grain, in which case the feed may comprise from about 35% to about 80% corn.

In feeds comprising mixed grains (e.g., comprising, for example, corn and wheat), the feed may comprise at least 10% corn.

Additionally or alternatively, the feed may also comprise at least one high fiber feed material and/or at least one by-product of at least one high fiber feed material to provide a high fiber feed. Examples of high fiber feed materials include: wheat, barley, rye, oats, by-products from cereals such as corn gluten meal, corn protein feed, wet cake, Distillers Dried Grains (DDG), distillers dried grains with Solubles (DDGs), wheat bran, wheat middlings, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp. Some protein sources may also be considered high fiber: proteins obtained from sources such as sunflower, lupin, fava bean and cotton. In one aspect, a feed as described herein comprises at least one high fiber material and/or at least one byproduct of at least one high fiber feed material selected from the group consisting of, for example: distillers dried grains with solubles (DDGS) (particularly cDDGS), wet cake, Distillers Dried Grains (DDG) (particularly cDDG), wheat bran, and wheat. In one embodiment, the feed of the invention comprises at least one high fiber material and/or at least one by-product of at least one high fiber feed material selected from the group consisting of: such as distillers dried grains with solubles (DDGS), particularly cdddgs, wheat bran, and wheat.

The feed may be one or more of: compound feeds and premixes, including pellets, pellets (nut) or (for livestock) cakes; crop or crop residue: corn, soybean, sorghum, oat, barley, coconut kernel, straw, husk, and beet residue; fish meal; meat meal and bone meal; molasses; oil cake and filter cake; an oligosaccharide; sugar-dip forage plants: ensiling the feed; sea grass; seeds and grains, intact or prepared by crushing, grinding, etc.; sprouted grain and beans; a yeast extract.

As used herein, the term "feed" encompasses pet foods in some embodiments. Pet food is a plant or animal material intended for consumption by a pet, such as dog food or cat food. Pet foods (e.g., dog and cat foods) can be in dry form (e.g., ground foods for dogs) or wet canned form. The cat food may contain the amino acid taurine.

The animal feed may also include fish food. Fish food usually contains large amounts of nutrients, trace elements and vitamins that are required to keep farmed fish in good health. The fish food may be in the form of small pieces, pellets or tablets. Compression into pellet form (some of which settle rapidly) is often used for larger fish or bottom feed species. Some fish foods also contain additives (such as beta carotene or sex hormones) to artificially enhance the color of ornamental fish.

In yet another aspect, the animal feed encompasses bird feed. Bird food includes food used in bird feeders and for feeding pet birds. Typically, bird feed consists of a variety of seeds, but also suet (beef or mutton fat) can be encompassed.

As used herein, the term "contacting" refers to the indirect or direct application of a trypsin-like serine protease (or a composition comprising a trypsin-like serine protease) to a product (e.g., a feed). Examples of application methods that may be used include, but are not limited to: treating the product in a material comprising the feed additive composition, applying directly by mixing the feed additive composition with the product, spraying the feed additive composition onto the surface of the product, or dipping the product into a formulation of the feed additive composition. In one embodiment, the feed additive composition of the invention is preferably mixed with a product (e.g. a feed). Alternatively, the feed additive composition may be included in the emulsion or original ingredients of the feed. For some applications it is important that the composition is made available or made available on the surface of the product to be influenced/treated. This allows the composition to impart performance benefits.

In some aspects, the thermostable serine protease is used for pretreatment of food or feed. For example, the feed has 10% -300% moisture mixed and incubated with protease at 5-80 ℃, preferably between 25-50 ℃, more preferably between 30-45 ℃ for 1 minute to 72 hours under aerobic conditions or 1 day to 2 months under anaerobic conditions. The pretreated material can be fed directly to the animal (so-called liquid feed). The pretreated material can also be steam granulated at elevated temperatures (60 ℃ to 120 ℃). The protease may be impregnated into the feed or food material by a vacuum sprayer.

The trypsin-like serine protease (or a composition comprising the trypsin-like serine protease) and a controlled amount of the enzyme may be used to spread, coat and/or impregnate a product (e.g., a feed or the original ingredients of a feed).

Preferably, the feed additive composition will be heat stable for heat treatment up to about 70 ℃, up to about 85 ℃, or up to about 95 ℃. The heat treatment may be performed for up to about 1 minute; up to about 5 minutes; up to about 10 minutes; up to about 30 minutes; up to about 60 minutes. The term "thermostable" means that at least about 75% of the enzyme components and/or DFM present/active in the additive prior to heating to a particular temperature are still present/active after cooling to room temperature. Preferably, at least about 80% of the protease component and/or DFM comprising one or more bacterial strains that is present and active in the additive prior to heating to the specified temperature is still present and active after cooling to room temperature. In a particularly preferred embodiment, the feed additive composition is homogenised to form a powder.

Alternatively, the feed additive composition is formulated into pellets as described in WO 2007/044968 (referred to as TPT pellets), which is incorporated herein by reference.

In another preferred embodiment, when the feed additive composition is formulated as granules, the granules comprise hydrated barrier salt sprayed onto a protein core. Such salt coatings have the advantage of providing improved thermostability, improved storage stability and protection from other feed additives that would otherwise adversely affect the at least one protease and/or DFM comprising one or more bacterial strains. Preferably, the salt used for the salt coating has a water activity of greater than 0.25 or a constant humidity of greater than 60% at 20 ℃. Preferably, the salt coating comprises Na₂SO₄。

The method of preparing the feed additive composition may also comprise the additional step of pelletizing the powder. The powder may be mixed with other components known in the art. The powder, or mixture containing the powder, may be forced through a die and the resulting strands cut into suitable pellets of different lengths.

The process for preparing a trypsin-like serine protease (or a composition comprising a trypsin-like serine protease) may further comprise the additional step of granulating the powder. The powder may be mixed with other components known in the art. The powder, or mixture containing the powder, may be forced through a die and the resulting strands cut into suitable pellets of different lengths.

