ES2349172T3 - POLYPEPTIDES AND NUCLEIC ACIDS CODING THEM. - Google Patents

Abstract

Nucleic acid construct comprising a nucleic acid sequence encoding polypeptides, whose polypeptides improve feed utilization by improving food conversion coefficient (FCR), and whose nucleic acid sequence (a) hybridizes under medium astringency conditions with ( i) nucleotides 124-378 of SEQ ID NO: 1, a sub-sequence of (i) of at least 100 nucleotides, and / or (iii) a complementary chain of (i), or (ii); and / or (b) has an identity degree with nucleotides 124-378 of SEQ ID NO: 1 of at least 48%, as determined by the align program using the default identity matrix, a penalty of - 16 for the first waste of a space and penalties of -4 for subsequent waste of a space; with the proviso that the nucleic acid sequence is not of SEQ ID NO: 3, are not nucleotides 601-978 of SEQ ID NO: 3, and are not nucleotides 1-381 of SEQ ID NO: 1 the polynucleotides being operatively linked to one or more control sequences that direct the production of the polypeptide in a Bacillus host cell, the DNA of whose host cell, when collected and used as a DNA standard in a PCR reaction with SEQ ID NOs: 6 and 7 as primers, leads to the generation of a PCR fragment of a size of approximately 0.4 kb.

Description

       \ global \ parskip0.940000 \ baselineskip
    

Polypeptides and nucleic acids that encode

Field of the Invention

The present invention relates to the non-therapeutic use of certain polypeptides isolated in feed, for example to improve the Food Conversion Coefficient (FCR) and / or to modulate the intestinal microflora. An example of a polypeptide of the invention is the so-called Bacillus licheniformis ATCC 14580 L12 protein, which has here the amino acid sequence of amino acids +1 to +85 of SEQ ID NO: 2 (as follows amino acids 1-85 of SEQ ID NO: 2). The invention also relates to the non-therapeutic probiotic use in Bacillus strains feed which produce L12 related proteins.

Background of the invention State of the art

WO 960739 discloses the use of a combination of (i) a xylanase, (ii) a protease, and optionally a β-glucanase in food additives. At additive the proportion of xylanase activity per g of additive nutritional with respect to activity β-glucanase per g of food additive is of 1: 0-0.25. In WO 960739 it is stated that the inclusion of this enzymatic food additive improves digestion of animal.

WO 03/093453 sets forth, in Example 1, the design of an improved Bacillus licheniformis host cell which does not produce a small extracellular protein encoded by nucleotides 601 to 978 of SEQ ID NO: 133 of WO 03/093453, having The protein amino acid sequence of amino acids 1-126 of SEQ ID NO: 134 of WO 03/093453. SEQ ID Nos. 133 and 134 of WO 03/093453 are included in this sequence listing as SEQ ID Nos. 3 and 4, respectively. The amino acid sequence 1-126 of SEQ ID NO: 4 here is identical to amino acids -41 to +85 of SEQ ID NO: 2 here. WO 03/093453 does not reveal an isolated polypeptide having amino acids 1-85 of SEQ ID NO: 2. WO 03/093453 also does not reveal an isolated nucleic acid sequence having nucleotides 124-378 of SEQ ID NO: 1.

The GenPept accession no. YP_081375 is a hypothetical protein BL00275 from Bacillus licheniformis ATCC 14580. GenPept accession no. YP_081375 is identical to amino acids -41 to +85 of SEQ ID NO: 2 here. The sequence of the hypothetical protein BL00275 of Bacillus licheniformis ATCC 14580 has also been introduced in UniProtKB / TrEMBL with the primary accession number Q65CU4. This sequence is also identical to amino acids -41 to +85 of SEQ ID NO: 2 here.

The nucleotide sequence encoding YP_081375 has the GenBank accession no. NC_006270. Accession GenBank no. NC_006270 is identical to nucleotides 1-381 of SEQ ID NO: 1 here.

The present inventors surprisingly discovered that polypeptides related to part of this hypothetical sequence of Bacillus licheniformis , ie amino acids 1-85 of SEQ ID NO: 2, have great potential for use in feed.

The SWISSPROT accession no. Q8DQM5 is a hypothetical 115 amino acid spr0600 protein from Streptococcus pneumoniae (ATCC strain BAA-255 / R6). The sequence is also described in "Genome of the bacterium Streptococcus pneumoniae strain R6" by Hoskins et al , J. Bacteriol. 183: 5709 (2001). Q8DQM5 does not comprise an amino acid sequence that has at least 33% identity with amino acids 1-85 of SEQ ID NO: 2.

Martinez et al , Microbiology (1999), vol. 145, p. 3155-3161 reveals, in Fig. 3, the 91 amino acid sequence deduced from precocteriocin lactococcin 972 with a predicted mature C-terminal portion of 66 amino acids. The percent identity of each of the mature prepolypeptides and polypeptides with amino acids 1-85 of SEQ ID NO: 2 is less than 33%.

A search of nucleotide databases with nucleotides 124-378 of SEQ ID NO: 1 (which encodes amino acids 1-85 of SEQ ID NO: 2) revealed a DNA fragment with 47% identity with nucleotides 124-378 of SEQ ID NO: 1. This fragment is part of a sequence resulting from a sequencing project still in progress. The organism from which the fragment is derived is called strain 10 of Bacillus (Geobacillus) stearothermophilus . The sequencing work is done at the University of Oklahoma (http://www.genome.ou.edu/.bstearo.html). There is no publication of any of the amino acid sequences potentially corresponding to this DNA fragment, and therefore no indication of any potential utility of any encoded amino acid sequence.

A probiotic product mixed with the food designated as BioPlus ™ 2B is for sale through Chr. Hansen A / S, 10-12 Boege Allé, DK-2970 Hoersholm, Denmark. The product contains a strain of Bacillus licheniformis which nevertheless does not produce an L12-related protein (see Example 6 here).

It is an objective of the present invention the provide special strains of Bacillus, as well as methods not Therapeutics for the use of polypeptides and strains of Bacillus in feed.

       \ newpage
    

       \ global \ parskip1.000000 \ baselineskip
    

Summary of the Invention

The present invention relates to the use not therapeutic of isolated polypeptides selected from the group that consists of: (a) a polypeptide having amino acids 1-126 of SEQ ID NO: 4 (b) a polypeptide comprising an amino acid sequence which has a degree Identity with amino acids 1-85 of SEQ ID NO: 2 of at least 33%; as determined by the program Needle, using the BLOSUM62 replacement matrix, a penalty of space opening of 10, and an extension penalty of space of 0.5 and (c) a polypeptide which is encoded by a nucleic acid sequence that hybridizes under conditions of astringency means with (i) nucleotides 124-378 of SEQ ID NO: 1, (ii) a sub-sequence of (i) of at least 100 nucleotides, or (iii) a complementary chain of (i), or (ii); (C) Inside I think

The present invention also relates to nucleic acid constructs, vectors, and host cells comprising nucleic acid sequences as well as methods to produce and use polypeptides in feed.

The present invention also relates to the use in I think of a strain of Bacillus which is positive in the test from Example 6 here, that is, the DNA from which, when collected and collected used as a DNA standard in a PCR reaction with SEQ IDs Nos: 6 and 7 as primers, leads to the generation of a fragment PCR size of approximately 0.4 kb.

Detailed description of the invention The Polypeptide, its Characteristics, and Use

The invention relates to non-therapeutic use in I think of a polypeptide selected from the group consisting in:

(to)

a polypeptide having amino acids 1-126 of the SEC ID NO: 4;

(b)

a polypeptide comprising an amino acid sequence with a degree Identity with amino acids 1-85 of SEQ ID NO: 2 of at least 33%, as determined by the Needle program, using the BLOSUM62 replacement matrix, a penalty of space opening of 10, and an extension penalty of 0.5 and space

(C)

a polypeptide which is encoded by an acid sequence nuclei that hybridizes under medium astringency conditions with (i) nucleotides 124-378 of SEQ ID NO: 1, (ii) a sub-sequence of (i) of at least 100 nucleotides, and / or (iii) a complementary chain of (i), or (ii) as well as the use of any of these in the preparation of a composition for use in feed. These polypeptides improve feed utilization by improving the food conversion coefficient (FCR), and / or modulating the intestinal microflora

A polypeptide to be used in the present invention may be a bacterial or fungal polypeptide. In a second particular embodiment, the polypeptide is a Gram positive bacterial polypeptide such as a Bacillus polypeptide or a variant thereof, for example a Bacillus licheniformis polypeptide for example derived from Bacillus licheniformis ATCC 14580, which is the type strain of Bacillus licheniformis and is available upon request through the American Type Culture Collection, ATCC. Preferred strains of Bacillus licheniformis are positive in the test of example 6 here, such as the following strains of Bacillus licheniformis : ATCC 14580 (= NCIB 9375), NCIMB 6346 (= DSM 8785), NCTC 1024, NCTC 1025, NCTC 2120, NCTC 7589, NCTC 9932, ATCC 21424, NCIMB 10689, and ATCC 53757.

The polypeptide improves feed utilization improving the food conversion coefficient (FCR), and / or modulating the intestinal microflora. In embodiments alternatives, the polypeptide improves the digestibility of the feed, and / or maintains the health of the animal helping proper digestion and / or maintaining the function of the immune system.

The FCR can be determined based on a broiler growth test comprising a first treatment in which the polypeptide is added to the feed at a concentration of 5 mg per kg of feed, and a second treatment (control) without the addition of Polypeptide in the feed, each treatment consisting of 8 groups of 6 male chickens, the chickens being fed with granules of a corn / SBM48 diet ad libitum , the FCR being calculated as the consumption of feed in g / bird in relation to weight gain in g / bird during days 8-29 of the test, improving the FCR of the first treatment in relation to the FCR of the second treatment. In a particular embodiment the growth test is a cage test. For more details, see Example 5.

The FCR can also be determined based on a broiler growth test comprising a first treatment in which the polypeptide is added to the feed in a concentration of (i) 2.5, (ii) 5.0, or ( iii) 7.5 mg per kg of feed, and a second treatment (control) without adding the polypeptide in the feed, each treatment consisting of 10 groups of 6 male chickens, the chickens being fed a corn / SBM48 diet ad libitum , the FCR was calculated as the feed intake in g / bird in relation to the weight gain in g / bird on day 8-29 of the test, improving the FCR for the first treatment in relation to the FCR of the second treatment. In a particular embodiment the growth test is a cage test. For more details, see example 9.

       \ newpage
    

       \ global \ parskip0.990000 \ baselineskip
    

FCR can also be determined based on a broiler growth test comprising a first treatment in which the polypeptide is added to the feed at a concentration of 7.5 mg per kg of feed, and a second treatment (control) without adding the polypeptide in the feed, each treatment consisting of 12 groups of 20 chickens (6 groups of 20 females, 6 groups of 20 males), feeding the chickens with granules from a diet based on wheat, rye and soybean meal ad libitum , the FCR was calculated as feed consumption in g / bird in relation to weight gain in g / bird during days 1-36 of the test, improving the FCR for the first treatment in relation to the FCR of the second treatment. In a first particular embodiment the chickens are housed in chicken coops with the floor covered with wood chips. In a second particular embodiment, the chickens are fed with an initiation diet in the period of days 1-22 and a fattening diet in the period of days 22-36, both based on wheat, rye and soybean meal. , and with a composition as shown in Table 9. For more details, see Example 12.

\hbox{en al menos un 3,1%, 3,2%,
3,3%, 3,4%, 3,5%, 3,6%, 3,7%,  o al menos un 3,8%.}

As is generally known, an improved FCR is lower than the control FCR. In particular embodiments, the FCR is improved (ie, reduced) compared to the control by at least 1.0%, preferably at least 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or at least 2.5%. In other particular embodiments, the FCR is improved (ie reduced) compared to the control by at least 2.6%, 2.7%, 2.8%, 2.9%, or at least 3.0% In other particular embodiments, the FCR is improved (ie reduced) compared to the control

 \ hbox {at least 3.1%, 3.2%,
3.3%, 3.4%, 3.5%, 3.6%, 3.7%, or at least 3.8%.} 

The term "intestine" as used here designates the gastrointestinal or digestive tract (also referred to like the digestive tract) and this refers to the organ system within multicellular animals that take food, digest it to extract energy and nutrients, and expel the remaining waste. The intestine can be divided into the upper gastrointestinal tract and lower, the first comprising the mouth and stomach, and the second understanding the intestines. The intestines can be divide into small and large intestine. The basic components of small intestine, however with differences between species animals, are duodenum, jejunum, and ileum. The basic components of Large intestine are blind intestine, colon and rectum (sewer).

The term "intestinal microflora" as it is used here refers to natural microbial cultures residing in the intestine and maintaining health aiding in digestion appropriate and / or maintaining the function of the immune system.

The term "modular" as used in this case in relation to the intestinal microflora usually means change, manipulate, alter, or adjust the function or state of it in a healthy animal with normal functions, it is say a non-therapeutic use. Modulation is in response to polypeptides and / or Bacillus strains of the invention.

The following are particular examples not limiting the effect of the modulation of the intestinal microflora obtained by the polypeptide of the invention (changes in comparison with a control without the polypeptide) - for more details, see the Example 8:

(i)

an increase in the total number of facultative anaerobic bacteria in vivo , for example in piglets and / or broilers, preferably determined after the cultivation of ileo-rectal and / or cecal contents, respectively, in Brucella agar supplemented with 5% vol / vol of sheep's blood after incubation in an anaerobic chamber at 37 ° C for five days;

(ii)

an increase in the number of Escherichia coli in vivo , for example in piglets and / or broilers, preferably determined after cultivation of ileo-rectal and / or cecal contents, respectively, in chromogenic Coli-ID media, aerobically, at 37 ° C for 24 hours;

(iii)

no substantial change, or an increase, in total lactic acid bacteria in vivo , for example in piglets and / or broilers, preferably determined after the cultivation of ileo-rectal and / or cecal contents, respectively, in MRS agar , in an anaerobic chamber, at 37 ° C for 48 hours;

(iv)

no substantial change in the total of Lactobacillus spp. in vivo , for example in piglets, preferably determined after the cultivation of ileo-rectal contents in Rogosa agar , in an anaerobic chamber at 37 ° C for 48 hours;

(v)

a reduction in the number of other Enterobacteriaceae (other than E. coli ) in vivo , for example in piglets, preferably determined after the cultivation of ileo-rectal contents in a chromogenic Coli-ID medium, aerobically, at 37 ° C for 24 hours;

(saw)

a reduction in the number of Enterococcus spp. in vivo , for example in piglets, preferably determined after cultivation of ileo-rectal contents in an Enterococci agar , aerobically, at 37 ° C for 48 hours; I

(vii)

a reduction in the frequency with which Clostridium perfringens occurs in vivo , for example in piglets, preferably determined after culturing ileo-rectal contents in TSN agar after heating the sample at 80 ° C for 10 min, in an anaerobic chamber at 46 ° C for 24 hours.

       \ newpage
    

       \ global \ parskip1.000000 \ baselineskip
    

In addition, also in relation to the effect of the modulation of the intestinal microflora, and with reference to a control without the polypeptide of the invention, the polypeptide preferably:

(iix)

it is more active in vitro against Gram positive cocci than against Gram negative bacteria, that is, exhibiting a stronger reduction of the former, for example for Gram positive cocci and Gram negative bacteria isolated from intestinal contents of piglets and / or chickens fattening

(ix)

it is more active in vitro against Clostridium spp., for example Clostridium perfringens , than against Gram-negative bacteria, that is, exhibiting a stronger reduction of the first, for example for Clostridium perfringens and Gram-negative bacteria isolated from intestinal piglet content and / or broilers;

(x)

does not substantially influence the in vitro growth of beneficial microorganisms, such as bacteria, for example, such as those isolated from intestinal contents of piglets and / or broilers; and / or (xi) substantially influences, for example, reduces in vitro growth of harmful microorganisms, such as bacteria, for example, such as those isolated from intestinal contents of piglets and / or broilers.

