Polypeptides having protease activity and nuclear acids encoding the same

FIELD: biotechnology.

SUBSTANCE: disclosed is isolated polypeptide being acid-proof metalloprotease isolated from Thermoascus aurantiacus. Described are strain Thermoascus aurantiacus CGMCC No 0670 for polypeptide production and method for polypeptide production using said strain. Disclosed is method for plant protein treatment to increase digestion value thereof by using said polypeptide.

EFFECT: protease of good acid resistance useful in feed production.

6 cl, 7 ex, 4 tbl

 

The technical field

The present invention relates to the selected polypeptides having by activity, and selected sequences of nucleic acids encoding these polypeptides. This invention relates to constructions based on nucleic acids, vectors and cells of the host containing the above nucleic acid sequences, and methods of producing and using these polypeptides.

Background of the invention

Cloning of the gene of penicillamine (plnC) from a strain of Penicillium citrinum described in article Matsumoto et al., in Biochim. Biophys. Acta 1218: 469 (1994). The sequence was included in the database EMBL number D25535.

In the application WO 97/46689 described below (coding) sequence of the protease isolated from specific strains of Aspergillus:

SEQ ID No.1 in the application WO 97/46689 is incomplete gene rern Aspergillus niger;

SEQ ID No.2 in the application WO 97/46689 is incomplete cDNA rern Aspergillus niger;

SEQ ID No.3 in the application WO 97/46689 is incomplete amino acid sequence RERN Aspergillus niger;

SEQ ID No.4 in the application WO 97/46689 is gene pepI Aspergillus nidulans;

SEQ ID No.5 in the application WO 97/46689 represents cDNA pepI Aspergillus nidulans; and

SEQ ID No.6 in the application WO 97/46689 represents the amino acid sequence of PEPI Aspergillus nidulans.

In article Ramesh et al., Gene 165(1):121-125 (1995)described the cloning and IP is the study of genes, encoding protease from strains of Aspergillus flavus and Aspergillus fumigatus. These sequences included in the database EMBL respectively numbered L7524 and U24146.

A sequence variant of a neutral protease, created on the basis of neutral protease II from a strain of Aspergillus oryzae as described in Japan patent 05-168479. Cloning of the parent neutral protease II Aspergillus oryzae as described in article Hiroki Tatsumi et al., Mol. Gen. Genet. (1991), 228, p. 97-103.

The Mature peptide part of the protease is identical to the Mature peptide part of the protease Thermoascus of the present invention 75.7 percent or less.

The nucleotide sequence corresponding to the Mature peptide parts of the above proteases, identical to the nucleotide sequence corresponding to the Mature peptide part of the protease Thermoascus of the present invention, 68,4% or less.

The aim of the present invention to provide an alternative proteases for use in animal feed.

A brief statement of the substance of the invention

The present invention relates to the selected polypeptides, by having an activity selected from the group consisting of:

(a) a polypeptide containing the amino acid sequence that is at least 80% identical to amino acids 178-177, -159-177 or +1-177 SEQ ID No.2;

(b) polypeptide, the code is generated by a sequence of nucleic acid, which it hybridises under conditions of low (degree) severity (hardness):

(i) the Mature protease, the coding part of the plasmid in the strain DSM 14652 Escherichia coli;

(ii) nucleotides 25-1089, 1-1089, 1-1344, 25-1344, 559-1344 or 559-1089 SEQ ID No.1;

(iii) suppositionally (i) or (ii)containing at least 100 nucleotides, or

(iv) a complementary chain sequence (i), (ii) or (iii);

(C) a variant polypeptide containing the amino acid sequence comprising amino acids-178-177, -159-177 or +1-177 SEQ ID No.2, including substitutions, deletions and/or insertions of one or more amino acids;

(d) allelic variants of the polypeptide (a) or (b);

(e) a fragment of the polypeptide (a), (b) or (d)having by activity.

SEQ ID No.1 represents the cDNA sequence of the protease from strain CGMCC No. 0670 Thermoascus aurantiacus (SEQ ID No.1), and SEQ ID No.2 is derived from her amino acid sequence.

The present invention relates also to selected sequences of nucleic acids encoding the polypeptides, and to the structures based on nucleic acids, vectors and cells of the host containing the nucleic acid sequences, as well as to methods of production and use of the polypeptides, in particular, in animal feed.

Brief description of drawings

The figure 1 shows the test results on engebi the Finance using protease strain CGMCC No. 0670 Thermoascus aurantiacus.

The figure 2 shows the temperature profile of the above tests.

The figure 3 shows the profile of the pH of the above tests.

The figure 4 shows the stability of the pH in the above test.

The figure 5 shows the results of tests on substrate specificity in the above test.