Optionally, the pelletizing step may include a steam treatment or conditioning stage that is performed prior to pellet formation. The mixture containing the powder may be placed in a conditioner, such as a mixer with steam injection. The mixture is heated in a conditioner to a specified temperature, e.g., from 60 ℃ to 100 ℃, and typical temperatures will be 70 ℃, 80 ℃, 85 ℃,90 ℃, or 95 ℃. Residence times can vary from a few seconds to several minutes and even hours. Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, and 1 hour. It will be appreciated that the thermostable serine protease enzyme (or a composition comprising a thermostable serine protease enzyme) described herein is suitable for addition to any suitable feed material.

The skilled person will appreciate that different animals will require different feeds, and even the same animal may require different feeds, depending on the purpose for which the animal is being raised.

Optionally, the feed may also contain additional minerals (such as, for example, calcium) and/or additional vitamins. In some embodiments, the feed is a corn soybean meal mixture.

The feed is typically produced in a feed mill where the raw materials are first ground to the appropriate particle size and then mixed with the appropriate additives. The feed may then be produced into a paste or pellets; the latter generally relates to a process by which the temperature is raised to a target level and then the feed is passed through a die to produce pellets of a particular size. The pellets were allowed to cool. Subsequently, liquid additives such as fats and enzymes may be added. The preparation of the feed may also involve additional steps including extrusion or expansion prior to granulation, in particular by suitable techniques which may include at least the use of steam.

The feed may be a feed for: monogastric animals, such as poultry (e.g., broiler chickens, laying hens, broiler breeders, broiler chickens, turkeys, ducks, geese, waterfowl) and swine (all age categories); ruminants such as cattle (e.g. cows or bulls (including calves)), horses, sheep, pets (e.g. dogs, cats) or fish (e.g. gastrofree fish, gastric fish, freshwater fish such as salmon, cod, trout and carp (e.g. koi), sea fish (e.g. sea bass) and crustaceans such as shrimp, mussel and scallop). Preferably, the feed is for poultry.

The feed additive composition and/or the feedstuffs comprising the same may be used in any suitable form. The feed additive composition can be used in solid or liquid formulations or as an alternative thereto. Examples of solid formulations include powders, pastes, macroparticles, capsules, pellets, tablets, dusts, and granules, which may be wettable, spray-dried, or freeze-dried. Examples of liquid formulations include, but are not limited to, aqueous, organic or aqueous-organic solutions, suspensions, and emulsions.

In some applications, the feed additive composition may be mixed with feed or administered in drinking water.

A feed additive composition comprising a protease as taught herein mixed with a feed acceptable carrier, diluent or excipient and (optionally) packaged.

The feed and/or feed additive composition may be mixed with at least one mineral and/or at least one vitamin. The compositions thus derived may be referred to herein as premixes.

In some embodiments, the trypsin-like serine protease may be present in the feed in the range of 1ppb (parts per billion) to 10% (w/w), based on pure enzyme protein. In some embodiments, the protease is present in the feed in the range of 1-100ppm (parts per million). A preferred dose may be 1-20g trypsin-like serine protease per tonne of feed product or feed composition, or a final dose of 1-20ppm trypsin-like serine protease in the final product.

Preferably, the trypsin-like serine protease may be present in the feed at least about 200PU/kg or at least about 300PU/kg feed or at least about 400PU/kg feed or at least about 500PU/kg feed or at least about 600PU/kg feed, or at least about 700PU/kg feed, at least about 800PU/kg feed, at least about 900PU/kg feed, or at least about 1000PU/kg feed, or at least about 1500PU/kg feed, or at least about 2000PU/kg feed, or at least about 2500PU/kg feed, or at least about 3000PU/kg feed, or at least about 3500PU/kg feed, or at least about 4000PU/kg feed, or at least about 4500PU/kg feed, or at least about 5000PU/kg feed.

In another aspect, the trypsin-like serine protease may be present in the feed at less than about 60,000PU/kg feed, or at less than about 70,000PU/kg feed, or at less than about 80,000PU/kg feed, or at less than about 90,000PU/kg feed, or at less than about 100,000PU/kg feed, or at less than about 200,000PU/kg feed, or at less than about 60000PU/kg feed, or at less than about 70000PU/kg feed.

Ranges can include, but are not limited to, any combination of the lower and upper ranges discussed above.

It will be appreciated that one Protease Unit (PU) is the amount of enzyme that releases 2.3 micrograms of phenolic compounds (expressed as tyrosine equivalents) from a casein substrate at 50 ℃ per minute at pH 10.0. This may be referred to as an assay to determine 1 PU.

The formulation comprising the trypsin-like serine protease and the compositions described herein may be prepared in any suitable manner to ensure that the formulation comprises the active enzyme. Such formulations may be in the form of a liquid, a dry powder or granules. Preferably, the feed additive composition may be in liquid form, and the liquid form may also be suitable for spray drying on feed pellets.

Dry powders or granules can be prepared by means known to those skilled in the art, such as high shear granulation, drum granulation, extrusion, spheronization, fluidized bed agglomeration, fluidized bed spraying.

The trypsin-like serine proteases and compositions described herein can be coated, e.g., encapsulated, within a capsule. In one embodiment, the coating protects the enzyme from heat and may be considered a heat shielding agent.

The feed additive compositions described herein are formulated as dry powders or granules, as described in WO 2007/044968 (referred to as TPT granules) or WO 1997/016076 or WO 1992/012645 (each of which is incorporated herein by reference).

In one embodiment, the feed additive composition may be formulated as a granule for a feed composition, the granule comprising: a core; an active agent; and at least one coating, the active agent of the particle remaining at least 50% active, at least 60% active, at least 70% active, at least 80% active after conditions selected from one or more of: a) a feed pelleting process, b) a steam heated feed pre-treatment process, c) storage, d) storage as an ingredient in an unpelletized mixture, and e) storage as an ingredient in a feed base mixture or feed premix comprising at least one compound selected from the group consisting of: trace minerals, organic acids, reducing sugars, vitamins, choline chloride, and compounds that produce acidic or basic feed base mixtures or feed premixes.