For details regarding the ways of realization (iix) - (xi), please see the last part of the Example 8.

In the embodiment (x), the beneficial microorganisms are preferably selected from Lactobacillus spp., Such as Lactobacillus salivarius DSM 18070, for which MIC 90 is at least 1,000 micrograms / ml, preferably at least 2,000, 3,000, 4,000, 4,500, 5,000, 5,500, 6,000, or at least 7,000 micrograms / ml, in particular at least 4,170 micrograms / ml.

Accordingly, the polypeptide has a MIC 90 against Lactobacillus salivarius DSM 18070 of at least 1,000 micrograms / ml, preferably at least 2,000, 3,000, 4,000, 4,500, 5,000, 5,500, 6,000, or at least 7,000 micrograms / ml, in particular at least 4,170 micrograms / ml.

In the embodiment (xi), the harmful microorganisms are preferably selected from Enterococcus, Stafilococcus, Clostridium (preferably Clostridium perfringens ); such as Enterococcus faecalis DSM 18047, for which MIC 90 is less than 4,000 micrograms / ml, preferably less than 3,000, 2,000, 1,000, 800, 600, 500, 400, 300, 200, 100, 90, 80, or less than 70 micrograms / ml, preferably a MIC 90 of 50-80, more preferably 60-70 micrograms / ml.

Accordingly, the polypeptide has a MIC 90 against Enterococcus faecalis DSM 18047 of less than 4,000 micrograms / ml, preferably less than 3,000, 2,000, 1,000, 800, 600, 500, 400, 300, 200, 100, 90, 80, or less than 70 micrograms / ml, preferably a MIC 90 of 50-80, more preferably 60-70 micrograms / ml.

MIC 90 designates the inhibitory concentration minimum required for growth inhibition of 90% of organisms. For these objectives, MIC_ {90} is defined as the concentration of the polypeptide that is required to reduce the crop density, determined as OD 595, by 90% in relation to a control without the polypeptide. OD_ {595} is determined after incubation under appropriate incubation conditions (which depend on the organism in question, as shown in Table 5 from Example 8). The determination of MIC_ {90} can be done by a method of dilution of broth by microtiter, using approximately 10 5 CFU / ml of the pure bacteria in broth tripticasa-soy, adding the polypeptide in series of dilutions of 2 layers, using water as a control. For more details, see Example 8. MIC_ {90} is indicated here generally in the microgram units of the pure polypeptide by ml (micrograms / ml).

In other particular embodiments, the polypeptide:

(xii)

has a MIC_90 for a Gram negative bacterium, such as a strain of Escherichia coli, Klebsiella pneumoniae, Salmonella enteritidis, salmonella typhimurium , or Proteus mirabilis , which is at least 50% greater than a MIC_ {90} for a Coconut Gram positive, such as a strain of Enterococcus or Staphylococcus, for example Enterococcus faecalis , preferably Enterococcus faecalis DSM 18047 - in preferred sub-embodiments thereof, the MIC 90 of the former is preferably at least 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or at least 500% higher than the MIC_ {90} of the latter.

In a fourth particular embodiment (see Example 11 for more details), the polypeptide in its form native (i.e. not denatured), it is not degraded by (i) Pepsin (pH 3.5, 40 ° C, 2 hours); (ii) pancreatin (pH 7.0, 40 ° C, 4 hours); and / or (iii) pepsin-followed-by-pancreatin (pepsin at pH 3.5, 40 ° C, 2 hours - followed by pancreatin at pH 7.0, 40 ° C, 4 hours) - for all embodiments (i) - (iii) as considered by SDS-PAGE (see Example 10).

In a fifth particular embodiment (see Example 7 for more details), the polypeptide has the following denaturation temperatures, as determined by Differential analysis calorimetry (DSC): (i) at least 54ºC at pH 2.5, (ii) at least 68 ° C at pH 4.0, and / or (iii) at least 59 ° C at pH 7.0; preferably (iv) 55 ° C at pH 2.5; (v) 69 ° C at pH 4.0; I saw) 60 ° C at pH 7.0.

Identity and Hybridization, Fragments and Variants

The relationship between two amino acid sequences It is described by the "identity" parameter.

For purposes of the present invention, the alignment of two amino acid sequences is determined using the Needle program from the EMBOSS package (http://emboss.org) version 2.8.0. The Needle program implements the alignment algorithm global described in Needleman, S. B. and Wunsch, C. D. (1970) J. mol. Biol. 48, 443-453. The replacement matrix used is BLOSUM62, the penalty of opening space is 10, and the Space extension penalty is 0.5.

The degree of identity between a sequence of amino acids for use according to the present invention ("sequence of the invention "; for example amino acids 1-85 of SEQ ID NO: 2, and a sequence of Different amino acids ("strange sequence") are calculated as the number of exact correspondences in an alignment of the two sequences, divided by the length of the "sequence of the invention "or the length of the" strange sequence ", which be shorter The result is expressed in identity in percentage.

An exact correspondence occurs when the "sequence of the invention" and the "strange sequence" they have identical amino acid residues in the same positions of the coating (in the alignment example below, represented by "I"). The length of a sequence is the number of amino acid residues in the sequence (eg the length of amino acids 1-85 of SEQ ID NO: 2 is 85).

In the example of purely hypothetical alignment from below, the coating is the amino acid sequence "HTWGER-NL" of Sequence 1; or the sequence of amino acids "HGWGEDANL" of Sequence 2. In the example a space is indicated by a "-".

Example of hypothetical alignment:

one

The percent identity of a sequence of amino acids of a polypeptide with, or towards, amino acids 1-85 of SEQ ID NO: 2 is determined i) by aligning two amino acid sequences using the Needle program, with the BLOSUM62 replacement matrix, an opening penalty of space of 10, and a space extension penalty of 0.5; ii) counting the number of exact correspondences in the alignment; iii) dividing the number of exact correspondences by length of the shortest of the two amino acid sequences, and iv) converting the result of the division of iii) into a percentage. In the hypothetical example above, the number of correspondences exact is 6, the length of the shortest of the two sequences of amino acids is 12, therefore the percentage of identity is 50%

As an alternative, the degree of identity between two amino acid sequences, as well as the degree of identity between two nucleotide sequences, is determined by the program "Align" which is an alignment Needleman-Wunsch (that is, a global alignment). The program is used for polypeptide alignment, as well as of nucleotide sequences. The default scoring matrix BLOSUM50 is used for polypeptide alignments, and the matrix of Default identity is used for nucleotide alignments. The penalty for the first remainder of a space is -10 for polypeptides and -16 for nucleotides. The penalties for other residues of a space are -2 for polypeptides, and -4 for nucleotides "Align" is part of the v20u6 version of the FASTA package (see W. R. Pearson and D. J. Lipman (1988), "Improved Tools for Biological Sequence Analysis" PNAS 85: 2444-2448, and W. R. Pearson (1990) "Rapid and Sensitive Sequence Comparison with FASTP and FASTA ", Methods in Enzymology 183: 63-98). FASTA protein alignments use the Smith-Waterman algorithm without limitation in space size (see "Smith-Waterman algorithm ", T. F. Smith and M. S. Waterman (1981) J. Mol. Biol. 147: 195-197). See also Myers and Miller, CABIOS (1989) 4: 11-17.

In preferred embodiments, the degree of identity for amino acids 1-85 of SEQ ID NO: 2 is at least 35%, or at least 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90% , 92%, 95%, 97%, or at least 99%. Polypeptides with any of these degrees of identity for amino acids 1-85 of SEQ ID NO: 2 are referred to as homologous polypeptides. In an alternative embodiment, the degree of identity for amino acids 1-85 of SEQ ID NO: 2 is
32%

In particular embodiments, the polypeptides comprise (or have, or consist of) a sequence of amino acids that differ by (i) 57, 55, 50, 45, 40, 35, 30, or 25 amino acids of amino acids 1-85 of SEQ ID NO: 2 or by (ii) 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 amino acids of amino acids 1-85 of SEQ ID NO: 2 or by (iii) 10, 9, 8, 7, 6, or 5 amino acids of the amino acids 1-85 of SEQ ID NO: 2. In another form of particular embodiment, the polypeptides comprise (or have, or they consist of) an amino acid sequence that differs by 4, 3, or 2 amino acids, or per 1 amino acid of the amino acids 1-85 of SEQ ID NO: 2.

A fragment of, for example, amino acids 1-85 of SEQ ID NO: 2 is a polypeptide having one or more amino acids deleted from the amino and / or carboxyl terminal of these amino acid sequences. In an embodiment a fragment contains at least 30, 35, 40, 45, 50, or at least 55 amino acids. In another embodiment a fragment contains the at least 65 amino acid residues, or at least 70 residues of amino acids, or at least 75 amino acid residues, or at least 80 amino acid residues, or at least 81 amino acid residues, or at least 82 amino acid residues, or at least 83 residues of amino acids, or at least 84 amino acid residues.

An allelic variant denotes any of the two or more alternative forms of a gene occupying the same chromosomal location Allelic variation arises naturally at through mutation, and can result in a polymorphism within populations Genetic mutations can be silent (no change in the encoded polypeptide) or they can encode polypeptides having altered amino acid sequences. A allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

The present invention also relates to the use of isolated polypeptides that are encoded by sequences of nucleic acids that hybridize under astringency conditions medium, or medium-high, or high, or very high with a nucleic acid probe that hybridizes under them conditions with (i) nucleotides 124-378 of the SEQ ID NO: 1, (ii) a sub-sequence of (i), or (iii) a chain complementary to (i), or (ii) (J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York). In one embodiment Particularly the nucleic acid probe is selected from among nucleic acid sequences of (i), (ii), or (iii) above.

The nucleotide subsequence 124-378 of SEQ ID NO: 1 may be at least 100 nucleotides, or in another embodiment of at least 50, 150, or 200 nucleotides

The nucleic acid sequence of nucleotides 124-378 of SEQ ID NO: 1 or a Subsequence thereof, like the amino acid sequence of amino acids 1-85 of SEQ ID NO: 2 or a fragment thereof, can be used to design an acid probe nucleic to identify and clone DNA encoding polypeptides having a potential for use in strains of different strains genus or species according to methods well known in the art. In in particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following Southern blotting standard procedures, in order to Identify and isolate the corresponding gene here. Such probes they can be considerably shorter than the entire sequence, but they should be at least 15, preferably at least 25, and more preferably at least 35 nucleotides in length. I also know They can use longer probes. Both DNA probes and RNA Probes are typically labeled to detect the gene corresponding (for example, with 32 P, 3 H, 35 S, biotin, or avidin). Such probes are hereby understood. invention.

Thus, a genomic library of DNA or cDNA prepared from such other organisms can be selected for DNA that hybridizes with the probes described above and that encode a polypeptide having the desired activity. Genomic or other DNA from such other organisms can be separated by agarose or polyacrylamide gel electrophoresis, or other techniques from separation. Library DNA or separate DNA can be transfer to and immobilize in nitrocellulose or other carrier material suitable. To identify a clone or DNA that is homologous to the SEC ID NO: 1 or a sub sequence thereof, the carrier material is used in a southern blot. For the purposes of the present invention, hybridization indicates that the nucleic acid sequence hybridizes to a labeled nucleic acid probe corresponding to the nucleic acid sequence shown in SEQ ID NO: 1, its chain complementary, or a subsequent sequence thereof, under conditions of astringency from very low to very high. The molecules to which the Hybridizing nucleic acid probe under these conditions is They detect using a radiographic film.

In a particular embodiment, the probe of nucleic acids is a sequence of nucleic acids that encodes amino acids 1-85 of SEQ ID NO: 2, or Subsequences thereof. In another embodiment, the probe of nucleic acid is from nucleotides 124-378 of SEQ ID NO: 1 (the mature polypeptide coding region of the SEQ ID NO: 1).

For long probes of at least 100 nucleotides in length, astringency conditions from very low to very high defined as prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 µg / ml of cut salmon sperm DNA and denatured, and either 25% formamide for very astringent low and low, 35% formamide for medium astringency and medium-high, or 50% formamide for astringency high and very high, following Southern blotting procedures standards.

For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each time for 15 minutes using 2 X SSC, 0.2% SDS preferably at least 45 ° C (very low astringency), more preferably at least 50 ° C (low astringency), more preferably at least 55 ° C (medium astringency), more preferably at least 60 ° C (astringency medium-high), even more preferably at least at 65 ° C (high astringency), and most preferably at least at 70ºC (very high astringency).

For short probes of approximately 15 nucleotides approximately 70 nucleotides in length, the astringency conditions are defined as prehybridization, hybridization, and post-hybridization washing between 5 ° C and 10 ° C below the T m calculated using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48: 1390) in 0.9 M NaCl, 0.09 M of Tris-HCl at pH 7.6, 6 mM EDTA, 0.5% of NP-40, 1X Denhardt solution, 1 mM of sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM of ATP, and 0.2 mg of yeast RNA per ml following procedures Southern blotting standard.

For short probes of approximately 15 nucleotides approximately 70 nucleotides in length, the carrier material is washed once in 6X SSC plus 0.1% SDS for 15 minutes and twice 15 minutes each using 6X of SSC at 5 ° C to 10 ° C below the calculated T m.

The present invention also relates to the use of variants of the polypeptide having an amino acid sequence of amino acids 1-85 of SEQ ID NO: 2 comprising a substitution, deletion, and / or insertion of one or more amino acids.

The amino acid sequences of Variant polypeptides may differ from the amino acid sequence.  of amino acids 1-85 of SEQ ID NO: 2 for one insertion or deletion of one or more amino acid residues and / or the substitution of one or more amino acid residues with different amino acid residues Preferably, the changes of amino acids are of a minor nature, since they are the conservative amino acid substitutions those that do not affect significantly to the development and / or activity of the protein; small deletions, typically from one to about 30 amino acids; amino extensions carboxyl-terminals, such as a residue of amino terminal methionine; a small peptide linker of up to about 20-25 residues; or a small extension that facilitates purification by changing the net charge or other function, such as a polyhistidine tract.

Examples of conservative substitutions are in the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), amino acids hydrophobic (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Therefore, for example, the invention relates to a polypeptide having, or comprising, a sequence like the one set forth in SEQ ID NO: 2, preferably the mature part of it, where Conservative amino acid substitutions comprise replacements, one by another, among the basic amino acids (arginine, lysine and histidine), between the acidic amino acids (glutamic acid and acid aspartic), among the polar amino acids (glutamine and asparagine), between hydrophobic amino acids (alanine, leucine, isoleucine, and valine), among the aromatic amino acids (phenylalanine, tryptophan and tyrosine), and among the small amino acids (glycine, alanine, serine, threonine and methionine), or any combination of themselves, or active fragment thereof.

As defined herein, a polypeptide "isolated" or "pure" is a polypeptide that is essentially free of other polypeptides, for example, at least one 80% pure, preferably at least 85%, 86%, 87%, 88%, 89%, or at minus 90% pure, more preferably at least 91%, 92%, 93%, 94%, 95%, or at least 96% pure, as determined by the SDS-PAGE (e.g., by Coomassie staining and subsequent scanning by methods known in the art - see Examples 2 and 4). Purity can also be determined by HPLC, preferably by RP-HPLC (e.g., using a Waters \ mu-Bondapak C18 column, Mobile Phase A: 0.1% TFA, Mobile Phase B: Acetonitrile + 0.1% TFA, detection at 280 nm - see Example 10). Purity of SDS-PAGE, like HPLC purity, is refers to the amount of the polypeptide of the invention, in relation to the amount of protein In alternative embodiments, the polypeptide can be at least 20%, 40%, 60%, or at least 70% pure.