Detailed description of the invention

The authors of the present invention unexpectedly discovered a new class of proteases that may be suitable for use in animal feed. The most well-known fodder proteases are serine-proteases, many of these proteases do not have sufficient acid to avoid destruction in the acidic environment of the stomach. Protease according to this invention are acid resistant metalloprotease isolated from Thermoascus aurantiacus. Mature polypeptide part of the specified protease contains amino acids 1-177 SEQ ID No.2. When performing in vitro, simulating digestion in animals with single-chamber stomach and fishes, it was found that protease Thermoascus aurantiacus can increase the amount of soluble and degradable protein and to increase the degree of protein hydrolysis. Homologous to a new polypeptide detected in Aspergillus oryzae (amino acids 177-353 SEQ ID No.11).

Polypeptides having by activity

Protease sometimes also referred to as a peptidase, proteases, peptide hydrolases or proteolytic enzymes. Protease can relate to Exo-type, whereby the hydrolysis of peptides occurs, starting from either end, or endo-type, in accordance with which the hydrolysis occurs within polypeptide chains (endopeptidase). Endopeptidase actively affecting peptide substrates, blocked N - and C-Terminus, which is typical for specificity under consideration protease.

The term "protease" used herein is meant an enzyme that hydrolyzes peptide bonds. A protease is any enzyme belonging to the group of enzymes EC 3.4 (including enzymes thirteen subclasses). EC-number refers to the nomenclature of enzymes presented in Enzyme Nomenclature 1992 NC-IUBMB, Academic Press, San Diego, California, including additions 1-5, published respectively in magazines Eur. J. Biochem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650. This item regularly and updated; see, for example, the Internet (WWW) on the website http://www.chem.gmw.ac.uk/iubmb/enzyme/index.html).

Proteases are classified with regard to their catalytic mechanism into the following groups: serine protease (S), cysteine protease, aspartic protease (A), metalloprotease (M) and unknown or not yet classified protease (U); see reference Handook of Proteolytic Enzymes, A.J. Barrett, N.D. Rawlings, J.F. Woessner (eds.), Academic Press (1998), in particular, General introduction.

In specific embodiments of the invention the protease according to this invention is selected from the group consisting of:

(a) proteases related to metalloendopeptidases EC 3.4.24;

(b) metalloprotease belonging to the group M, the above reference;

(C) metalloprotease, not yet assigned to any class (symbol: Clan MX) or related to one of the classes MA, MB, MC, MD, ME, MF, MG, MH (discussed on pp. 989-991 the above reference);

(d) other collections metalloprotease (discussed on pp. 1448-1452 the above reference);

(e) metalloprotease fragment HEXXH;

(f) metalloprotease fragment HEFTH;

(g) metalloprotease related to one of the families M3, M26, M27, M32, M, M35, m, M41, M or m (discussed on pp. 1448-1452 the above reference); and

(h) metalloprotease, belonging to the family M35 (discussed on pp. 1492-1495 the above reference).

In other specific embodiments of the invention metalloprotease are hydrolases, in which nucleophilic effect on the peptide bond is mediated by a water molecule activated by divalent cation metal. Examples of cations of divalent metals are zinc, cobalt or manganese. Metal ion can derivatise the desired position of the ligand amino acids. The number of ligands may be equal to five, four, three, two, one or zero. In a specific embodiment of the invention the number of ligands is two or three, preferably three.

To determine whether this protease by metalloproteases or not, you should refer to the above reference to a set of principles. Such a determination can be made for all types of proteases, regardless, is this protease to natural or wild type, is genetically engineered or synthetic.

The protease activity can be measured using any analysis that uses a substrate having a peptide bond, the corresponding specificity of the investigational protease. The pH and temperature of the analysis must also be aligned with the investigational protease. Examples of pH values for the analysis are pH 6, 7, 8, 9, 10 or 11. Examples of temperatures for analysis are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70 or 80°C.

An example of a substrate for a protease is casein, in particular casein, stitched with Surinam (AZCL-casein). Two analyses of protease described in example 1, of which the so-called analysis using AZCL-casein is preferred to achieve the objectives of the present invention.

There are no restrictions relating to the origin of the protease filed with the invention. Thus, the term "protease" includes not only protease natural or wild-type obtained from microorganisms of any kind, but also any mutants, variants, fragments, etc. with by activity, as well as synthetic protease, such as inverted protease and consensus protease. Such genetically engineered protease can be obtained by methods known in this field, for example, by using site-directed mutagenesis, polymerase chain reaction (PCR) (using the PCR fragment containing the desired mutation as a nucleating when performing PCR reactions) or non-specific mutagenesis. Obtaining consensus proteins are described, for example, in European patent EP 897985. The term "obtained from" as applied to this source means that the polypeptide encoded by the nucleic acid sequence produced by the source or by a cell in which the nucleic acid sequence. In a preferred embodiment of the invention the polypeptide is secreted outside the cell.