With respect to the particles, at least one coating may comprise a moisture hydrating material comprising at least 55% w/w of the particle; and/or at least one coating may comprise two coatings. The two coatings may be a moisture hydrating coating and a moisture resistant coating. In some embodiments, the moisture hydrating coating may be 25% w/w to 60% w/w of the particle and the moisture barrier coating may be 2% w/w to 15% w/w of the particle. The moisture hydrating coating may be selected from the group consisting of inorganic salts, sucrose, starch, and maltodextrin, and the moisture resistant coating may be selected from the group consisting of polymers, gums, whey, and starch.

The granules can be prepared using a feed granulation process and the feed pretreatment process can be carried out at 70 ℃ to 95 ℃ (e.g., at 85 ℃ to 95 ℃) for up to several minutes.

The feed additive composition may be formulated as a granule for animal feed, the granule comprising: a core; an active agent, the active agent of the granules remaining at least 80% active after storage and after a steam heated granulation process of the granules as a component; a moisture resistant coating; and a moisture hydrating coating of at least 25% w/w of the granule, the granule having a water activity of less than 0.5 prior to the steam heated granulation process.

The particles may have a moisture resistant coating selected from polymers and gums and the moisture hydrating material may be an inorganic salt. The moisture hydrating coating may be 25% w/w to 45% w/w of the particle and the moisture barrier coating may be 2% w/w to 10% w/w of the particle.

The granules can be prepared using a steam heated pelleting process, which can be carried out at 85 ℃ to 95 ℃ for up to several minutes.

Alternatively, the composition is in a liquid formulation suitable for consumption, preferably such liquid consumer product contains one or more of the following: buffers, salts, sorbitol and/or glycerol.

In addition, the feed additive composition may be formulated by applying (e.g., spraying) the one or more enzymes onto a carrier substrate (e.g., ground wheat).

In one embodiment, the feed additive composition may be formulated as a premix. By way of example only, the premix may comprise one or more feed components, such as one or more minerals and/or one or more vitamins.

In one embodiment, a direct fed microbial ("DFM") and/or thermostable serine protease is formulated with at least one physiologically acceptable carrier selected from at least one of the following: maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat component, sucrose, starch, Na₂SO₄Talc, PVA, sorbitol, benzoate, sorbate, glycerol, sucrose, propylene glycol, 1, 3-propanediol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formate, and mixtures thereof.

Non-limiting examples of the compositions and methods disclosed herein include:

1. an isolated polypeptide having serine protease activity, selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence having at least 91% identity to the amino acid sequence of SEQ ID NO. 22;

b) a polypeptide comprising an amino acid sequence having at least 94% identity to the amino acid sequence of SEQ ID NO. 23;

c) a polypeptide comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO. 24;

d) a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO. 25.

2. An isolated polypeptide having serine protease activity and comprising a predicted precursor amino acid sequence selected from the group consisting of: 3, SEQ ID NO; 6, SEQ ID NO; 9, SEQ ID NO; and SEQ ID NO 12.

3. An isolated polypeptide having serine protease activity and comprising a protease catalytic region selected from the group consisting of:

a) a protease catalytic region having an amino acid sequence with at least 96% identity to the amino acid sequence of SEQ ID NO. 18;

b) an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO. 19;

c) 20, the amino acid sequence of SEQ ID NO;

d) an amino acid sequence having at least 91% identity to the amino acid sequence of SEQ ID NO. 21;

4. a recombinant construct comprising a control sequence functional in a production host operably linked to a nucleotide sequence encoding at least one polypeptide having serine protease activity as described in examples 1-3.

5. The recombinant construct of embodiment 5, wherein said host is selected from the group consisting of: fungi, bacteria and algae.

6. A method for producing at least one polypeptide having a serine protease, the method comprising:

(a) transforming a production host with the recombinant construct as described in example 4; and

(b) cultivating the production host of step (a) under conditions to produce at least one polypeptide having serine protease activity.

7. The method of example 6, wherein the serine protease is optionally recovered from the production host.

8. A culture supernatant comprising a serine protease obtained by the method of any one of examples 6 or 7.

9. A recombinant microbial production host for expressing at least one polypeptide comprising a recombinant construct as described in example 4.

10. The production host of embodiment 9, wherein the host is selected from the group consisting of: bacteria, fungi and algae.

11. An animal feed comprising at least one polypeptide according to any one of examples 1-3, wherein the polypeptide is present in an amount of 1-20g per ton of feed.

12. The animal feed of embodiment 11, further comprising at least one direct fed microbial.

13. The animal feed of embodiment 11 or 12, further comprising at least one additional enzyme.

14. A feed, feedstuff, feed additive composition or premix comprising at least one polypeptide as described in any of examples 1-3.

15. The feed, feed additive composition or premix of example 14 further comprising at least one direct fed microbial.

16. A feed, feed additive composition or premix according to example 14 or 15 further comprising at least one additional enzyme.

17. A feed additive composition according to any one of embodiments 14-17 wherein the composition further comprises at least one component selected from the group consisting of: proteins, peptides, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methyl paraben, and propyl paraben.

18. A granulated feed additive composition for use in animal feed, the granulated feed additive composition comprising at least one polypeptide as in any of embodiments 1-3, wherein the granulated feed additive composition comprises granules produced by a method selected from the group consisting of: high shear granulation, drum granulation, extrusion, spheronization, fluidized bed agglomeration, fluidized bed spraying, spray drying, freeze drying, granulation, spray cooling, rotary disk atomization, agglomeration, tableting, and combinations thereof.

19. A granulated feed additive composition according to embodiment 18 wherein the mean diameter of the granules is greater than 50 microns and less than 2000 microns.