The amount of protein can be determined by any method known in the art, for example the method Kjeldahl (A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC), and the Amount of the polypeptide of the invention can be determined by SDS-PAGE and subsequent scanning, also by methods known in the art.

The polypeptides encoded by sequences of nucleic acids also include fused polypeptides or divisible fusion polypeptides to which other polypeptides are merge into the N-terminal or the C-terminal of the polypeptide or fragment thereof. A fused polypeptide is produced by fusion of a sequence of nucleic acids (or a part thereof) encoding another polypeptide to a nucleic acid sequence (or a part of the same) encoding a polypeptide to be used according to the present invention. Techniques for producing fusion polypeptides are known. in the art, and include linking the coding sequences encoding the polypeptides so that they do not suffer changes in the reading frame and that polypeptide expression merged is under the control of the same (s) promoter (s) and terminator.

In a specific embodiment, the polypeptide is a hypoallergenic variant, designed to invoke a reduced immune response when exposed to animals, including man. The term "immune response" should be understood as any reaction by the immune system of an animal exposed to the polypeptide. One type of immune response is an allergic response leading to increased levels of IgE in the exposed animal. Hypoallergenic variants can be prepared using techniques known in the art. For example, the polypeptide can be conjugated to protective portions of polymer fractions or epitopes of the polypeptide involved in an immunological response. Polymer conjugation may involve in vitro chemical coupling of the polymer to the polypeptide, for example as described in WO 96/17929, WO98 / 30682, WO98 / 35026, and / or WO99 / 00489. The conjugation may additionally or alternatively involve the in vivo coupling of polymers to the polypeptide. Such conjugation can be achieved by genetic engineering of the nucleotide sequence by encoding the polypeptide. Another way of providing hypoallergenic variants is by genetic engineering of the nucleotide sequence by encoding the polypeptide to cause the polypeptides to self-oligomerize, causing the polypeptide monomers to protect the epitopes of other polypeptide monomers and thus reducing the antigenicity of oligomers Such products and their preparation are described for example in WO 96/16177. The epitopes involved in an immune response can be identified by several methods such as the phage display method described in WO 00/26230 and WO 01/83559, or the randomized approach described in EP 561907. Once an epitope has been identified, its amino acid sequence can be altered to produce altered immunological properties of the polypeptide by known genetic manipulation techniques such as site-directed mutagenesis (see for example WO 00/26230, WO 00/26354 and / or WO 00/22103) and / or the conjugation of a polymer can be carried out in sufficient proximity to the epitope so that the polymer protects the epitope.

Bacterial strains; Probiotic Bacillus strains

The invention relates to non-therapeutic use in I think of a strain of Bacillus, the DNA from which, when harvested and is used as a DNA standard in a PCR reaction with SEQ IDs NOs: 6 and 7 as primers, leads to the generation of a fragment PCR size of approximately 0.4 kb. This test is for identify strains with an L12 type gene, see Example 6 here. These Bacillus strains can also be used in the preparation of a composition for non-therapeutic use in feed. These uses, by For example, they are intended to improve the coefficient of food conversion (FCR), and / or modulate microflora intestinal.

In a first particular embodiment, Bacillus strain is a probiotic microorganism. The term "probiotic" generally refers to a non-pathogenic bacteria given as food to animals, including birds, as a way of prevent colonization by pathogenic bacteria. The basic concept is to encourage colonization of mucous surfaces as a way of blocking colonization by serious pathogens. Probiotics they can also be defined as living, or liveable, microorganisms, that beneficially affect the intestinal balance of beings healthy humans and animals with normal functioning, preferably thus increasing the weight gain and / or improving the food conversion

In a second particular embodiment, Bacillus strain is used in the form of spores. Spores can be exospores or, preferably, endospores. An endospore is any spore that occurs within an organism (usually a bacterium). Endospores can survive periods of environmental stress, and are therefore capable of survive the passage of an aggressive (acidic) tract environment bowel of the upper gastro, while only exercising your effect once it reaches the intestines, where they will form normal vegetative cells.

In a third particular embodiment, the PCR fragment, purified and sequenced encodes a amino acid sequence which has at least 33% identity with amino acids 1-85 of SEQ ID NO: 2. The particular embodiments of the first aspect of the invention (the non-therapeutic use in polypeptide feed) also they are applicable to this aspect of the invention.

In other particular embodiments, the Bacillus strain is a strain of Bacillus licheniformis , preferably selected from the following strains of Bacillus licheniformis : ATCC 14580 (= NCIB 9375), NCIMB 6346 (= DSM 8785), NCTC 1024, NCTC 1025, NCTC 2120, NCTC 7589, NCTC 9932, ATCC 21424, NCIMB 10689, and ATCC 53757. A preferred subgroup includes Bacillus licheniformis ATCC 14580 (= NCIB 9375), and Bacillus licheniformis NCIMB 6346 (= DSM 8785).

In other particular embodiments, the Bacillus strain for use according to the invention is not (i) Bacillus licheniformis DSMZ 5749, (ii) is not Bacillus subtilis DSMZ 5750, and / or (iii) is not Bacillus licheniformis FERM BP- 266 The strains of (i) and (ii) are included in the product BioPlus ™ 2B, see, for example, EP 1472933. The strain of (iii) is described in GB 2138023.

We can find a classification and taxonomic identification of bacterial references in the Manual of Systematic Bacteriology (Bergey Manual (1986), vol 2; ISBN0-683-0783; see for example section 13 on p. 1104, "Endospore forming Gram positive rods and cocci";"GenusBacillus" on p. 1105; gender description on pages 1105-1129; description of the individual species Bacillus on p. 1130-1138, for example in Bacillus licheniformis on p. 1132). Alternatively, the well-known 16SrRNA sequence analysis can be used (see for example Johansen et al. , Int. J. Syst. Bacteriol, 1999, 49, 1231-1240, in particular the "Methods" section on p. 1.233, 2a column); or you can consult taxonomy experts, for example from DSMZ or other recognized depository institutes.

Bacillus strains, such as Bacillus licheniformis strains, are known in the art and are available through, for example, crop collections such as the ATTC mentioned above, or can be isolated from nature. Live, or liveable, Bacillus cell preparations can be prepared in the manner known in the art. Examples of such cells are vegetative cells, and spores such as endospores. In one embodiment, a fermentation extract of the Bacillus strain is used, for example in the form of a spray-dried fermentation solution.

The test of example 6 is a PCR reaction, in this example carried out with DNA isolated from several strains of Bacillus licheniformis . In a particular embodiment of this test, the DNA used as a standard for the PCR reaction is chromosomal DNA that can be isolated by methods known in the art. The test result of Example 6 is positive when a PCR fragment of the appropriate size is obtained. In Example 6, the appropriate size is indicated as 0.4 kb. In a particular embodiment, the appropriate size is between 0.35 kb and 0.44 kb (= 350 bp-440 bp). In alternative embodiments, the appropriate size is 330-430 bp, 340-420 bp, 350-410 bp, 360-400 bp, 370-390 bp, or 385-395 bp. The size of the coding sequence (CDS) of SEQ ID NO: 1 is approximately 380 bp (i.e. 378 bp).

Nucleic acid sequences

The invention also relates to the use not therapeutic of a polypeptide encoded by a sequence of isolated nucleic acids comprising an acid sequence polypeptide coding nuclei, which hybridizes under average astringency conditions with (i) nucleotides 124-378 of SEQ ID NO: 1, (ii) a sub sequence of (i) of at least 100 nucleotides, and / or (iii) a complementary chain of (i), or (ii).

Identity and hybridization are defined in a previous section.

In a first particular embodiment, the nucleic acid sequence encodes a polypeptide, the use which improves feed utilization by improving the coefficient of food conversion (FCR), and / or modulating microflora intestinal.

A particular nucleic acid sequence is nucleotides 124-378 of SEQ ID NO: 1, this one corresponding to the mature polypeptide coding region. Other particular nucleic acid sequences are those that encode the polypeptide of amino acids 1-85 of any of SEQ ID NOs: 2, 8, 9, and 10.

The present invention also includes the use non-therapeutic fragment of SEQ ID NO: 2 encoded by nucleic acid sequences comprising a sequence of nucleic acids encoding a polypeptide having the sequence of amino acids of amino acids 1-85 of SEQ ID NO: 2, which differ from the corresponding parts of the SEC ID NO: 1 with the help of the degeneracy of the genetic code. This includes the sub-sequences of SEQ ID NO: 1 which encode fragments of SEQ ID NO: 2.

A sub sequence of SEQ ID NO: 1 is a nucleic acid sequence comprised by SEQ ID NO: 1 except that one or more nucleotides of the 5 'and / or 3' end are has deleted. Preferably, a sub sequence it contains at least 150 nucleotides, more preferably at least 165, 180, 195, 210, 225, 240, 270, 285, 300, 315, 330, or more Preferably at least 345 nucleotides.

The present invention also relates to the use non-therapeutic polypeptide encoded by sequences of nucleotides which comprise a nucleic acid sequence encoding a polypeptide and which have a degree of nucleotide identity 124-378 of SEQ ID NO: 1 of at least 48%. To determine the degree of identity of nucleotides, the "align" program is used reference above.

In preferred embodiments, the degree of identity for nucleotides 124-378 of SEQ ID NO: 1 is at least 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, or at least 99% In alternative embodiments, the degree of identity for nucleotides 124-378 of SEQ ID NO: 1 is at least 32%, or at least 35%, 37%, 40%, 42%, 45%, or at minus 47%.

Such nucleotides also include sequences from mutant nucleic acids comprising at least one mutation in nucleotides 124-378 of SEQ ID NO: 1, in the which the mutant nucleic acid sequence encodes a polypeptide that (i) consists of amino acids 1-85 of SEQ ID NO: 2, or (ii) is a variant of the sequence of (i), where the variant comprises a substitution, deletion, and / or insertion of one or more amino acids, or (iii) is an allelic variant of the sequence of (i), or (iv) is a fragment of the sequence of (i).

Techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. Cloning of the nucleic acid sequences of such genomic DNA can be performed, for example, using the well-known polymerase chain reaction (PCR) or the selection of antibodies from expression libraries to detect cloned DNA fragments with shared structural characteristics. . See, for example, Innis et al ., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and amplification based on a nucleic acid sequence (NASBA) can be used. The nucleic acid sequence can be cloned from a strain of Bacillus, preferably Bacillus licheniformis , or other or a related organism and thus, for example, can be an allelic or species variant of the polypeptide coding region of The nucleic acid sequence.

The term "isolated nucleic acid sequence" as used herein refers to a nucleic acid sequence that is essentially free of other nucleic acid sequences, for example, at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90% pure as determined by agarose electrophoresis. For example, an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to reposition the nucleic acid sequence from its natural location to a different site where it will reproduce. The cloning procedures may involve cleavage and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where it is they will replicate multiple copies or clones of the nucleic acid sequence. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic, or any combination of the
same.

Modification of a nucleic acid sequence encoding a polypeptide used in the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to forms of unnatural origin of the polypeptide. These polypeptides may differ somewhat in their creation of the polypeptide isolated from its native source, for example, variants that differ in thermostability, pH stability, stability towards digestive enzymes, allergenicity, or the like. The variant sequence may be constructed based on the nucleic acid sequence presented as the coding part of the polypeptide of SEQ ID NO: 1, for example, a sub-sequence thereof, and / or by introduction of nucleotide substitutions that do not they give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but corresponding to the codon use of the host organism destined for the production of the polypeptide, or by introduction of nucleotide substitutions which can give rise to a sequence of different amino acids. For a general description of nucleotide substitution see, for example, Ford et al ., 1991, Protein Expression and Purification 2: 95-107. Hypoallergenic polypeptides can be prepared for example be prepared as described above.

The present invention also relates to the non-therapeutic use of a polypeptide encoded by isolated nucleic acid sequences comprising a nucleic acid sequence encoding a polypeptide and which hybridizes under medium astringency conditions, preferably medium-high astringency conditions, more preferably high astringency conditions, and more preferably very high astringency conditions with a nucleic acid probe that hybridizes under the same conditions with the nucleic acid sequence of SEQ ID NO: 1 or its complementary chain; or allelic variants and sub-sequences thereof (Sambrook et al ., 1989, supra ), as defined herein.

The present invention also relates to the use non-therapeutic polypeptide encoded by acid sequences isolated nuclei produced by (a) hybridization of a low DNA medium, medium-high astringency conditions, high, or very high with (i) nucleotide nucleotides 124-378 of SEQ ID NO: 1, (ii) a sub sequence of (i), or (iii) a complementary chain of (i), or (ii); and by (b) isolating from the nucleic acid sequence. The sub sequence is preferably a sequence of at least 100 nucleotides such as  a sequence encoding a polypeptide fragment.

The present invention further relates to methods to produce a mutant nucleic acid sequence, comprising the introduction of at least one mutation in the Mature polypeptide coding sequence of SEQ ID NO: 1 or a sub sequence thereof, where the acid sequence mutant nucleic encodes a polypeptide consisting of the amino acids 1-85 of SEQ ID NO: 2 or a fragment of the same.

The introduction of a mutation in the sequence of nucleic acids to exchange one nucleotide for another nucleotide can be performed by directed mutagenesis using any of the methods known in the art. Is particularly useful is the procedure that uses a DNA vector supercoiled double-stranded with an insert of interest and two synthetic primers containing the desired mutation. The primers oligonucleotides, each complementary to opposite strands of the vector, extend during cyclic temperature variation by Pfu DNA polymerase. In the incorporation of the primers, a mutated plasmid containing bevel cuts is generated. Behind the cyclic temperature variation, the product is treated with Dpnl which is specific for methylated and hemimethylated DNA to digest the parental DNA pattern and to select synthesized DNA containing mutation Other procedures can also be used. known in the art.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising an acid sequence nuclei operably linked to one or more control sequences which direct the expression of the coding sequence in a host of adequate expression. Suitable expression guests are cells Bacillus host, the DNA of which, when collected and used as a DNA standard in a PCR reaction with SEQ ID NOs: 6 and 7 as primers, as described in Example 6, leads to generation of a PCR fragment of a size of approximately 0.4 kb

Examples of suitable host cells are mentioned in the section below entitled "Host cells". The expression shall be understood as including any phase involved in the polypeptide production including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

       \ newpage
    

       \ global \ parskip0.880000 \ baselineskip
    

"Nucleic acid construct" is defined here as a nucleic acid molecule, either unique or double stranded, which has been isolated from a gene of natural origin or is has modified to contain combined nucleic acid segments and superimposed in such a way that otherwise they would not exist in the nature. The term nucleic acid construct is synonymous with term expression cassette when the nucleic acid construct it contains all the control sequences required for the expression of a coding sequence for a polypeptide to be used for the non-therapeutic purposes of the present invention. He term "coding sequence" is defined herein as a nucleic acid sequence that directly specifies the amino acid sequence of your protein product. The limits of the coding sequence is usually determined by a site  of ribosome binding (prokaryotes) or by the ATG start codon (eukaryotes) located just upstream of the frame of open reading at the 5 'end of the mRNA and a sequence of transcription terminator located just downstream of the open reading frame at the 3 'end of the mRNA. A sequence encoder may include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

An isolated nucleic acid sequence encoding a polypeptide to be used for non-therapeutic purposes  of the present invention can be manipulated in a variety of ways to provide polypeptide expression. The manipulation of the nucleic acid sequence before its insertion into a vector may be desirable or necessary depending of the expression vector. The techniques to modify sequences of nucleic acids using recombinant DNA methods are known Fine in the art.