20. A feed additive composition according to any one of embodiments 14-17 wherein the composition is in liquid form.

21. The feed additive composition of embodiment 20 wherein the composition is in a liquid form suitable for spray drying on feed pellets.

Examples of the invention

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton et al, DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY [ DICTIONARY OF microbiology AND MOLECULAR BIOLOGY ],2 nd edition, John Wiley AND Sons [ John Willi-Ginko, Inc. ], New York (1994), AND Hale AND Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY [ DICTIONARY OF Huppe Corolins ], Harper Perennial [ Huppe PERMAN, N.Y. (1991) provide the skilled artisan with a general DICTIONARY OF many OF the terms used in this disclosure.

The present disclosure is further defined in the examples below. It should be understood that these examples, while indicating certain embodiments, are given by way of illustration only. From the above discussion and the examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt it to various usages and conditions.

Example 1

Cloning of trypsin-type serine proteases from Streptomyces species

Four fungal strains (streptomyces species C004, streptomyces species C009, streptomyces species C001 and streptomyces species S055) were selected as potential sources of enzymes, and these strains could be used in a variety of industrial applications. Chromosomal DNA was isolated from the four strains and sequenced using Illumina' next generation sequencing technology. After annotation of the four streptomyces species above, the gene encoding the trypsin-like serine protease was identified; and identifying the nucleotide or amino acid sequence thereof.

All 4 protein genes had N-terminal signal peptides, as predicted by SignalP software version 4.0 (Nordahl Petersen et al, (2011) Nature Methods [ Nature Methods ]8:785-786), indicating that they all secrete enzymes.

The nucleotide sequence of the SspCPPro 29 gene isolated from Streptomyces species C009 is shown in SEQ ID NO: 1. The predicted signal sequence for the SspPro 29 precursor protein is shown in SEQ ID NO 2. The amino acid sequence of the SspPro 29 precursor protein is shown in SEQ ID NO 3.

The nucleotide sequence of the SspCPPro 33 gene isolated from Streptomyces species C001 is shown in SEQ ID NO 4. The predicted signal sequence for the SspPro 33 precursor protein is shown in SEQ ID NO 5. The amino acid sequence of the SspPro 33 precursor protein is shown in SEQ ID NO 6.

The nucleotide sequence of the SspCPPro 23 gene isolated from Streptomyces species C003 is shown in SEQ ID NO 7. The predicted signal sequence for the SspPro 23 precursor protein is shown in SEQ ID NO 8. The amino acid sequence of the SspPro 23 precursor protein is shown in SEQ ID NO 9.

The nucleotide sequence of the SsppCPro 59 gene isolated from Streptomyces species C055 is shown in SEQ ID NO: 10. The predicted signal sequence for the SspPro 59 precursor protein is shown in SEQ ID NO 11. The amino acid sequence of the SspPro 59 precursor protein is shown in SEQ ID NO 12. Based on the signal sequence predictions, the full-length amino acid sequence was predicted as follows: SspCPI 29(SEQ ID NO: 22); SspCPI 33(SEQ ID NO: 23); SspCPI 23(SEQ ID NO: 24); and SspCPI 59(SEQ ID NO: 25).

Example 2

Expression of trypsin-type serine protease from Streptomyces species

The DNA sequences encoding the mature forms of the propeptide of the trypsin homologues SspCPPro 23, SspC29, SspC33 and SspC59 of Streptomyces species (precursor Protein minus signal sequence) were synthesized by the Jersey company (general) (Shanghai, China) and each inserted into the Bacillus subtilis expression vector p2JM 103I (Vogtentanz, Protein Expr purify [ Protein expression and purification ],55:40-52,2007). The resulting plasmids were designated pGX384 (AprE-SspCPPro 23), pGX390 (AprE-SspCPPro 29), pGX394 (AprE-SspCPPro 33) and pGX738 (AprE-SspCPPro 59). The synthetic gene has an alternative start codon (GTG).

FIG. 1 provides a plasmid map of pGX390 (AprE-SspCPI 29), three additional plasmids having similar composition except for the inserted gene encoding each serine protease target Gene (GOI). The nucleotide sequences of the synthesized AprE-SspCPPro 23, AprE-SspCPPro 29, AprE-SspCPPro 33 and AprE-SspCPPro 59 genes are shown as SEQ ID NO 13, 14, 15 and 16, respectively. Ligation of the genes encoding each GOI into a linearized expression vector resulted in the addition of three codons (encoding residues Ala-Gly-Lys) between the 3 'end of the sequence encoding the bacillus subtilis AprE signal and the 5' end of the sequence encoding the propeptide-maturation sequence. The aprE signal sequence (SEQ ID NO:17) is used to direct secretion of the recombinant protein in B.subtilis.

The expression plasmid is then transformed into the appropriate Bacillus subtilisBacillus cells, and the transformed cells were cultured on Luria Agar (Luria Agar) plates supplemented with 5ppm chloramphenicol and 1.2% skim milk (catalog No. 232100, Difco). Colonies forming the most pronounced halo were picked and used to inoculate liquid cultures. MOPS-based defined Medium (supplemented with 5mM CaCl) was used₂) The fermentation was carried out in 250mL shake flasks.

To purify the SspCPro29 and SspCPro33 proteases, clear supernatants from shake flask cultures were subjected to column chromatography using hydrophobic interaction and ion exchange resins. The resulting active protein fractions were then pooled, concentrated via a 10K Amicon Ultra device, and stored at-20 ℃ in 40% glycerol until use.

Various sequence regions of SspCPro29, SspCPro23, SspCPro33, and SspCPro59 were further analyzed using protein sequence annotation of the Streptomyces griseus serine protease Streptomyces protease C (Uniprot accession number P52320) to identify putative amino acid residues comprising the catalytic domains of these proteases. Streptomyces protease C is represented as a 457 residue polypeptide comprising a signal sequence (residues 1-34), a propeptide region (residues 35-202) and a mature chain (residues 203 to 457).

The mature chain is further composed of a catalytic domain (residues 203 to 393, SEQ ID NO:39), a linker (residue 394-413) and a chitin binding region (residue 415-457). Based on this information, the catalytic domains of SspCPro29, SspCPro23, SspCPro33, and SspCPro59 are predicted to be: respectively SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20 and SEQ ID NO 21.

Example 3

Proteolytic Activity of Trypsin-type serine proteases of Streptomyces species

The proteolytic activity of the purified SspcPro29 and SspcPro33 was measured in 50mM HEPES buffer (pH 8) using Suc-Ala-Ala-Pro-Phe-pNA (AAPF-pNA, Cat. No. L-1400.0250, BACHEM) as a substrate. Using commercial productsSamples of proAct protease (DSM) were used as reference. Before the reaction, the reaction solution is subjected to a reaction,the enzyme was diluted to a specific concentration with purified water (Millipore). AAPF-pNA was dissolved in dimethyl sulfoxide (DMSO, catalogue number STBD2470V, Sigma (Sigma)) to a final concentration of 10 mM.

To start the reaction, 5 μ L of AAPF-pNA was first mixed with 85 μ L of HEPES buffer in a non-binding 96-well microtiter plate (96-MTP) (Corning Life Sciences, #3641) and incubated at 40 ℃ for 5min at 600rpm in a thermostatic mixer (Eppendorf), then 10 μ L of diluted enzyme (or purified H only) was added₂O as blank control). After incubation in a thermostatic mixer at 40 ℃ and 600rpm for 10min, the reaction plate was read directly at 410nm using SpectraMax 190. By starting from enzyme A₄₁₀Minus A of blank control₄₁₀To calculate net A₄₁₀And then plotted against different protein concentrations (from 0.02ppm to 0.3125 ppm). Each value is the average of three replicates.

Proteolytic activity on AAPF-pNA substrate is shown as neat A in FIG. 2₄₁₀. The proteolytic activity of SspCPro23 and SsCPro59 was measured using clear supernatants from shake flask cultures. Clarified culture supernatants of B.subtilis cells transformed with p2JM103BBI (lacking the protease gene) were used as vector controls. Before the reaction, the supernatant was diluted 200-fold with purified water. The assay procedure was performed as described above and by A from the enzyme sample₄₁₀Minus A of vehicle control₄₁₀To calculate net A₄₁₀. Each value is the average of three replicates. Proteolytic activity is shown as neat A in Table 1₄₁₀SsppCPro 23 and SsppCPro 59 are indicated as active proteases.

Example 4

pH profile of Trypsin-type serine proteases of Streptomyces species

The pH profile of the trypsin homologue was studied in 25mM glycine/sodium acetate/HEPES buffer at different pH values (ranging from pH 3 to 10) with AAPF-pNA as substrate. Prior to the assay, 85. mu.L of 25mM glycine/sodium acetate/HEPES buffer having a specific pH was first mixed with 5. mu.L of 10mM AAPF-pNA in 96-MTP and then with 10. mu.L of purified water. The reaction was then started by adding diluted enzyme (0.2ppm for purified SspCPro29 and SspCPro33, or 200-fold dilution of clarified supernatant of SspCPro23 and SspCPro 59). Water or supernatant from the vehicle control (200-fold dilution) was used as a blank for purified or unpurified enzyme, respectively. The reaction was carried out and analyzed as described in example 3. The enzyme activity at each pH was reported as relative activity, with the enzyme activity at the optimum pH set to 100%. The pH values tested for the purified enzymes (SspCPro29 and SspCPro33) were 3, 4, 5, 6, 7, 8,9, and 10; and 3, 5.5, 8,9, 10 for the unpurified enzymes (SspcPro23 and SspPro 59). Each value is the average of three replicates.

As shown in FIG. 3, all trypsin homologues are alkaline proteases.

Example 5

Temperature profile of trypsin-type serine protease from Streptomyces species

The temperature profile of the trypsin homologue was analyzed in 50mM HEPES buffer (pH 8) using the AAPF-pNA assay. Before the reaction, 85. mu.L of 50mM HEPES buffer pH 8.0 and 5. mu.L of 10mM AAPF-pNA were added to a 200. mu.L PCR tube, which was then subsequently incubated in a Peltier thermal cycler (Bio Rad) at the desired temperature (30 ℃ to 80 ℃) for 5 min. After incubation, 10 μ L of enzyme sample (0.2ppm for purified SspCPro29 and SspCPro33, or clear supernatant (200-fold dilution) for SspCPro23 and SspCPro59) was added to the substrate to initiate the reaction. Water alone or supernatant from vehicle control (200-fold dilution) was added as a blank for purified or unpurified enzyme, respectively. After incubation in a Peltier thermal cycler for 10min at different temperatures, 80. mu.L of the reaction mixture was transferred to a fresh 96-MTP and the absorbance read at 410 nm. The activity is reported as relative activity, with the activity at the optimal temperature set to 100%. The temperatures tested for the purified enzymes (SspcPro29 and SspcPro33) were 30 ℃,35 ℃,40 ℃, 45 ℃, 50 ℃,55 ℃,60 ℃, 65 ℃, 70 ℃,75 ℃ and 80 ℃; and the unpurified proteins SspcPro23 and SspcPro59 were tested at 35 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, and 70 deg.C. Each value reported is the average of three replicates. The results are shown in FIG. 4.

Example 6

Hydrolysis of corn soybean meal by trypsin-type serine protease of streptomyces species.