The term "control sequences" is defined here by including all the components that are necessary or advantageous for the expression of a polypeptide to be used for non-therapeutic objectives of the present invention. Each sequence control can be native or foreign to the acid sequence nucleic acids encoding the polypeptide. Such control sequences include, but are not limited to, a sequence of polyadenylation, sequence of propeptides, promoter, sequence of signal peptides, and transcription terminator, leader. As minimum, control sequences include a promoter, and stop signals Transcriptional and translational. The control sequences are can provide with linkers for the purpose of introducing specific restriction sites to facilitate the ligation of control sequences with the coding region of the sequence of nucleic acids encoding a polypeptide. The term "operably linked" is defined here as a configuration in which a control sequence is properly placed in a position relative to the sequence coding of the DNA sequence so that the sequence of control directs the expression of a polypeptide.

The promoter sequence, that is a sequence of nucleic acids that is recognized by a host cell for nucleic acid sequence expression, contains sequences of transcriptional controls that mediate the expression of polypeptide. The promoter can be any acid sequence nuclei that show transcriptional activity in the cell host of choice including mutant promoters, truncated, and hybrids, and can be obtained from genes that encode extracellular or intracellular polypeptides either homologous or heterologists to the host cell.

Examples of promoters suitable for directing the transcription of the nucleic acid constructs of the present invention in a Bacillus licheniformis host cell that are positive in the test of Example 6 here are promoters obtained from the Bacillus licheniformis alpha-amylase gene. (amyl), the Bacillus stearothermophilus (amyM) maltogenic amylase gene, the Bacillus amyloliquefaciens alpha-amylase (amyQ) gene, a CriIIIA promoter (see WO 99/43835), as well as the endogenous L12 promoter of any of the specific Bacillus licheniformis host cells mentioned below.

The transcription terminator sequence is say a sequence recognized by a host cell to end The transcript is operatively linked to the 3 'terminal of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice is You can use in the present invention.

Preferred terminators for the aforementioned Bacillus licheniformis host cells are the terminators of the Bacillus licheniformis alpha-amylase gene (amyl), and the endogenous L12 terminator of any of these host cells.

The control sequence can also be a proper guide sequence, an untranslated region of an mRNA that is important for translation by the host cell. Sequence guide is operatively linked to terminal 5 'of the sequence of nucleic acids encoding the polypeptide. Any sequence guide that is functional in the host cell of choice can be use in the present invention.

The coding region of the signal peptide encodes an amino acid sequence linked to the amino terminal of a polypeptide that directs the encoded polypeptide in the pathway cell secretor. In a preferred embodiment, the coding region of the signal peptide are nucleotides 1-123 of SEQ ID NO: 1 coding the amino acids -41 to -1 of SEQ ID NO: 2.

According to the SignaIP Version 3.0 software, the Predicted signal peptide of SEQ ID NO: 2 is amino acids -41 to -2. This means that the predicted mature protein begins in the amino acid -1 of SEQ ID NO: 2, that is, Ala. However, according to Example 2 here, the N-terminal protein mature begins with amino acid +1 of SEQ ID NO: 2, that is, Trp, which means that the signal peptide part encompasses the amino acids -41 to -1 of SEQ ID NO: 2, which is an amino acid Longer than predicted.

       \ newpage
    

       \ global \ parskip1.000000 \ baselineskip
    

Therefore amino acids -1 to +85 of the SEC ID NO: 2 are an alternative mature form of the L12 protein, the use of which for non-therapeutic purposes in feed is also part of the present invention. Therefore, any claim and any statement here in reference to the amino acids 1-85 of SEQ ID NO: 2 can therefore either also, or alternatively, refer to amino acids -1 to +85 of SEQ ID NO: 2. The same happens for any claim and any statement here referring to the part Corresponding to SEQ ID NO: 1: Nucleotides 121-378 of SEQ ID NO: 1 may also refer to of, or as an alternative to, nucleotides 124-378 of SEQ ID NO: 1.

Also, any reference here to the part signal of SEQ ID NO: 2 may also, or alternatively, be for amino acids -41 to -2 of SEQ ID NO: 2. The part corresponding to SEQ ID NO: 1 is the nucleotides 1-120 of SEQ ID NO: 1. Any claim and any balance here in reference to the signal peptide part it can therefore also, or alternatively, refer to the nucleotides 1-120 of SEQ ID NO: 1.

The SignaIP V. 3.0 method is described in Bendtsen et al in the Journal of Molecular Biology 2004, 340 (4), p. 783-95. See also Nielsen et al in Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (ISMB 6), AAAI Press, Menlo Park, California, pp 122-130, 1998 (V. 2.0); and Nielsen et al in Protein Engineering 10, 1-6 (1997) (V. 1.1).

Expression vectors

The present invention also relates to recombinant expression vectors comprising a construct of nucleic acids, such as recombinant expression vectors comprising a nucleic acid sequence encoding a polypeptide to be used according to the present invention, a promoter, and Transcriptional and translational stop signals. The different nucleic acid and control sequences described above can be joined to produce an expression vector recombinant that may include one or more restriction sites convenient to allow the insertion or replacement of the nucleic acid sequence encoding the polypeptide in such sites. Alternatively, the nucleic acid sequence is can express by insertion of the nucleic acid sequence or a nucleic acid construct comprising the sequence in a appropriate vector for expression. In the creation of the vector of expression, the coding sequence is located in the vector of such so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be submitted conveniently to recombinant DNA procedures and can cause the expression of the nucleic acid sequence. The Vector choice will typically depend on the compatibility of the vector with the host cell into which the vector will be introduced. Vectors can be linear or circular plasmids closed.

The vector can be a replication vector autonomous, that is, a vector that exists as an entity extrachromosomal, the replication of which is independent of the chromosomal replication, for example, a plasmid, an element extrachromosomal, a minichromosome, or an artificial chromosome. He vector can contain any means to ensure the self-replication Alternatively, the vector may be one that, introduced into the host cell, it integrates into the genome and it replicates with the chromosome (s) in which / which has been integrated. In addition, a single vector or plasmid or two or more vectors or plasmids that contain the total DNA together at introduce into the genome of the host cell, or a transposon.

The vectors of the present invention preferably contain one or more selectable markers that allow easy selection of transformed cells. A selectable marker is a gene the product of which provides biocidal or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the Bacillus subtilis or Bacillus licheniformis dal genes, or markers that confer antibiotic resistance such as ampicillin resistance, kanamycin, chloramphenicol or tetracycline.

The vectors of the present invention contain preferably an element (s) that allows (n) the stable integration of the vector into the genome of the host cell or autonomous replication of the vector in the cell genome independent.

For integration into the host cell genome, the vector may be based on the nucleic acid sequence by encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional nucleic acid sequences to direct integration by homologous recombination into the genome of the host cell. Additional nucleic acid sequences allow the vector to integrate into the genome of the host cell in one (s)
precise location (s) on the chromosome (s). To increase the likelihood of integration in a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous to the corresponding target sequence to improve the probability of homologous recombination. The integrational elements can be any sequence that is homologous to the target sequence in the genome of the host cell. In addition, the integrational elements may not be encoding or encoding nucleic acid sequences. Instead, the vector can be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication allowing the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACiC177, and pACiC184 allowing replication in E. coli , and pUB110, pE194, pTA1060, and pAMβ1 allowing replication in Bacillus. The origin of replication can be one having a mutation that makes its thermosensitive operation in the host cell (see, for example, Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75: 1433).

You can insert more than one copy of one nucleic acid sequence in the host cell to increase the Production of the genetic product. You can get an increase in the number of copies of the nucleic acid sequence integrating the minus an additional copy of the sequence in the cell genome host or including a selectable marker gene amplifiable with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and therefore additional copies of the nucleic acid sequence, you can select by culturing the cells in the presence of the agent selectable appropriate.

The methods used to link the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, for example, Sambrook et al ., 1989, supra ).

The polypeptide can also be coexpressed together. with at least one other component of interest to the feed, such as a polypeptide with a desired enzymatic activity for use in I think. The polypeptide and at least one other polypeptide can be co-express from different vectors, from a vector, or using A mixture of both techniques.

Host cells

The invention also relates to the use for non-therapeutic targets in feed from a host cell of Recombinant Bacillus comprising an acid construct nuclei of the invention and / or an expression vector of the invention. The DNA of the host cell, when collected and used as a DNA standard in a PCR reaction with SEQ ID NOs: 6 and 7 as primers, leads to the generation of a PCR fragment of a size of approximately 0.4 kb.

In a particular embodiment, the PCR fragment, purified and ordered encodes a sequence of amino acids having at least 33% identity with amino acids 1-85 of SEQ ID NO: 2.

The particular embodiments (in particular those described in the sections entitled "The Polypeptide, its Characteristics, and Use "e" Identity and Hybridization, Fragments and Variants ") are also applicable to the host cells of the invention. For example, in other forms of particular embodiments of the Bacillus host cell of the invention, the PCR fragment, purified and ordered, encodes a amino acid sequence which has a degree of identity with amino acids 1-85 of SEQ ID NO: 2 of at least one 35%, or at least 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, or at least 99%.

In other particular embodiments, the Bacillus host cell is a Bacillus licheniformis host cell, preferably selected from the following Bacillus licheniformis strains: ATCC 14580 (= NCIB 9375), NCIMB 6346 (= DSM 8785), NCTC 1024 , NCTC 1025, NCTC 2120, NCTC 7589, NCTC 9932, ATCC 21424, NCIMB 10689, and ATCC 53757. A preferred subgroup includes Bacillus licheniformis ATCC 14580 (= NCIB 9375), and Bacillus licheniformis NCIMB 6346 (= DSM 8785).

These host cells are advantageously used in recombinant production of polypeptides. A vector comprising a nucleic acid sequence encoding a polypeptide to be used according to the present invention is introduced into the host cell so that the vector is maintained as an integral chromosomal or as an extrachromosomal vector that doubles as described above. The term "host cell" comprises any descendant of the parental cell that is not identical to the parental cell due to mutations that occur during replication.

The introduction of a vector into a cell bacterial host can, for example, be effected by transformation  protoplast (see, for example, Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), using cells competent (see, for example, Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, for example, Shigekawa and Dower, 1988 Biotechniques 6: 742-751), or conjugation (see, for example, Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).

Production methods

The invention also relates to methods to produce a polypeptide to be used according to the invention, the method comprising (a) culturing a recombinant host cell of the invention to produce a supernatant comprising the polypeptide; and (b) recover the polypeptide.

       \ newpage
    

       \ global \ parskip0.910000 \ baselineskip
    

The invention further relates to a method for the production of such a polypeptide comprising (a) cultivating a Bacillus strain, the DNA from which, when collected and used as a DNA standard in a PCR reaction with SEQ ID NOs: 6 and 7 as primers, leads to the generation of a PCR fragment of a size of about 0.4 kb; and (b) recover the polypeptide.

Examples of host cells are mentioned in the previous section entitled "Host cells".

The present invention also relates to methods for producing a polypeptide to be used according to the present invention comprising (a) culturing a recombinant host cell of Bacillus licheniformis ATCC 14580 (= NCIB 9375) or Bacillus licheniformis NCIMB 6346 (= DSM 8785) under auspicious conditions for the production of the polypeptide, wherein the host cell comprises a mutant nucleic acid sequence comprising at least one mutation in nucleotides 124-378 of SEQ ID NO: 1, where the mutant nucleic acid sequence encodes a polypeptide that (i) consists of amino acids 1-85 of SEQ ID NO: 2, or (ii) is a variant of the sequence of (i), where the variant comprises a substitution, deletion, and / or insertion of one or more amino acids, or (iii) is an allelic variant of the sequence of (i), or (iv) is a fragment of the sequence of (i).

In the production methods of this invention, cells are grown in a suitable nutrient medium for the production of the polypeptide using methods known in the technique. For example, the cell can be cultured by culture in stirring flask, small-scale or large-scale fermentation (including continuous fermentations, in flow, in flow discontinuous, in repeated discontinuous flow, or in solid state) in laboratory or industrial fermenters in a suitable and low environment conditions allowing the polypeptide to be expressed and / or isolated. He crop is grown in a suitable nutrient medium comprising sources of nitrogen and carbon and inorganic salts, using procedures known in the art. Suitable means are available through commercial suppliers or can be prepared according to published compositions (e.g., in American catalogs Type Culture Collection). If the polypeptide is secreted in the medium nutritious, it can be recovered directly from the environment. If he polypeptide is not secreted, it can be recovered from cell lysates

Polypeptides can be detected using methods known in the art that are specific to polypeptides These detection methods may include the use of specific antibodies; gel analysis SDS-PAGE revealing a molecular weight band relative, Mr, of approximately 12 kDa; and / or the determination of the N-terminal sequence, in purified samples and / or in the band of a 12 kDa Mr, as a clipping of the gel SDS-PAGE. In this case, the N-terminal should preferably have a identity with SEQ ID NO: 5 of at least 33%, or at least 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, or at minus 99%, the percentage of identity being determined as generally described above).

The resulting polypeptide can be recovered by methods known in the art. For example, the polypeptide is can recover from the nutrient medium by procedures conventional including, but not limited to, centrifuged, filtration (such as ultrafiltration and / or diafiltration), extraction, spray drying, evaporation, or precipitation.

The polypeptides can be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic chromatoenfoque, and size exclusion), electrophoretic procedures (e.g., isoelectric focusing) preparatory), differential solubility (e.g. precipitation of ammonium sulfate), SDS-PAGE, or extraction (see, for example, Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

Compositions and Uses

In a further aspect, the present invention refers to compositions comprising a polypeptide and / or a Bacillus strain of the present invention.

Polypeptide compositions can be prepare according to methods known in the art and can be in the form of a liquid or a dry composition. For example, the Polypeptide composition may be in the form of a granulate or a microgranulate. The polypeptides to be included in the composition are they can stabilize according to methods known in the art.

Particular examples of compositions of the invention are as follows:

An additive for I think comprising (a) i) a polypeptide having amino acids 1-126 of SEQ ID NO: 4 and / or ii) a polypeptide comprising an amino acid sequence that has a degree of identity with amino acids 1-85 of SEQ ID NO: 2 of at least 33%, as determined by the Needle program, using the BLOSUM62 replacement matrix, a penalty of space opening of 10, and an extension penalty of 0.5 space; and (b) at least one fat-soluble vitamin, (c) at less a water-soluble vitamin, (d) at least one trace element, and / or (e) at least one macromineral;

a composition of feed having a crude protein content of 50 to 800 g / kg and comprising i) a polypeptide having amino acids 1-126 of SEQ ID NO: 4 and / or ii) a polypeptide comprising an amino acid sequence which has a degree Identity with amino acids 1-85 of SEQ ID NO: 2 of at least 33%, as determined by the program Needle, using the BLOSUM62 replacement matrix, a penalty of space opening of 10, and an extension penalty of 0.5 space;

an additive for I think comprising (a) a strain of Bacillus as defined in the section entitled "Bacterial strains; Bacillus strains probiotics "; and (b) at least one fat-soluble vitamin, (c) at less a water-soluble vitamin, (d) at least one trace element, and / or (e) at least one macromineral; Y

a composition of feed having a crude protein content of 50 to 800 g / kg and comprising a strain of Bacillus as defined in the section entitled "Bacterial strains; Bacillus strains probiotics. "

The so-called premixes are examples of feed additives of the invention. A premix designates a preferably uniform mixture of one or more micro-ingredients with diluent and / or carrier. The premixes are used to facilitate uniform dispersion of Micro-ingredients in a larger mixture.

Examples of preferred uses are given above the invention (in the sections entitled "The Polypeptide, its Characteristics, and Uses "and" Bacterial strains; Strains of Probiotic Bacillus "), and more details are given below.

I think

The term animal includes all animals. Examples of animals are non-ruminants, and ruminants. The ruminant animals include, for example, animals such as sheep, goats, and cattle, for example cow as cattle beef and dairy cow In one embodiment In particular, the animal is a non-ruminant animal. Animals not Ruminants include mono-gastric animals; by example the pig or pig (including, but not limited to, piglets, growth pigs, and pigs); birds such as turkeys, ducks and chickens (including but not limited to broilers, laying hens); fish (including but not limited to salmon, trout, tilapia, catfish and carp); and crustaceans (including but not limited to prawn and crayfish).

The term feed or feed composition means any compound, preparation, mixture, or composition suitable for, or intended for ingestion by an animal.

In the use according to the invention the polypeptide and / or Bacillus strain can be given to the animal before, after, or simultaneously with the diet. It is preferred last.

In a particular embodiment, the polypeptide, in the form in which it is added to the feed, or being included in a feed additive, it is well defined. The term well defined means that the polypeptide preparation is at minus 50% pure, determined as previously described, or by Size exclusion chromatography (see example 12 of WO 01/58275). In other particular embodiments the Well defined polypeptide preparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure.

A well defined polypeptide preparation is advantageous For example, it is much easier to dose correctly in the feed a polypeptide that is essentially safe from interfere or contaminate other polypeptides. The term dose correctly refers in particular to the objective of obtaining consistent and consistent results, and the ability to optimize the dosage based on the desired effect.

For use in feed, however, the polypeptide does not need to be so pure; this one for example can include other polypeptides such as feed enzymes, in which case this could be termed as a preparation of polypeptides

The polypeptide preparation can be (a) add directly to the feed (or use directly in a process of protein treatment), or (b) it can be used in production of one or more intermediate compositions such as additives food or premixes that will be added later to the feed (or will be used in a treatment process). Degree of purity described above refers to the purity of the preparation of Original polypeptides, whether used according to (a) or (b) above.

Polypeptide preparations with purities of this order of magnitude are obtainable in particular using methods production recombinants, while these are not so easily obtainable and also undergo variation by lots much larger when the polypeptide is produced by methods of traditional fermentation.

In the present context, the term coefficient of food conversion, or FCR, is used as a synonym for term food conversion. The FCR is calculated as consumption of feed in g / animal in relation to weight gain in g / animal, see for example Table 2 in Example 5.

The polypeptide can be added to the food in Either way, being in the form of a relatively polypeptide pure, or in combination with other components intended for addition to feed, that is in the form of feed additives, such as the so-called premixes for feed.

Apart from the Bacillus polypeptide and / or strain, feed additives of the invention contain at least one fat-soluble vitamin, and / or at least one water-soluble vitamin, and / or at least one trace element, and / or at least one macromineral.

       \ newpage
    

       \ global \ parskip1.000000 \ baselineskip
    

Other optional additive ingredients for I think they are coloring agents, for example carotenoids such as beta-carotene, astaxanthin, and lutein; compounds flavorings; stabilizers; antimicrobial peptides; acids polyunsaturated fats; reactive oxygen generating species; and / or at least one enzyme selected from phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4.-.-), phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (EC 3.1.4.3); phospholipase D (EC 3.1.4.4); amylase such as, for example, alpha-amylase (EC 3.2.1.1); I beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6). In a particular embodiment these other enzymes are fine defined (as defined above for the preparations of polypeptides).

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A, Tritrpticin, Protegrin-1, Tanatine, Defensin, Lactoferrin, Lactoferricin, and ovispirin such as novispirin (Robert Lehren, 2000), Plectasins, and Statins, including the compounds and polypeptides described in WO 03/044049 and WO 03/048148, as well as variants or fragments of the foregoing that retain antimicrobial activity.

Examples of antifungicidal polypeptides (AFPs) are the Aspergillus giganteus , and Aspergillus niger peptides, as well as variants and fragments thereof that retain antifungal activity, as described in WO 94/01459 and WO 02/090384.

Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentanoic acid and gamma-linolenic acid.

Examples of reactive species generating oxygen are chemicals such as perborate, persulfate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a synthetase

Normally water soluble lipo- e vitamins, like trace elements are part of a so-called premix intended for addition to feed, while Macrominerals are usually added separately to feed. Any of these types of composition, when enriched with a polypeptide or a strain of Bacillus of the invention, is an additive for feed of the invention.

In a particular embodiment, the feed additive of the invention is intended to be included (or prescribed as having to be included) in diets or food of animals at levels of 0.01 to 10.0%; more particularly 0.05 5.0%; or 0.2 to 1.0% (% meaning g of additive per 100 g of I think). This is particularly true for premixes.

The following are non-exclusive lists of Examples of these components:

Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin K, for example vitamin K3

Examples of water-soluble vitamins are vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and pantothenate, for example Ca-D-pantothenate.

Examples of trace elements are manganese, zinc, iron, copper, iodine, selenium, and cobalt.

Examples of macrominerals are calcium, phosphorus and sodium.

The nutritional requirements of these components (exemplified with birds and piglets / pigs) are cataloged in the Table A of WO 01/58275. Nutritive requirement means that these components should be provided in the diet in indicated concentrations.

As an alternative, the feed additive of the invention comprises at least one of the individual components specific in Table A of WO 01/58275. At least one means any of, one or more of, one, or two, or three, or four, etc. up to a total of thirteen, or up to a total of fifteen components individual. More specifically, this at least one component individual is included in the additive of the invention in an amount such to provide a concentration in food in the range indicated in column four, or column five, or column six of Table A.

Feed compositions or diets for Animals have a relatively high protein content. The Poultry and pig diets may be characterized as indicated in Table B of WO 01/58275, columns 2-3. Diets for fish they can be characterized as indicated in the column 4 of this Table B. In addition such diets for fish normally They have a crude fat content of 200-310 g / kg

A feed composition according to the invention It has a crude protein content of 50-800 g / kg, and also comprises at least one polypeptide and / or at least one strain of Bacillus as described and / or claimed here.

In addition, or as an alternative (to the content of raw protein indicated above), the feed composition of the invention has a metabolizable energy content of 10-30 MJ / kg; and / or a calcium content of 0.1-200 g / kg; and / or an available phosphorus content 0.1-200 g / kg; and / or a methionine content of 0.1-100 g / kg; and / or one more methionine content 0.1-150 g / kg cysteine; and / or a lysine content 0.5-50 g / kg.

In particular embodiments, the metabolizable energy content, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and / or lysine is inside of any of ranges 2, 3, 4 or 5 in Table B of WO 01/58275 (R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25, that is, Crude protein (g / kg) = N (g / kg) x 6.25. The nitrogen content is determined by the method Kjeldahl (A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC).

The metabolizable energy can be calculated based on the NRC publication Nutrient requirements in swine (nutrient requirements in pigs of the NRC publication) ninth 1988 corrected edition, subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council (subcommission on nutrition in pigs, commission on nutrition in pigs, agriculture board, council of national investigation). National Academy Press, Washington, D.C., P. 2-6, and the European Table of Energy Values for poultry feed, Spelderholt research center of birds and extension, 7361 DA Beekbergen, The Netherlands. Grafisch Bedrijf Ponsen & Looijen BV, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, phosphorus available and amino acids in diets for whole animals are calculates based on food tables such as the Veevoedertabel of 1997, gegevens on chemische samenstelling, verteerbaarheid in voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the feed composition of the invention contains at least one Vegetable protein or protein source. Can also contain animal protein, such as meat and bone meal, and / or meal fish, typically in an amount of 0-25%. He term vegetable protein as used in this case refers to any compound, composition, preparation or mixture that includes at least one protein derived from or originated from a vegetable, including modified proteins and protein derivatives. In forms of particular embodiment, the protein content of the Vegetable protein is at least 10, 20, 30, 40, 50, or 60% (p / p).

Vegetable proteins may be derived from sources of vegetable protein, such as legumes and cereals, for example plant materials of the Fabaceae families (Leguminosae), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy flour, lupine flour and rapeseed meal.

In a particular embodiment, the Vegetable protein source is material from one or more plants of the Fabaceae family, for example soybean, lupine, pea, or Bean.

In another particular embodiment, the Vegetable protein source is material from one or more plants of the Chenopodiaceae family, for example beet, sugar beet, spinach or quinoa

Other examples of vegetable protein sources they are rapeseed, sunflower seed, cottonseed, and cabbage.

Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oats, corn (cob), rice, triticale, and sorghum.

In other particular embodiments, the feed composition of the invention contains a 0-80% corn; and / or 0-80% of sorghum; and / or 0-70% wheat; and / or a 0-70% barley; and / or 0-30% of oats; and / or 0-30% rye; and / or a 0-40% soy flour; and / or a 0-25% fishmeal; and / or a 0-25% meat and bone meal; and / or a 0-20% whey.

Animal diets can be manufactured by example as pureed food (not granulated) or granulated food. Typically, the crushed food materials are mixed and add sufficient amounts of essential vitamins and minerals according to the specifications for the species in question. They polypeptide (s) and / or Bacillus strain can be added as solid or liquid formulations. For example, a formulation Solid polypeptide is typically added before or during the stage of mixing; and a liquid polypeptide preparation is added typically after the granulation stage. Polypeptide It can also be incorporated as a feed additive or premix

The final polypeptide concentration in the diet is in the range of 0.01-200 mg of protein per kg of diet, for example in the range of 0.1-20 mg of protein per kg of animal diet.

The Bacillus strain and / or strain should be added of course in an effective amount, that is in a adequate amount to improve feed conversion.

It is contemplated herein that the polypeptide be administered in one or more of the following amounts (rangps of dosage): 0.01-200, 0.01-100, 0.5-100, 1-50, 5-100, 10-100, 0.05-50, 1-10 or 0.10-10, all these ranges being in mg of polypeptide protein per kg (ppm) of feed. In a form of particular embodiment, the dosage range is 1-9, 1-8, 2-7, 2-6, or 2-5 ppm. Range Examples Particularly preferred dosage are; 0.5-15.0, 1.0-12.5, 1.5-10.0, and 2.5-7.5 ppm. The amount of the polypeptide is determined as described for the L12 protein in Example 10.

It is contemplated herein that the strain of Bacillus is administered in one or more of the following amounts (dosage ranges): 10 E2-14, 10 E4-12, 10 E6-10, 10 E7-9, preferably 10 E8 CFU / g of feed (the designation E means exponent, i.e., for example, 10 E2-14 means 10 2 -10 14).

To determine the mg of protein from polypeptide per kg of feed, the polypeptide is purified from of the feed composition, and the dosage in mg of polypeptide protein per kg of food, for example as described in Example 10. The same principles apply to determine mg of polypeptide protein in additives food.

Deposit of biological material

The following biological material, which is isolated from intestinal contents of broilers, as described in Example 8, has been deposited according to the conditions of the Budapest treaty with the DSMZ (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany) and has received The following registration numbers:

2

Deposits were made by Novozymes A / S, Krogshoejvej 36; DK-2880, Denmark.

Examples

Example one

Fermentation in a shaker flask of Bacillus licheniformis

Bacillus licheniformis ATCC 14580 was propagated overnight at 37 ° C in TY agar medium (TY solidified broth with 2% agar) and inoculated in a shake flask containing 100 ml of TY broth with the following composition: tryptone: 20 g / l, yeast extract: 5 g / l, FeCl 2, 4H 2 O: 7 mg / l, MnCl 2, 4H 2 O: 1 mg / l, MgSO 4, 7H 2 O: 15 mg / l, pH 7.3. The shake flask was incubated at 37 ° C for 20 hours with a stirring speed of 225 rpm. The cells were removed by centrifugation.

SDS polyacrylamide gel electrophoresis of the supernatant revealed a band of a relative molecular weight of 12 kDa corresponding to the desired polypeptide. The polypeptide is called as "the L12 protein" due to its molecular weight relative of 12 kDa, per SDS-PAGE (however, the Theoretical molecular weight can be calculated at 9591.56 Da).

Example 2

Purification of the fermentation in a shaker flask of Bacillus licheniformis

The fermentation broth of Example 1 is centrifuged (20,000 x g, 20 min) and the supernatants were decanted Carefully precipitate. The combined supernatants are filtered through a Seitz EKS plate to remove cells of Bacillus. The EKS filtrate was ultrafiltered and diafiltered in Filtron 3k trimmed cassettes to concentrate proteins and reduce the conductivity in the filtering of Seitz EKS. The concentrate  Filtron was filtered through another Seitz EKS plate.

The pH of the concentrate was adjusted to pH 4.5 with a 20% CH 3 COOH. Something (not L12 protein) in the solution is precipiton during pH adjustment and this precipitate was removed by filtration through a Seitz K-250 plate. He K-250 filtrate was applied to a 100 ml column of SP-sepharose FF equilibrated in 20 mM CH 3 COOH,  50 mM of H 3 BO 3, 1 mM of CaCl 2, and adjusted to pH 4.5 with NaOH After washing the column extensively with the equilibration buffer, the L12 protein was eluted with a gradient of Linear NaCl (0 - -> 0.5M) in the same buffer. The protein L12 containing fractions was identified by analysis of SDS-PAGE and these fractions were grouped and diluted 10 times with demineralized water to reduce set conductivity. The set was added to a column 8 ml SOURCE S balanced in the same equilibration buffer (20 mM of CH 3 COOH, 50 mM of H 3 BO 3, 1 mM of CaCl 2, adjusted to pH 4.5 with NaOH) and after washing the column extensively with the equilibration buffer, the L12 protein is eluted with a gradient of linear NaCl (0 - -> 1.0M) in The same buffer.

Fractions of the column were analyzed by SDS-PAGE analysis and L12 protein containing fractions were grouped, the pH was adjusted to pH 8 with 3% NaOH and the Grouping was left in the cold chamber until the next day. He adjusting to pH 8 caused the L12 protein to precipitate, and the day following the precipitate of L12 was collected by centrifugation (5000 x g, 10 min).

The L12 protein precipitate was washed with 20 mM Tris / HCl, at pH 8 to increase the purity of L12 protein hasty After a second spin (5000 x g, 10 min) the L12 protein precipitate dissolved in a minimum volume of (20 mM CH 3 COOH, 50 mM H 3 BO 3, 1 mM CaCl2, 100 mM NaCl, adjusted to pH 4.5 with NaOH) and added to a Superdex 75 300 ml size exclusion column balanced in the same buffer.

The Superdex 75 column was eluted with the same buffer and column fractions were analyzed by Analysis SDS-PAGE. The fractions, where you only saw one Coomassie spotted SDS-PAGE gel band, were grouped as the purified L12 protein preparation. Was added glycerol to L12 fractions grouped to a concentration 50% final (w / w).

L12 protein formulated from glycerol is Stored cold in a refrigerator.

The preparation was essentially pure as it determined on a stained SDS-PAGE gel of Coomassie (a band).

features

The relative molecular weight as determined by SDS-PAGE was: Mr = 12 kDa.

Sequence N-terminal was:

WVNPGYHYQYPSEGG (SEQ ID NO: 5)

Example 3

Bacillus licheniformis discontinuous flow fermentation

A discontinuous flow fermentation process of Bacillus licheniformis ATCC 14580 was carried out as described below. All media were sterilized by methods known in the art. Unless described otherwise, tap water was used. The ingredient concentrations referred to in the recipes below are before any inoculation.