The degree of hydrolysis of corn soybean meal (with a mixture of 40% soybean meal and 60% corn flour) by trypsin homologues was evaluated using OPA (o-phthalaldehyde) or BCA (bisquinolinecarboxylic acid) detection assays as described below to measure the amount of newly produced N-terminal amine groups or soluble peptides released into the supernatant after the enzymatic reaction, respectively. To perform the assay, 140 μ L of corn soybean meal substrate (10% (w/w) corn soybean meal slurry suspended in MES pH 6 buffer) was mixed in 96-MTP with 20 μ L of diluted purified enzyme samples (SspPro 29 and SspPro 33) or with 60 μ L of clarified supernatant (SspPro 23 and SspPro 59). After incubation in an incubator at 40 ℃ for 2hr, the plates were centrifuged at 3700rpm for 15min at 4 ℃. The resulting supernatant was diluted 10-fold in water and the subsequent reaction product detection was performed using OPA and BCA assays. In the case of a purified protease, the protease is,samples of ProAct protease (DSM) were included as a commercial reference protease and water was used as the (no enzyme) blank. For unpurified, supernatant from the vector control was used as a blank control.

By mixing 30mL of 2% trisodium phosphate buffer (pH 11), 800. mu.L of 4% OPA (catalog No. P1378, Sigma dissolved in 96% ethanol), 1mL of 3.52% dithiothreitol (catalog No. D0632, Sigma) and 8.2mL of H₂O to prepare OPA reagent. The reaction was initiated by adding 10. mu.L of 10X diluted protease reaction to 175. mu.L of OPA reagent in 96-MTP (catalog No. 3635, Corning Life sciences). After incubation at 20 ℃ for 2min, a spectrophotometer was used at 340nm (A)₃₄₀) The absorbance of the resulting solution was measured. By feeding from each eggA of the protease reaction₃₄₀Minus A of blank control (water for SspcPro29 and SspcPro 33; supernatant from vector control for SspPro 23 and SspPro 59)₃₄₀To calculate net A₃₄₀To measure the degree of hydrolysis of corn soybean meal by each protease sample. The results are shown in fig. 5A and table 2.

The BCA reaction was performed by mixing 10 μ L of diluted supernatant with 200 μ L BCA reagent according to manufacturer's instructions. Incubation was performed in a thermostatic mixer at 37 ℃ for 30min and the reaction product was measured in a spectrophotometer as the end-point absorbance reading at 562 nm. By A from each protease reactant₅₆₂Minus A of blank control (water for SspcPro29 and SspcPro 33; supernatant from vector control for SspPro 23 and SspPro 59)₅₆₂To calculate net A₅₆₂To measure the degree of hydrolysis of corn soybean meal by each protease sample. The results are shown in fig. 5B and table 2.

Example 7

Pepsin stability of trypsin-type serine proteases of Streptomyces species

The pepsin stability of the trypsin homologues was analyzed by incubating the trypsin homologues with pepsin (sigma, catalog No. P7000) in 50mM sodium acetate buffer (pH 3.0) and performing residual activity measurements using AAPF-pNA as substrate. First mixing the trypsin homolog and the pepsin in a ratio (w/w) of 1:0, 1:25, 1:250 or 1:2500, wherein the dosage of the trypsin homolog is 20 ppm; and the resulting mixture was then incubated at 37 ℃ for 30 min. At the same time, 20ppm aliquots of each trypsin homologue were kept on ice as untreated controls. For residual activity measurements, 5. mu.L of 10mM AAPF-pNA was mixed with 85. mu.L of HEPES buffer (50mM, pH 8.0) in 96-MTP, followed by addition of 10. mu.L of a mixture diluted in purified water (0.2ppm or H only)₂O as blank control). The reaction was carried out and analyzed as described in example 3.

Table 3 shows the residual enzyme activity after pepsin treatment, wherein the activity of the untreated sample kept on ice was set to 100%.

Example 8

Stability of SspcPro29 protease under feed pelleting conditions

The granulation conditions were as follows: 62.5g of a concentrated solution of SspCPI 29 protease consisting of 38.37g of protein was diluted to 600mL in tap water and mixed with 120kg of corn soybean feed (60% corn, 31.5% soybean flour, 4.0% soybean oil, 0.4% salt, 0.2% DL-methionine, 1.16% limestone, 1.46% calcium phosphate and 1.25% vitamin and mineral mix by weight). The mixture was granulated at 90 ℃ or 95 ℃ for 30 seconds. A mixture of similarly prepared enzyme sample and corn soybean feed (mash feed) without pelleting served as a control. The extraction conditions were as follows: 1g of the pellet-pressed feed or pasty feed was ground with a magnetic stir bar in a 10mL beaker at room temperature (22 ℃), mixed with 10mL of buffer (100mM Tris buffer, containing 1% SDS pH 10) for 10min, and then centrifuged at 4000rpm for 10min using a bench top centrifuge. The supernatant was filtered through a glass fiber filter. The filtrate was used directly in the enzyme activity assay. The enzyme activity was measured under the following conditions: 0.18mL of 0.1M Tricine buffer (pH 9.75, containing 1% SDS), 1. mu.L of enzyme feed extract and 20. mu.L of LAAPF-pNA substrate (10mg/mL in DMSO) were mixed for 1min at 900 rpm. The samples were incubated at 30 ℃ for 120min with continuous shaking.

The extent of the reaction was determined by measuring the absorbance at 410nm in a spectrophotometer. The results are shown in Table 4.

Example 9

Hydrolysis and solubilization of proteins in corn soybean feed with SspcPro29 protease and SspPro 33 protease

The reaction contained 140 μ L of 10% (w/w) corn soybean feed slurry (Yu S, Cowieson a, Gilbert C, Plumstead P, Dalsgaard S., Interactions of phytate and myo-inositolphonate esters (IP1-5) including IP5 isomers with a di-ethyl protein and irony and inhibition of pepsin, [ phytate and inositol phosphate including IP5 isomers (IP1-5) interaction with dietary protein and iron and inhibition of pepsin ] j.anim.sci. [ journal of zoology ]2012,90: 1824-; giving final concentrations of 0, 250, 500, 750, 1000 and 1250ppm of 20 μ L protease in 50mM sodium acetate (pH 3.0) for corn soybean feed; and 10. mu.L of pepsin (Sigma P7000 dissolved in water at 1.69 mg/ml). Plates were incubated in an iEMS incubator/shaker (Thermoscientific) at 1150rpm for 45min at 40 ℃. At the end of the incubation, 34 μ L porcine pancreatin (sigma P7545, 0.4636mg/mL in 1M sodium bicarbonate) was added and the plates were further incubated in an iEMS incubator/shaker at 1150rpm for 60min at 40 ℃. After incubation, plates were centrifuged at 4000rpm for 15min at 5 ℃. Transfer 10 μ L of supernatant to a new 96-well MTP containing 90 μ L of water (10x dilution). OPA (proteolysis) and BCA (protein lysis) values were determined for the 10-fold diluted supernatants at 340nm and 562nm, respectively.