Media

LB agar: 10 g / l casein peptone (such as, Fluka catalog No. 95039, tryptic casein digestion); 5 g / l yeast extract (manufactured by autolysis of Saccharomyces cerevisiae , for example catalog No. 9512 of Organotechnie SA, 27, avenue Jean Mermoz, F-93120 La Courneuve, France); 10 g / l sodium chloride; 12 g / l Bacto-agar (LB-agar (Miller), Merck catalog No. 110283) adjusted to pH 7.0 +/- 0.2.

M-9 buffer: Di-Sodiumhydrogen phosphate, 2H 2 O 8.8 g / l; potassium dihydrogen phosphate 3 g / l; 4 g / l sodium chloride; sulfate magnesium, 7H 2 O 0.2 g / l (water is used in this buffer deionized).

PRK-50: 110 g / l semolina soy; Di-Sodiohydrogen phosphate, 2H 2 O 5 g / l; antifoam (such as, for example, Struktol SB2121, Schill & Seilacher Hamburg, Germany) 1 ml / l; pH adjusted to 8.0 with NaOH / H 3 PO 4 before sterilization.

Composition medium: Tryptone (hydrolyzate of Casein such as, for example, pancreatic digestion of Triptone Bacto ™ from casein catalog no. 211699) 30 g / l; sulfate magnesium, 7H 2 O 4 g / l; di-potassium dihydrogen phosphate 7 g / l; gave- sodium hydrogen phosphate, 2H 2 O 7 g / l; di-ammonium sulfate 4 g / l; citric acid 0.78 g / l; vitamins (thiamine-dichloride 34.2 mg / l; riboflavin 2.9 mg / l; nicotinic acid 23 mg / l; calcium D-pantothenate 28.5 mg / l; pyridoxal-HCl 5.7 mg / l; D-biotin 1.1 mg / l; folic acid 2.9 mg / l); trace metals (MnSO4, H2O 39.2 mg / l; FeSO 4, 7H 2 O 157 mg / l; CuSO_ {4}, 5H2 O 15.6 mg / l; ZnCl2 15.6 mg / l); antifoam (Struktol SB2121, see above) 1.25 ml / L; pH adjusted to 6.0 with NaOH / H 3 PO4 before of sterilization. 24 ml / L of monopropylene glycol was added (MPG) 28 and 47 hours after inoculation (i.e. after approximately 1 and 2 days, respectively), were added in total 48 ml / l of MPG. Food medium: Glucose, 1H 2 O 820 g / l.

Fermentation procedure

Bacillus licheniformis ATCC 14580 was grown in LB agar bias for one day at 37 ° C. The agar was then washed with M-9 buffer, and the optical density (OD) at 650 nm of the resulting cell suspension was measured. The inoculum shake flasks (with 100 ml of PRK-50 medium) were inoculated with an inoculum of OD (650 nm) x ml cell suspension = 0.1 (which means that the required amount of inoculum in ml is obtained by dividing 0.1 by OD (650 nm) of the inoculum cell suspension). The shake flasks were incubated at 37 ° C at 300 rpm for 20 hr.

The fermenters used were fermenters standard lab equipped with a temperature regulation system, pH control with ammonia water and phosphoric acid, electrode Dissolved oxygen to measure> 20% oxygen saturation throughout the fermentation.

Fermentation in the main fermenter (fermentation tank) was started by inoculating the fermenter main with the growth culture of a shake flask inoculum The inoculated volume was 10% of the composition medium (80 ml for 720 ml of the composition medium, resulting in 800 ml of initial broth after inoculation).

The fermentation parameters were: temperature 41 ° C; pH between 6.8 and 7.2 (using ammonia water and acid phosphoric, control 6.8 (ammonia water), 7.2 phosphoric acid). Aeration: 1.5 liter / min / kg of the weight of the fermentation broth, agitation: 1500 r.p.m.

Food medium of the following was added way: Initial feed rate 0.05 g / min / kg at the beginning of fermentation, increasing linearly to 0.16 g / min / kg after 8 hours, and remaining at 0.16 g / min / kg until the end of the fermentation (by reference to the initial weight of the broth of fermentation, just after inoculation).

After 3 days (70 hours) the broth of Fermentation was collected and purified as described in Example 4 down.

Example 4

Purification of the fermentation in discontinuous flow of Bacillus licheniformis

\hbox{fue de Mr = 12 kDa. La secuencia
 N-terminal fue: WNVPGYHYQY (aminoácidos
1-10 de la SEC ID NO:5).}

The fermentation broth of Example 3 is centrifuged (20,000 x g, 20 min) and the supernatants were decanted Carefully precipitate. The combined supernatants are filtered through a Seitz K-250 plate and then through a Seitz EKS plate to eliminate the rest of Bacillus host cells. EKS filtering conductivity It was 10 mS / cm. 100 ml of EKS filtrate was diluted 10x in 20 mM of CH 3 COOH, 50 mM of H 3 BO 3, 1 mM of CaCl 2, is adjusted to pH 4.5 with NaOH and the pH of the diluted EKS filtrate was adjusted to pH 4.5 with 20% CH 3 COOH. EKS filtering diluted was added to a column of SP-sepharose FF of 19 ml balanced in 20 mM CH 3 COOH, 50 mM of H 3 BO 3, 1 mM CaCl 2, and adjusted to pH 4.5 with NaOH. After washing the column extensively with the buffer balanced, the L12 protein was eluted with a NaCl gradient linear (0 - -> 0.5M) in the same buffer. Fractions containing the L12 protein were identified by analysis SDS-PAGE and were grouped and diluted 10 times with demineralized water to reduce the conductivity of the group. The pool was added to a SOURCE S 8 ml column balanced in the same equilibration buffer (20 mM of CH 3 COOH, 50 mM H 3 BO 3, 1 mM CaCl 2, adjusted  at pH 4.5 with NaOH) and after column washing extensively with the equilibration buffer, the L12 protein is eluted with a linear NaCl gradient (0 - -> 1.0M) in the same buffer Fractions of the column were analyzed by analysis SDS-PAGE and protein containing fractions L12 were grouped and added to a size exclusion column Superdex 75 of 120 ml balanced in 20 mM CH 3 COOH, 50 mM of H 3 BO 3, 100 mM NaCl, 1 mM CaCl 2, adjusted to pH 4.5 with NaOH. The Superdex 75 column was eluted with the same buffer and column fractions were analyzed by analysis SDS-PAGE. The fractions that originated a band strong at 12 kDa on the stained SDS-PAGE gel of coomassie were grouped as the preparation of L12 protein purified. The preparation was at least 90% pure as observed on a spotted SDS-PAGE gel of coomassie, and the relative molecular weight as determined by SDS-PAGE

 \ hbox {was Mr = 12 kDa. Sequence
 N-terminal was: WNVPGYHYQY (amino acids
1-10 of SEQ ID NO: 5).} 

Example 5

Feed performance in vivo (cage test)

The yield of L12 protein in feed is evaluated in a broiler growth test with following parameters:

Growth test:

Day 8 to day 29 (day 0 = day of hatch)

Treatments:

A: Control;

quad

B: 5 mg of purified L12 protein per kg of I think

Subsistence allowance:

Corn / diet SBM48 (see feed composition, Table 1)

After mixing the food it was granulated to approximately 70 ° C (3 x 25 mM). The L12 protein was diluted in 500 ml of water and sprayed on the granules. For the treatment of control the same amount of water was sprayed on the granules

Replicas:

8 groups of 6 male chickens, ROSS PM3, per treatment

Feeding:

Granules ad libitum .

TABLE 1 Feed composition of the diet experimental

       \ vskip1.000000 \ baselineskip
    

6

       \ newpage
    

       \ global \ parskip0.890000 \ baselineskip
    

TABLE 2 Performance of male broilers; average ± stdev

8

Example 6

Bacillus strains with L12 type genes, as identified by PCR

Genes similar to the gene encoding L12 protein (SEQ ID NO: 1) were identified in a number of other Bacillus licheniformis strains by PCR. DNA for use as a standard for the PCR reaction was isolated from eleven different Bacillus licheniformis strains grown overnight at 37 ° C in TY agar plates (for the formula, see Example 1). An inoculation tube with cells from each strain was suspended in 0.1 ml of H2O and boiled for 10 min, centrifuged, and 5 microliters of each supernatant was used as a DNA standard in PCR reactions as It is described below.

PCR reactions were carried out in "Pure TaqTM Ready-To GoTM PCR Beads" Amersham Pure TaqTM Ready-To GoTM PCR) Biosciences: 5 microliters of DNA standard + 2 x 1 microliters of primer Pep481 (SEQ ID NO: 6) and Pep482 (SEQ ID NO: 7) + 18 microliters of H2O.

PCR program: 1) 95 ° C 3 min; 2) 95 ° C 10 sec; 3) 65ºC 30 sec -1ºC pr. cycle; 4) 72 ° C 1 min; 5) Go To 2) 9 times; 6) 95 ° C 10 sec; 7) 55 ° C 30 sec; 8) 72 ° C 1 min; 9) Go To 6) 19 times; 10) 72 ° C 5 min; 11) 4 ° C always, which means that after the stage 10) the temperature is reduced to 4 ° C.

Primers

Pep481

AATTACGCGTGTTGGTGCGATAGTAGTAACG-3 ' (SEQ ID NO: 6)

Pep482

TTAAGAATTCGAATGAAAGAGGAGGAATG-3 ' (SEQ ID NO: 7)

The 0.4 kb PCR fragment resulting from five positive strains (positive meaning giving a band of DNA of the appropriate size) was purified and used in an experiment of DNA sequencing, using again as sequence primers primers Pep481 (SEQ ID NO: 6) and Pep482 (SEQ ID NO: 7).

Three of the five positive strains gave the same DNA sequence: Bacillus licheniformis ATCC 14580, Bacillus licheniformis NCIMB 6346 (= DSM 8785) and the Bacillus licheniformis 712 strain, resulting in the amino acid sequence of SEQ ID NO: 2. In the changes in the DNA of the Bacillus licheniformis 470 strain, two amino acid changes resulted (SEQ ID NO: 9), however there was no change in the mature peptide. Fifteen amino acid changes resulted in DNA changes in the Bacillus licheniformis 009 strain (SEQ ID NO: 8), eight of which occurred in the mature peptide. In addition, a consensus sequence (SEQ ID NO: 10) was derived from SEQ ID NOs: 2, 8 and 9.

Note that, in this experiment, the nucleotides encoding the seven C-terminal amino acids of SEQ ID NO: 2 is included in the Pep481 primer (SEQ ID NO: 6), and seven C-terminal amino acid residues of the SEQ ID NOs: 8-9 may therefore not be correct. Notwithstanding the accuracy of SEQ ID NOs: 8-9 confirmed later.

A strain of Bacillus licheniformis that was isolated from the probiotic product added in the designated feed BioPlus?
2B (offered by Chr. Hansen A / S, 10-12 Boege Allé, DK-2970 Hoersholm, Denmark) was included among the eleven strains tested but was negative.

In addition, another 44 strains of Bacillus licheniformis were tested as described above. A positive PCR response was obtained in 27 of these strains. Examples of additional publicly available Bacillus licheniformis strains that turned out to be L12 positive have the following deposit numbers: NCTC 1024, NCTC 1025, NCTC 2120, NCTC 7589, NCTC 9932, ATCC 21424, NCIMB 10689, ATCC 53757. NCTC is the National Collection of Type Cultures. ATCC is the American Type Culture Collection. NCIMB is the National Collection of Industrial, Marine and Food Bacteria.

Example 7

Thermostability

Differential analysis calorimetry (DSC) was used to determine the temperature stability of the L12 protein at pH 2.5, 4.0 and 7.0. Purified L12 was dialyzed in a concentration of approximately 2 mg / ml overnight at 4 ° C with an appropriate buffer and was carried out with an instrument VP-DSC (MicroCal) with a constant scan rate 1.5ºC / min between 20 and 90ºC. Data manipulation was performed using the Microcal Origin software (version 4.10), and the temperature of denaturation was defined as temperature at the apex of the maximum enthalpy value. It was discovered that L12 had a denaturation temperature of 55 ° C in 10 mM citric acid, 50 mM sodium chloride, at pH 2.5. In 10 mM sodium acetate, 50 mM of NaCl, at pH 4.0, L12 was denatured at 69 ° C, and in 10 mM of sodium phosphate, 50 mM NaCl, at pH 7.0 the temperature of denaturation was 60 ° C.

Example 8

Modulation of intestinal microflora in vivo and in vitro

The influence of the L12 protein on the intestinal microflora was evaluated a) in vivo , in piglets and fattening pigs; eb) in vitro , in microorganisms isolated from the intestinal microflora.

Study in piglets

This study, which was conducted according to French legal regulations for animal experiments alive, was done to evaluate the effect of 5 ppm of L12 (5 mg of L12 purified per kg of feed) in intestinal microflora of sucker

Ten piglets (hybrids of Large-White, Landrace, and Piétrain, obtained from GAEC Leclerc, Ostheim, France) of an initial body weight of 25.3 ± 1.3 kg underwent anastomosis ileo-rectal (connecting the terminal ileum to the end of the rectum, diverting the blind intestine and colon). In such pigs, the terminal ileum microflora can be collected in the level of anus and is representative of the bacterial population of all consecutive digestive parts of the intestines. After of surgery, during recovery, and during experimental period the animals were placed in metabolic cages allowing simple sampling of content ileo-rectal.

During the six-week experimental period, the piglets were fed each and alternatively (in a double latin square design, to reduce the effect of variation individual and also any potential influence of the sequence of treatments) with a basic diet supplemented or not with test compounds The diets were composed of the Following way:

Diet A: KLIBA, available through Provimi-Kliba, Kaiseraugst, Switzerland, with 18% soybean meal, 53% corn, 13% barley, 6% oatmeal, 5.4% wheat bran, 1% of soybean oil, 3.6% of minerals, vitamins and amino acids synthetic (p / p).

Diet E: Diet A with the addition of 5 mg / kg of the L12 protein.

Diets B, C and D they included other test compounds with no relevance to the present invention

Test compounds (including L12) are incorporated in the diets in a Buhlen mixer (Buhlen, Aschwill, Switzerland). The experimental diets were prepared and administered to the animals in a crushed form.

Experimental diets were allowed to animals at the level of 2 kg per day distributed in two meals same at 8:00 and 15:30.

Content samples were taken ileo-rectal of each animal in the last two days of each treatment period, and the concentrations were determined of dry substance and the different ingredients of the microflora

The animals showed no symptoms of Toxicosis or disease during the experiment. Your increase of Daily weight during observation was 1.14 +/- 0.1 kg which is a very good zootechnical performance. At the end of the experiment euthanized animals by lethal injection after reassure them

The dry matter content of the samples determined after drying overnight at 105 ° C, 1 g of sample, according to the standard procedure of the Association of Official Analytical Chemists (AOAC) (1009) (Association of Official Analytical Chemists (1990). (Official methods of analysis. 15th edition. Association of Official Analytical Chemists. Arlington)

The microflora analysis was performed from the Following way:

Immediately after emission, 10 g of ileo-rectal content were transferred and homogenized in flasks containing 100 ml of peptonado broth of sodium chloride (Merck, Darmstadt, Germany, catalog no. 10582).

Having passed less than 5 min between broadcasts and content removal ileal which was quickly transferred to an anaerobic chamber (AES Cheminex, Combourg, France). Subsequently, the samples are serially diluted in 10 stages using chloride-chopped broth sodium from 10 - 1 to 10 - 8 (p / vol). All bacterial counts were achieved with two replication plates.

The total of optional anaerobic accounts represents the average number of colonies that grew on Brucella agar (Merck, Darmstadt, Germany, catalog No. 10490) supplemented with sheep's blood (5% vol / vol, supplied by AES Cheminex, Combourg, France ). The plates were incubated in an anaerobic cabinet at 37 ° C for 5 days.

Lactic acid bacteria were listed in MRS agar (Merck, Darmstadt, Germany, catalog No. 110660) and Lactobacillus spp. in Rogosa agar (Merck, Darmstadt, Germany, catalog no. 105413). Both plates were incubated in an anaerobic vessel at 37 ° C for 48 h.

Enterobacteriacae were counted in VRBD agar (Merck, Darmstadt, Germany, catalog No. 110275). Escherichia coli and other Enterobacteriacae were analyzed in a Coli-ID chromogenic medium (BioMérieux, Marcy l'Etoile, France, catalog No. 42017). This medium contains two chromogenic substrates: One for the detection of beta D-glucuronidase ( E. coli ) producing pink colonies, and one for the detection of galactosidase (other Enterobacteriaceae) producing blue colonies. Both plates were incubated aerobically at 37 ° C for 24 h.

Enterococcus spp. It was evaluated in Enterococci agar (Merck, Darmstadt, Germany, catalog No. 65009), and Staphylococcus spp. in Baird Parken agar (AES Cheminex, Combourg, France, catalog No. AEB150302) after aerobic incubation at 37 ° C for 48 h.

After heating the sample in a water bath at 80 ° C for 10 min, the Clostridium perfringens was isolated using TSN agar (BioMérieux, Marcy l'Etoile, France, catalog No. 51048) incubated in an anaerobic chamber at 46 ° C for 24 hours.

Identities of representative colonies are also confirmed by microscopic examination after Gram staining and the biochemical test using the appropriate API system (BioMérieux, Marcy l'Etoile, France).

For each test group, the analysis Statistical data involved the calculation of the mean deviation and mean standard as well as a variation analysis followed Student's "t" Test to assess the importance of differences between the groups.

The respective bacterial accounts (number of colony forming units (CFU) per gram of dry substance of ileal content (DM), +/- the standard deviation (SD) are presented in table 3 below, which also shows the amendment in the respective bacterial counts caused by the addition of 5 ppm of L12, in relation to control.

TABLE 3 Bacterial microflora content Ileo-rectal sucker

9

       \ newpage
    

       \ global \ parskip1.000000 \ baselineskip
    

10

In this trial, no differences were found Significant bacterial counts were only observed numerical effects, but sometimes these were very marked.

A positive effect of L12 was seen in the mean of the total facultative anaerobic bacteria, where counts increased by 49% in relation to control.

A neutral effect of L12 was seen in the total lactic acid bacteria and in the total Lactobacillus spp., Which are representative of the beneficial bacteria of the intestinal microbiota. The same goes for the total Enterobacteriaceae. The main component of the population of Enterobacteriaceae in the microflora of the piglet, Escherichia coli , increased by 19% while the group of other Enterobacteriaceae (different from E. coli ) which are potentially pathogenic was greatly reduced.

A negative effect of L12 was seen in the population of Enterococcus spp.

Clostridium perfringens was found in thirteen of the twenty control samples (Diet A), while this was only found in six of the twenty samples with diet supplemented with L12 (Diet E). Therefore, this population of pathogenic bacteria was strongly reduced (by 99%) with L12.

Study with broilers

In the broiler growth test described in example 5, the effect of L12 (5 mg was also evaluated of purified L12 per kg of food) in the intestinal microflora of Broilers.

The microflora analysis of twelve animals per group was performed as described above (the study of sucker), using cecal content, after slaughter Broilers, as initial material.

The results of the population accounts bacterial in the cecal content of broilers is shown in Table 4.

TABLE 4 Bacterial content of chicken cecal microflora fattening

eleven

A positive effect of L12 on the flora was seen total facultative anaerobic which statistically increased from Significantly at 178%.

This variation could be explained by the observed (digital) increase in the total number of lactic acid bacteria observed in the group supplemented with L12. The total lactic acid bacteria represents the main population of the total anaerobic facultative anaerobic flora and are mainly composed of Lactobacillus species that play an important role in the benefits imparted by the normal microbiota for the health of the host.
ped.

Escherichia coli accounts increased numerically, but not significantly statistically, in the group supplemented with L12. This is consistent with the increase in the counts of the other two groups of microorganisms. All of the animals involved in this experiment had a healthy and balanced cecal microflora and no diarrheal disease was observed in the animals during the test, suggesting that it was the diner coli escherichia strains that could have increased.

       \ vskip1.000000 \ baselineskip
    

Modulation of intestinal microflora in vitro

The effect of L12 on the microflora was subsequently evaluated in vitro . Bacteria representative of beneficial and harmful microorganisms of the intestinal microbiota were isolated from the intestinal contents of the piglet and the broiler. Their identities were confirmed by microscopic examination after Gram staining and biochemical testing using the appropriate API system (BioMérieux, Marcy l'Etoile, France).

The effect of L12 on the bacterial growth of these bacteria was determined by the method of dilution in broth by microtiter by measuring the absorbance at 595 nm. All these in vitro tests were performed in 96-well sterilized plates (Falcon 353072 microtiter plates, Becton Dickinson Labware, Meylan, France) in a final volume of 110 microliters as follows: 100 microliters of suspension containing approximately 10 5 CFU / mL of the pure bacteria in tryptic soy broth (Merck, Darmstadt, Germany, catalog No. 105459) were added to 10 microliters of water containing L12 in serial 2-stage dilutions or 10 microliters of water as a control. Growth inhibition was determined by measuring the absorbance at 595 nm with a Multiskan Ascent (TermoLabsystems, Helsinki, Finland) after the appropriate incubation time at the appropriate temperature for the optimal growth of pure bacteria, see Table 5.

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

TABLE 5 Incubation conditions

       \ vskip1.000000 \ baselineskip
    

12

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

The reduction in crop density is determined by subtracting the OD595 from the test culture after 24 or 48 hours (according to Table 5) from OD 595 of the culture of control. The reduction in crop density was normalized expressing this as a percentage of OD 595 of the crop of control and MIC 90 corresponded to the concentration of L12 required to reduce crop density by 90%, meaning inhibit growth in 90% of the tested organism (MIC_ {90} being defined as the Inhibitory Concentration Minimum required to inhibit the growth of 90% of organisms).

The results of these evaluations are shown in Table 6.

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

TABLE 6 MIC_ {90} against bacteria isolated from content piglet and broiler bowel

       \ vskip1.000000 \ baselineskip
    

13

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

L12 is more active against Gram positive cocci and Clostridia isolated from the pig and chicken content of fattening that gram-negative bacteria isolated from same.

These results explained at least partially the influence of L12 on the microbiota gastrointestinal:

The observed effect of L12 in vitro on Enterococcus faecalis and Enterococcus faecium confirmed the observed reduction of Enterococcus spp. observed in vivo in piglets.

The observed effect of L12 in vitro on the Lactobacillus species confirmed the results of in vivo studies of piglets and broilers that L12 has no negative influence in vivo on the Lactobacillus species of pigs and birds.

The observed effect of L12 in vitro on Clostridium perfringens isolated from broiler chicken intestinal content confirmed the strong reduction of this population observed in vivo in piglets.

       \ newpage
    

Example 9

Feed performance in vivo (cage test) - different dosages

The yield of L12 protein in feed is evaluated in a fattening chicken growth test with the following parameters:

Proof increase:

day 8 to day 29 (day 0 = day incubation)

Treatments:

A: control; B: 2.5, 5.0, and 7.5 mg of L12 protein purified per kg of feed

Subsistence allowance:

 Corn / diet SBM48 (see feed composition, Table 7).

       \ vskip1.000000 \ baselineskip
    

After mixing the feed it was granulated to approximately 70 ° C (3 x 25 mm). L12 protein dissolved in 500 ml of water and sprayed onto the granules. For him control treatment the same amount of water was sprayed on the granules.

Replica:

10 groups of 6 male chickens, ROSS PM3, per treatment

Feeding:

Granules ad libitum .

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

(Table goes to page next)

TABLE 7 Feed composition of the diet experimental

14

TABLE 8 Performance of male broilers; average ± stdev

16

Example 10

Concentration determination

An L12 protein preparation was used purified as a standard of L12 protein. It was prepared as it described in Example 4, and was formulated with glycerol as described in Example 2. The purity was more than 96%, as measured by HPLC (using a Waters 2690 separation module and a UV detector Waters 2487, detecting at 280 nm, using columns ACE C18 5 um 100A 150x3.0 mm and Waters \ mu-Bondapak C18 20x3.9 mm (safety device column), a speed of flow rate of 0.5 ml / min, an injection volume of 10 microliters, mobile phase A: H2 O 18M_ + 0.1% TFA (Acetic acid Trifluor), phase mobile B acetonitrile + 0.1% TFA).

The concentration of L12 of the standard was 3.6 mg of pure protein / ml per, as determined by Analysis of Amino acids (as described below).

The purity and concentration of several others L12 samples were determined by a gel method of SDS-PAGE as also described below, by Reference to this standard.

       \ vskip1.000000 \ baselineskip
    

Amino Acid Analysis (AAA) - concentration of the standard of L12 protein

The peptide bonds of the standard sample of L12 protein underwent acid hydrolysis, followed by separation and quantification of the amino acids released in a Biochrom 20 Plus Amino Acid Analyzer, available commercially through Bie & Berntsen A / S, Sandbaekvej 5-7, DK-2610 Roedovre, Denmark, according to the manufacturer's instructions. For acid hydrolysis, the Protein sample was dried in a vacuum centrifuge, solved in 18.5% (vol / vol) HCl + 0.1% (vol / vol) phenol and incubated for 16 hr at 110 ° C. After incubation, the sample was dried another Once in the vacuum centrifuge, it was solved in a loading buffer (0.2 M Na-Citrate, at pH 2.2) and loaded onto the Biochrom 20 Plus Amino Acid Analyzer.

For quantification, the hydrolyzed sample was loaded onto a cation exchange resin column UltroPac No. 8, in the form of sodium, which is commercially available through Bie & Berntsen A / S, catalog no. 80-2104-15. PH buffers variable (pH 1 to pH 8) and ionic strength were pumped through the column according to the manufacturer's instructions referred to above, to separate the different amino acids. The temperature of the column was precisely controlled, also according to the instructions from the manufacturer (from 53ºC to 92ºC and again at 53ºC) to ensure The separation required. The column eluent was mixed with ninhydrin reagent (Bie & Berntsen, catalog no. 80-2038-07) and the mixture was passed to Through the high temperature reaction coil of the Analyzer of amino acids. In the reaction coil, the ninhydrin reacted with the amino acids to form colored compounds, the amount of which it was directly proportional to the amount of amino acids present.

       \ vskip1.000000 \ baselineskip
    

Concentration of L12 protein samples

The concentration of several samples of L12 is determined by the following procedure (all Novex products Referrals are commercially available through Invitrogen, see www.invitrogen.com):

50 microliters of L12 solution (0.1-1.0 mg of L12 per ml) were mixed with 5 1% (w / v) microliters of EDTA + 10 6% microliters of PMSF + 10 0.5M microliters of DTT + 25 microliters of sample buffer NuPage LDS (Novex NP0007) in an Eppendorf tube, and the tube is heated at 95 ° C for 5 minutes. 10 microliters of sample at 10% pre-melted Tris-Bis gel (NP0301 BOX of NOVEX). Electrophoresis was performed with a buffer displacement month (month = acid 2- (N-morpholino) ethan sulfonic; NOVEX NP0002) + antioxidant (NOVEX NP0005) a a constant voltage of 200 V according to the instructions of the maker. After electrophoresis, the gel was stirred gently in 10% acetic acid + 50% EtOH for 10 minutes The gel was then gently stirred with a solution of Colloidal Blue coloration (NOVEX 46-7016) during  a minimum of three hours and washed by gentle agitation for 2 to 4 hours with distilled water with different changes of distilled water. The wet gel was scanned with a BioRad calibrated densitometer Calibrated Densitometer GS-800 equipped with the Quantity One software (version 4.6.0, BioRad) and L12 protein are quantified according to the manufacturer's instructions. The standard of L12 described above was used as a standard, that is, in three-four dilutions in the range of 0.1-1.0 mg / ml.

       \ vskip1.000000 \ baselineskip
    

Example eleven

Stability with pepsin and pancreatin

Native L12 : Purified L12 protein, diluted to 1 mg / ml in a 10 mM Tris / CH 3 COOH buffer at pH 4.5.

Denatured L12 : A sample of purified L12 (1 mg / ml in a 10 mM Tris / CH 3 COOH buffer at pH 4.5) was boiled for 5 minutes to denature the protein.

Pepsin : swine pepsin (Sigma P-7000, 453 units / mg of solid). 30,000 units / ml were dissolved in 10 mM succinic acid at pH 3.0.

Pancreatin : Swine pancreatin (Sigma P-7545, 8xUSP). 80 mg / ml were suspended in 0.5 m Na-phosphate buffer pH 7.0.

Buffer at pH 3 : 10 mM succinic acid at pH 3.0.

Buffer at pH 7 : 0.5 M Na-phosphate buffer at pH 7.0.

The following samples were prepared:

       \ vskip1.000000 \ baselineskip
    

1) L12 incubated with Pepsin and Pancreatin

278 microliters of native or denatured L12 + 100 microliters of pepsin solution + 122 microliters of buffer at pH 3 was incubated for 2 hours at 40 ° C with stirring, the pH It was measured in 3.5. Then 400 microliters of buffer was added to pH 7 + 100 microliters of pancreatin suspension and then incubated for 4 hours at 40 ° C with stirring, the pH was measured in 7.0.

       \ vskip1.000000 \ baselineskip
    

2) L12 incubated with Pepsin

278 microliters of native or denatured L12 + 100 microliters of pepsin solution + 122 microliters of buffer at pH 3 was incubated for 2 hours at 40 ° C with stirring (the pH was measured in 3.5). Then 500 microliters of buffer was added to pH 7 (pH was measured in 7.0).

       \ vskip1.000000 \ baselineskip
    

3) L12 incubated with Pancreatin

278 microliters of native or denatured L12 + 222 microliters of buffer at pH 3 were incubated for 2 hours at 40 ° C with stirring. Then 400 microliters of buffer was added at pH 7 + 100 microliters of pancreatin suspension followed by its incubation for 4 hours at 40 ° C with stirring.

       \ vskip1.000000 \ baselineskip
    

4) L12 control sample

278 microliters of native or denatured L12 + 100 microliters of pepsin solution + 122 microliters of pH 3 buffer + 400 microliters pH 7 buffer + 100 microliters of pancreatin suspension were mixed in ice and kept cold

       \ vskip1.000000 \ baselineskip
    

5) Pepsin control sample

100 microliters of pepsin solution + 400 pH 3 buffer microliters were incubated for 2 hours at 40 ° C with stirring (the pH was measured in 3.5). Then 500 were added pH 7 microliters of buffer.

       \ vskip1.000000 \ baselineskip
    

6) Pancreatin control sample

500 microliters of buffer at pH 3 + 400 pH 7 microliter buffer + 100 microliter suspension Pancreatin was incubated for 4 hours at 40 ° C with shaking (pH it was measured in 7.0).

       \ vskip1.000000 \ baselineskip
    

7) Control sample of Pepsin + Pancreatin

100 microliters of pepsin solution + 400 microliter of buffer at pH 3 were incubated for 2 hours at 40 ° C with stirring (the pH was measured at 3.5). Then 400 microliters of pH 7 buffer + 100 microliters of pancreatin suspension was added followed by incubation for 4 hours at 40 ° C with stirring (the pH was measured in
7.0).

All samples were analyzed by SDS-PAGE as described in Example 10. According to the SDS-PAGE, the native L12 was not degraded by pepsin, pancreatin or a pepsin treatment followed by pancreatin Denatured L12 was extensively degraded by pepsin, pancreatin or a pepsin treatment followed by pancreatin since no band of L12 was detected in the SDS gel in the corresponding samples.