Proteolysis with the reagent o-phthalaldehyde (OPA) was performed essentially as described previously (with minor modifications) (P.M. NIELSEN, D.PETERSEN and C.DAMBMANN, Improved method for determining Improved degree of proteolysis in foods, J.food Sci. [ journal of food science ]66:642-646, 2001). OPA reagent was freshly prepared by mixing 30mL of trisodium phosphate (na3po4.12h2o, 2% w/v in water, with pH adjusted to pH 11), 0.8mL of OPA (0.4g of o-phthalaldehyde 97% (OPA) in 10mL of 96% ethanol, stored at-20 ℃), 1mL of DTT solution (0.352g of DL-Dithiothreitol (DTT) 99% in 10mL of water) and to a final volume of 40mL of water. The reagents were kept in the dark and used immediately after preparation. 20 μ L of 10 Xdiluted supernatant was mixed with 175 μ L of OPA reagent for 5 seconds and read accurately after 2min at 340 nm.

Protein solubilization was measured by using Pierce BCA protein assay kit (catalog No. 23225 from seemer feishel technologies). mu.L of the supernatant was mixed with 200. mu.L of BCA reagent (prepared by mixing 50mL BCA reagent A with 1mL BCA reagent B before use according to the manufacturer's instructions). The mixture was incubated at 37 ℃ for 30min, and then the absorbance at 562nm was measured.

Tables 5.1 and 5.2 show that in the presence of both pepsin and pancreatin, proteolysis and protein solubilization in corn soybean feed (respectively) increased with increasing dosages of SspCPro29 and SspCPro33 proteases from 0 to 1250 ppm. Respective correlation coefficients (R) of hydrolysis and dissolution²) 0.90 and 0.97.

Example 10

Cleaning Performance of SspcPro29 and SspcPro33 in ADW

The cleaning performance of SspCPro29 and SspCPro33 was tested using PA-S-38 (toned egg yolk, matured by heating) microsamples (CFT-tulathree (Vlaardingen), the netherlands) at pH 10.3 using model Automatic Dishwashing (ADW) detergent. To prepare a rinsed PAS38 sample, 180. mu.L of 10mM CAPS buffer (pH 11) was added to 96-MTP containing PAS38 sample. The plates were sealed and incubated in an iEMs incubator at 60 ℃, 1100rpm for 30 min. After incubation, the buffer was removed and purified H was used₂O rinse the sample. The panels were air dried before being used for performance measurements.

The purified protease sample was incubated with 0.1mM CaCl₂And 0.005%Diluted to 200ppm in 10mM NaCl. The test was carried out in 3g/L of GSM-B detergent. The composition of the GSM-B detergent (in weight percent) was as follows: 30% sodium citrate dehydrate, 25% sodium disilicate (Protil A, Corning (Cognis)), 12% maleic/acrylic acid copolymer sodium salt (maleic/acrylic acid copolymer sodium salt) ((Co.))CP5BASF), 5% sodium perborate monohydrate, 2% TAED, 2% linear fatty alcohol ethoxylate, and anhydrous sodium carbonate to 100%. 190. mu.L aliquots of GSM-B detergent were added to 96-MTP containing 1 rinsed PAS38 mini-sample per well and the reaction was started by adding 10. mu.L of diluted enzyme (or just dilution solution as a blank). The 96-MTP was sealed and placed in an incubator/shaker at 40 ℃ and 1150rpm for 30 min. After incubation, 100 μ L of wash from each well was transferred to a new 96-MTP and its absorbance was measured at 405nm using a spectrophotometer. Protease activity on PAS38 model stains is reported as neat A₄₀₅(by A from the enzyme-treated sample₄₀₅Minus A of blank control₄₀₅)。

The dose response of PAS38 mini-samples to SspCPro29 and SspCPro33 using GSM-B detergent at pH 10.3 is shown in figure 6.

Example 11

Cleaning Performance of SspcPro29 and SspcPro33 under clothing conditions

EMPA-116 (cotton soiled with blood/milk/ink) microsamples (obtained from CFT tulip, the netherlands) were used to test the cleaning performance of SspCPro29 and SspCPro33 proteases in liquid and powder laundry detergents at either pH 8.0 or pH 10.0. Prior to testing, a commercial liquid detergent (Tide Clean) was usedProcter of Baojie Co&Gamble), usa) was incubated at 95 ℃ for 1 hour to inactivate enzymes present in the detergent. The heat-treated detergent was further diluted with 5mM HEPES (pH 8.0) to a final concentration of 0.788 g/L. Will be provided withWater hardness of buffered liquid detergents adjusted to 100ppm Ca²⁺:Mg²⁺(3:1 ratio). For buffered powder detergent formulations, commercial detergents (A), (B), (C), (Procter, china) was dissolved to reach 2g/L in water (water hardness 100ppm) and heated in a microwave oven to boil only to inactivate the enzyme. Proteolytic assays were subsequently performed to confirm inactivation of proteases in commercial detergents.