       \ vskip1.000000 \ baselineskip
    

Example 12

Feed performance in vivo (pen test)

This example evaluates the effects of protein L12 in the growth performance of broilers at over a full 36-day growth cycle.

A performance test of growth with broilers from day 1 to day 36.

The animals were housed in pens whose ground He had covered himself with shavings. During the initial period (days 1-22) and during the cultivation period (days 22-36) the chickens received a diet based on wheat, rye and soy flour (for composition see Table 9). Along with a control treatment not supplemented, it was included L12 protein at a dosage of 7.5 mg of protein per kg of food.

Test increase:

Day 1 to day 36 (day 0 = day incubation)

Subsistence allowance:

Wheat / rye / SBM48 diet (see composition food in Table 9)

Feeding:

Granules ad libitum .

       \ vskip1.000000 \ baselineskip
    

After mixing, the feed was granulated at approximately 70 ° C (3 x 25 mm). L12 protein dissolved in 800 ml of water for the initial feed and 1200 ml of water for the crop feed, respectively, and was sprayed on granules For the control treatment it was sprayed amount of water without the L12 protein on the granules, respectively.

Treatments:

Control, 7.5 mg of L12 protein per kg of I think

Replicas:

6 groups of 20 chickens by sex and treatment (6 groups of 20 females, 6 groups of 20 males).

TABLE 9 Feed composition of the diet experimental

17

TABLE 10 Performance of broilers; average \ pm stdev

19

The L12 protein improved the weight gain of chickens slightly (Table 10), however this effect was not significant Feed consumption of chickens receiving the L12 protein was no different from that of control chickens. He food conversion coefficient (FCR) improved significantly by the L12 protein compared to the control, that is, 2.7%.

       \ newpage
    

Example 13

I think and additive Feed additive

A feed additive is prepared by adding 25 g of a coated T granulate comprising the L12 protein purified in an amount of 20 g / kg (prepared as described in Example 3 of EP 569468 B1, however with a coating of approx. 7% hydrogenated palm oil and approx. 13% of CaCO_ {3}) to the following premix (per kilo of premix):

       \ vskip1.000000 \ baselineskip
    

twenty

       \ newpage
    

I think

A culture diet is prepared for chickens fattening having the following composition (%, w / w) mixing the ingredients. Wheat, rye and SBM 48 are available through Moulin Moderne Hirsinque, Hirsingue, France. After mixing, the feed is granulated at a desired temperature, for example at approximately 70 ° C (3 x 25 mm).

twenty-one

The resulting feed comprises 5.0 mg of purified L12 protein per kg (5 ppm).

Additional feed and feed additive compositions are prepared in the same manner, however replacing 25 g of L12 CT granulate coated with kg of the premix with 10 13 colony forming units (CFU), preferably in the form of endospores, of Bacillus licheniformis ATCC 14580, which results in a feed with approximately 10 8 CFU per g of the feed composition.

       \ vskip1.000000 \ baselineskip
    

References cited in the description This list of references cited by the applicant has been collected exclusively for reader information. Do not It is part of the European patent document. It has been made with the greatest diligence; the EPO however does not assume any responsibility for possible errors or omissions Patent documents cited in the description

WO 960739 A [0002]

WO 0022103 A [0064]

WO 03093453 A [0003]

EP 1472933 A [0070]

WO 9617929 A [0064]

GB 2138023 A [0070]

WO 9830682 A [0064]

WO 9943835 A [0096]

WO 9835026 A [0064]

WO 0158275 A [0137] [0157]

WO 9900489 A [0064]

{} \ hskip0.17cm [0158] [0159] [0162]

WO 9616177 A [0064]

WO 03044049 A [0146]

WO 0026230 A [0064]

WO 03048148 A [0146]

WO 0183559 A [0064]

WO 9401459 A [0147]

EP 561907 A [0064]

WO 02090384 A [0147]

WO 0026354 A [0064]

EP 569468 B1 [0277]

Bibliography outside the patent cited in the description

Martinez et al . Microbiology, 1999 , vol. 145, 3155-3161 [0008]

Needleman , SB Wunsch , CD J. Mol. Biol., 1970 , vol. 48, 443-453 [0038]

WR Pearson DJ Lipman Improved Tools for Biological Sequence Analysis PNAS, 1988 , vol. 85, 2444-2448 [0044]

WR Pearson Rapid and Sensitive Sequence Comparison with FASTP and FASTA Methods in Enzymology, 1990 , vol. 183, 63-98 [0044]

TF Smith MS Waterman Smith-Waterman algorithm J. Mol. Biol., 1981 , vol. 147, 195-197 [0044]

Myers Miller CABIOS, 1989 , vol. 4, 11-17 [0044]

Bolton McCarthy Proceedings of the National Academy of Sciences USA, 1962 , vol. 48, 1390- [0056]

Official Methods of Analysis Association of Official Analytical Chemists 1984 . [0062] [0163]

Bergey's Manual of Systematic Bacteriology 1986 . vol. 2, [0071]

Johansen et al . Int. J. Syst. Bacteriol, 1999 , vol. 49, 1231-1240 [0071]

Innis et al . PCR: A Guide to Methods and Application Academic Press 1990 . [0083]

Bendtsen et al . Journal of Molecular Biology, 2004 , vol. 340, no. 4,783-95 [0104]

Nielsen et al . Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (ISMB 6) AAAI Press 1998 . vol. 2.0, 122-130 [0104]

Nielsen et al . Protein Engineering, 1997 , vol. 10, 1-6 [0104]

Ehrlich Proceedings of the National Academy of Sciences USA, 1978 , vol. 75, 1433- [0111]

Chang Cohen Molecular General Genetics, 1979 , vol. 168, 111-115 [0120]

Young Spizizin Journal of Bacteriology, 1961 , vol. 81, 823-829 [0120]

Dubnau Davidoff-Abelson Journal of Molecular Biology, 1971 , vol. 56, 209-221 [0120]

Shigekawa Dower Biotechniques, 1988 , vol. 6, 742-751 [0120]

Koehler Thorne Journal of Bacteriology, 1987 , vol. 169, 5771-5278 [0120]

Journal Officiel des Communautés Européennes, vol. L 130, 53-54 [0253] [0275]

       \ global \ parskip0.000000 \ baselineskip
    

<110> Novozymes A / S

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<120> Polypeptides

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<130> 10799.204-WO

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<160> 10

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<170> PatentIn version 3.3

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 1

         \ vskip0.400000 \ baselineskip
      

<211> 381

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Bacillus licheniformis ATCC 14580

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> CDS

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (378)

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> mat_peptide

         \ vskip0.400000 \ baselineskip
      

<222> (124) .. (378)

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 1

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,8cm
      

22

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 2

         \ vskip0.400000 \ baselineskip
      

<211> 126

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Bacillus licheniformis ATCC 14580

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 2

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,8cm
      

2. 3

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 3

         \ vskip0.400000 \ baselineskip
      

<211> 1581

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Bacillus licheniformis

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> CDS

         \ vskip0.400000 \ baselineskip
      

<222> (601) .. (978)

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 3

         \ hskip0,8cm
      

24

         \ vskip0.400000 \ baselineskip
      

<210> 4

         \ vskip0.400000 \ baselineskip
      

<211> 126

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Bacillus licheniformis

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 4

         \ hskip0,8cm
      

25

         \ vskip0.400000 \ baselineskip
      

<210> 5

         \ vskip0.400000 \ baselineskip
      

<211> 15

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Bacillus licheniformis ATCC 14580

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> MISC_FEATURE

         \ vskip0.400000 \ baselineskip
      

<223> N-terminal

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 5

         \ hskip0,8cm
      

26

         \ vskip0.400000 \ baselineskip
      

<210> 6

         \ vskip0.400000 \ baselineskip
      

<211> 31

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Primer Peptide 481

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> Primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 6

         \ hskip0,8cm
      

27

         \ vskip0.400000 \ baselineskip
      

<210> 7

         \ vskip0.400000 \ baselineskip
      

<211> 29

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Primer Peptide 482

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_feature

         \ vskip0.400000 \ baselineskip
      

<223> Primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 7

         \ hskip0,8cm
      

28

         \ vskip0.400000 \ baselineskip
      

<210> 8

         \ vskip0.400000 \ baselineskip
      

<211> 125

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Bacillus licheniformis strain 009

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> mat_peptide

         \ vskip0.400000 \ baselineskip
      

<222> (41) .. (125)

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 8

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,8cm
      

29

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<210> 9

         \ vskip0.400000 \ baselineskip
      

<211> 126

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Bacillus licheniformis strain 470

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> mat_peptide

         \ vskip0.400000 \ baselineskip
      

<222> (42) .. (126)

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 9

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,8cm
      

30

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 10

         \ vskip0.400000 \ baselineskip
      

<211> 126

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Consensus sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> mat_peptide

         \ vskip0.400000 \ baselineskip
      

<222> (42) .. (126)

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 10

         \ hskip0,8cm
      

31

Claims (10)

1. Nucleic acid construct comprising a nucleic acid sequence encoding polypeptides, whose polypeptides improve feed utilization by improving food conversion coefficient (FCR), and whose sequence of nucleic acids

(to)

hybridizes under conditions of astringency means with (i) nucleotides 124-378 of SEQ ID NO: 1, a sub-sequence of (i) of at least 100 nucleotides, and / or (iii) a complementary chain of (i), or (ii); I

(b)

has a degree of identity with nucleotides 124-378 of SEQ ID NO: 1 of at least one 48%, as determined by the align program using the matrix of default identity, a penalty of -16 for the first residue of a space and penalties of -4 for later waste from a space;

with the proviso that the acid sequence Nuclei not of SEQ ID NO: 3, other than nucleotides 601-978 of SEQ ID NO: 3, and not nucleotides 1-381 of SEQ ID NO: 1 the polynucleotides being operably linked to one or more control sequences that direct the production of the polypeptide in a host cell of Bacillus, the DNA of whose host cell, when collected and used as a DNA standard in a PCR reaction with SEQ ID NOs: 6 and 7 as primers, it leads to the generation of a PCR fragment of a size of approximately 0.4 kb. 2. Recombinant expression vector comprising the nucleic acid construct according to claim
one. 3. Recombinant Bacillus host cell comprising the nucleic acid construct according to the claim 1 or the vector according to claim 2, the DNA of whose host cell, when collected and used as a DNA standard in a PCR reaction with SEQ ID NOs: 6 and 7 as primers, leads to the generation of a PCR fragment of a size of approximately 0.4 kb. 4. Method for the production of a polypeptide encoded by the nucleic acid sequence encoding polypeptides of the construct according to claim 1, whose polypeptides improve feed utilization by improving food conversion coefficient (FCR), comprising the method (a) culturing a recombinant host cell according to the claim 3 to produce a supernatant comprising the polypeptide; and (b) recover the polypeptide. 5. Method for the production of a polypeptide according to claim 4, the method comprising (a) cultivating a strain of Bacillus, the DNA from which, when collected and used as a DNA standard in a PCR reaction with SEQ ID NOs: 6 and 7 as primers, it leads to the generation of a PCR fragment of a size of approximately 0.4 kb, whose PCR fragment encodes an amino acid sequence with at least 33% identity with amino acids 1-85 of SEQ ID NO: 2, as determined by the Needle program, using the replacement matrix BLOSUM62, a space opening penalty of 10, and a space extension penalty of 0.5 and (b) recover the polypeptide. 6. Use in feed for non-therapeutic purposes of a polypeptide, which improves feed utilization improving the food conversion coefficient (FCR), selected from the group consisting of:

(to)

a polypeptide having amino acids 1-126 of the SEC ID NO: 4;

(b)

a polypeptide comprising an amino acid sequence which has a degree of identity with amino acids 1-85 of SEQ ID NO: 2 of at least 33%, as determined by the program Needle, using the BLOSUM62 replacement matrix, a penalty of space opening of 10, and an extension penalty of 0.5 and space

(C)

a polypeptide which is encoded by an acid sequence nuclei that hybridizes under medium astringency conditions with (i) nucleotides 124-378 of SEQ ID NO: 1, (ii) a sub-sequence of (i) of at least 100 nucleotides, and / or (iii) a complementary chain of (i), or (ii).

7. Use in the preparation of a composition for use in feed of a polypeptide, which improves the feed utilization improving the conversion coefficient of food (FCR), selected from the group consisting of:

(to)

a polypeptide having amino acids 1-126 of the SEC ID NO: 4;

(b)

a polypeptide comprising an amino acid sequence having a degree of identity with amino acids 1-85 of the SEQ ID NO: 2 of at least 33%, as determined by the program Needle, using the BLOSUM62 replacement matrix, a penalty of space opening of 10, and an extension penalty of 0.5 and space

         \ newpage
      

(C)

a polypeptide which is encoded by an acid sequence nuclei that hybridizes under medium astringency conditions with (i) nucleotides 124-378 of SEQ ID NO: 1, (ii) a sub-sequence of (i) of at least 100 nucleotides, and / or (iii) a complementary chain of (i), or (ii).

8. Non-therapeutic method to improve food conversion coefficient (FCR), where a polypeptide, which improves feed utilization by improving the coefficient of food conversion (FCR), is added to the feed, being selected the polypeptide from the group consisting of:

(to)

a polypeptide having amino acids 1-126 of the SEC ID NO: 4;

(b)

a polypeptide comprising an amino acid sequence having a degree of identity with amino acids 1-85 of the SEQ ID NO: 2 of at least 33%, as determined by the program Needle, using the BLOSUM62 replacement matrix, a penalty of space opening of 10, and an extension penalty of 0.5 and space

(C)

a polypeptide that is encoded by an acid sequence nuclei that hybridizes under medium astringency conditions with (i) nucleotides 124-378 of SEQ ID NO: 1, (ii) a sub-sequence of (i) of at least 100 nucleotides, and / or (iii) a complementary chain of (i), or (ii).

9. A feed additive comprising a polypeptide, which improves feed utilization by improving food conversion coefficient (FCR), and at least one additional component selected from the group consisting of fat-soluble vitamins, water-soluble vitamins, trace elements, and macrominerals, where the polypeptide is selected from the group that consists in:

(to)

a polypeptide having amino acids 1-126 of the SEC ID NO: 4;

(b)

a polypeptide comprising an amino acid sequence having a degree of identity with amino acids 1-85 of the SEQ ID NO: 2 of at least 33%, as determined by the program Needle, using the BLOSUM62 replacement matrix, a penalty of space opening of 10, and an extension penalty of 0.5 and space

(C)

a polypeptide which is encoded by an acid sequence nuclei that hybridizes under medium astringency conditions with (i) nucleotides 124-378 of SEQ ID NO: 1, (ii) a sub-sequence of (i) of at least 100 nucleotides, and / or (iii) a complementary chain of (i), or (ii).

10. A feed composition with a content crude protein of 50 to 800 g / kg and comprising a polypeptide, which improves feed utilization by improving the coefficient of food conversion (FCR), selected from the group consisting in:

(to)

a polypeptide having amino acids 1-126 of the SEC ID NO: 4;

(b)

a polypeptide comprising an amino acid sequence having a degree of identity with amino acids 1-85 of the SEQ ID NO: 2 of at least 33%, as determined by the program Needle, using the BLOSUM62 replacement matrix, a penalty of space opening of 10, and an extension penalty of 0.5 and space

(C)

a polypeptide which is encoded by an acid sequence nuclei that hybridizes under medium astringency conditions with (i) nucleotides 124-378 of SEQ ID NO: 1, (ii) a sub-sequence of (i) of at least 100 nucleotides, and / or (iii) a complementary chain of (i), or (ii).