Prior to testing, the EMPA-116 microsamples were rinsed with water and air dried. To initiate the reaction, 190 μ L of buffered detergent was added to 96-MTP wells containing rinsed EMPA-116 mini-specimens followed by 10 μ L of diluted enzyme (or H)₂O as blank control). The 96-MTP was sealed and incubated for 20min in an iEMs at 32 ℃ and a thermostatic mixer at 16 ℃ respectively. After incubation, 100 μ L of wash from each well was transferred to a new 96-MTP and the absorbance was measured at 600nm using a spectrophotometer. By A from an enzyme-treated sample₆₀₀Minus A of blank control₆₀₀To calculate net A₆₀₀. Dose response curves performed on EMPA-116 mini-samples in liquid and powder laundry detergents at 16 ℃ and 32 ℃ for SspCPro29 and SspCPro 33. BPN' Y217L protease (SEQ ID NO:40) was used as a reference for HDL assessment, and GG36 protease (SEQ ID NO:41) was used as a reference for HDD assessment.

Fig. 7, 8 show the cleaning performance results using the HDL detergent at 16 ℃ and 32 ℃, and fig. 9 and 10 show the cleaning performance results using the HDD detergent at 16 ℃ and 32 ℃.

Example 12

Analysis of protein sequence of predicted Trypsin-type serine protease of full-Length Streptomyces species

Querying the Patent (Public and Genome Quest Patent) database (with search parameters set to default values) for Public and genomic queries, using predicted full-length amino acid sequences of SspPro 29(SEQ ID NO:22), SspPro 33(SEQ ID NO:23), SspPro 23(SEQ ID NO:24), and SspPro 59(SEQ ID NO:25), the relevant proteins were identified by BLAST search (Altschul et al, Nucleic Acids Res [ Nucleic Acids research ],25:3389-402,1997), and the subsets are shown in tables 6A and 6B (SspPro 29), respectively; tables 7A and 7B (SspCPro 33); in tables 8A and 8B (SspCPI 23); and tables 9A and 9B (SspCPI 59). Percent Identity (PID) of the two search sets was defined as the number of identical residues divided by the number of aligned residues in the pairwise alignment. The values on the table labeled "sequence length" correspond to the length (in amino acids) of the proteins referenced by the listed accession numbers, while "alignment length" is for the sequences used for alignment and PID calculations.

SspCPI 29(SEQ ID NO: 22); SspCPI 33(SEQ ID NO: 23); SspCPI 23(SEQ ID NO: 24); and SsppCPro 59(SEQ ID NO:25) as well as other Streptomyces species serine proteases: WP _064069271(SEQ ID NO: 26); WP _043225562(SEQ ID NO: 27); WP _024756173(SEQ ID NO: 28); WP _030548298(SEQ ID NO: 29); WP _005320871(SEQ ID NO: 30); WP _055639793(SEQ ID NO: 31); WO 2015048332-44360(SEQ ID NO: 32); WO 2015048332-44127(SEQ ID NO: 33); WP _030313004(SEQ ID NO: 34); WP _030212164(SEQ ID NO: 35); WP _030749137(SEQ ID NO: 36); WP _031004112(SEQ ID NO: 37); and WP _026277977(SEQ ID NO:38) were aligned using the MUSCLE program from Geneius software (biomaterials Ltd.) (Robert C. Edgar. MUSCLE: multiple sequence alignment with high accuracy and high throughput [ MUSCLE: multiple sequence alignment with high precision and high throughput ], Nucl. acids Res. [ nucleic acid research ] (2004)32(5):1792 and 1797) using default parameters. Multiple sequence alignments of the overlapping regions are shown in figure 11.

Example 13

Protein sequence analysis of the predicted catalytic domain of trypsin-type serine proteases of Streptomyces species

Query the patent databases against public and genomic (with search parameters set to default values), using the predicted catalytic domain sequences of SspPro 29(SEQ ID NO:18), SspPro 33(SEQ ID NO:19), SspPro 23(SEQ ID NO:20), and SspPro 59(SEQ ID NO:21), the relevant proteins were identified by BLAST search (Altschul et al, Nucleic Acids Res [ Nucleic Acids research ],25:3389-402,1997), and the subsets are shown in tables 10A and 10B, respectively (SspPro 29); tables 11A and 11B (SspPro 33); tables 12A and 12B (SspCPro 23); and tables 13A and 13B (SspCPI 59).

SspCPPro 29(SEQ ID NO: 18; aa 213-403 of SEQ ID NO: 3) was performed as described above; SspCPI 33(SEQ ID NO:19, aa204-394 of SEQ ID NO: 6); SspCPI 23(SEQ ID NO:20, aa201-391 of SEQ ID NO: 9); SspCPI 59(SEQ ID NO:21, aa 206 of SEQ ID NO: 12-); WP _064069271(SEQ ID NO:42, aa204 and 394 of SEQ ID NO: 26); WP _043225562(SEQ ID NO:43, aa204-394 of SEQ ID NO: 27); WP _024756173(SEQ ID NO:44, aa201-391 of SEQ ID NO: 28); WP _030548298(SEQ ID NO:45, aa 207 and 397 of SEQ ID NO: 29); WP _005320871(SEQ ID NO:46, aa204-394 of SEQ ID NO: 30); amino acid residue 138-328 of WP _029386953(SEQ ID NO: 47); WP _026277977(SEQ ID NO:48, aa 207 and 397 of SEQ ID NO: 38); amino acid residues 208-398 of WP _044383230(SEQ ID NO: 49); amino acid residue 193-383 of WP-069630550 (SEQ ID NO: 50); WP _055639793(SEQ ID NO:51, aa201-391 of SEQ ID NO: 31); amino acid residue 211-401 of WP _053699044(SEQ ID NO: 52); alignment of the predicted catalytic domain sequences of amino acid residues 205 and 395 of WP _031135572(SEQ ID NO:53) is shown in FIG. 12. The predicted catalytic domain consensus sequence from FIG. 12 is shown in SEQ ID NO 54. For positions in the consensus sequence, multiple amino acids are considered, which are depicted using the X ═ I or L and IUPAC codes: b ═ D or N.