In a specific embodiment of the invention, the protease is kabullendim option, causing a reduced immunological response when exposed to animals, including humans. The term "immunological response" refers to any of the reaction the human immune system of the animal under the action of this protease. One type of immunological reaction is an allergic reaction that causes increased levels of IgE have been subjected to such impact animal. Labelengine options can be obtained by methods known in this field. For example, a protease can be conjugated with polymers that protect parts or epitopes protease, causing an immunological reaction. Conjugation with polymers may include chemical linking of the polymer with protease in vitro, as described, for example, in applications WO 96/17929, WO 98/30682, WO 98/35026 and/or WO 99/00489. Algae may additionally or alternatively enable the linking of the polymers with the protease in vivo. Such conjugation may be achieved by the creation of methods of genetic engineering nucleotide sequence that encodes a protease, the introduction of protease consensus sequences encoding additional sites of glycosylation and expression of proteases in the host, capable to glycosylate protease, as described, for example, in the application WO 00/26354. Another way to get labellling option is to create methods of genetic engineering nucleotide sequence that encodes a protease that causes autoaugmentation protease, which one protease monomers can protect epitopes of other monomers protease and reduce, so that the antigenicity of the oligomers. Such products and obtaining them are described, for example, in the application WO 96/16177. Epitopes inducing an immunological response can be identified by various methods such as a method for the detection of phages described in applications WO 00/26230 and WO 01/83559, or nonspecific approach described in EP No. 561907. After identification of the epitope of its amino acid sequence can be changed by obtaining modified immunological properties of protease, using known methods of manipulating genes, such as siteprovides mutagenesis (see, for example, applications WO 00/26230, WO 00/26354 and/or WO 00/22103), and/or conjugase polymer in sufficient proximity to the epitope to protect the epitope.

The present invention relates to the selected polypeptides containing an amino acid sequence that is identical to amino acids 178-177, -159-177 or preferably amino acids 1-177 (Mature polypeptide) SEQ ID No.2 at least about 64%, or at least about 65%, at least about 70% or at least about 75%, at least about 76%, or at least about 77%, at least about 78%, or at least about 79%, at least about 80% or at least about 82%, at least about 85%, or at least about 90%, or at least about 95%, or is about, at least about 97%, and has by activity (hereinafter "homologous polypeptides"). In specific embodiments of the invention the polypeptides according to this invention (i) have or (ii) consist of amino acid sequences with the above degree of identity.

Protease Thermoascus aurantiacus, Mature polypeptide which contains the amino acids 1-177 SEQ ID No.2, undoubtedly, is one example of a polypeptide according to this invention.

Another polypeptide according to this invention, derived from Aspergillus oryzae, contains SEQ ID No.11, or amino acids-23-353, -23-374, -23-397, 1-353, 1-374, 1-397, 177-353, 177-374 or 177-397 and is encoded by SEQ ID No.10 or her nucleotides 2-1129, 2-1195, 2-1267, 71-1129, 71-1195, 71-1267, 599-1129, 599-1195 or 599-1267.

In accordance with the purposes of the present invention, the degree of identity of two amino acid sequences, as well as the degree of identity between two nucleotide sequences can be determined by using the program "Align", which is a comparison of Needlman-Wunsch (i.e. global mapping). This program is used to map the polypeptide and nucleotide sequences. For comparison of polypeptides using a matrix calculation by default BLOSUM50 and for matching nucleotides are used, the identity matrix by default. For the first balance circuit beg keetsa -12 points for polypetides and -16 points for nucleotides. Over the next remnants of the circuit is calculated -2 points for polypeptides and -4 points for nucleotides.

The program "Align" is part of the FASTA software package version v20u6 (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). In the mapper proteins used FASTA algorithm Smith-Waterman without limit circuit (see Smith-Waterman algorithm", T.F. Smith and M.S. Waterman (1981) J. Mol. Biol. 147:195-197).

In a specific embodiment of the invention the homologous polypeptides have an amino acid sequence that differs forty, thirty-five, thirty, twenty five, twenty or fifteen amino acids. In another embodiment of the invention the homologous polypeptides have an amino acid sequence that differs by ten, nine, eight, seven, six or five amino acids. In another specific embodiment of the invention homologous polypeptides differ in four, three or two amino acids, or by one amino acid from amino acids-178-177, -159-177 or +1-177 SEQ ID No.2.

In specific embodiments of the invention, the polypeptides of the present invention (a) or (b) consist of

i) amino acid sequences consisting of amino acids-178-177, -159-177 or +1-177 SEQ ID N.2;

ii) the amino acid sequence consisting of amino acids-23-353, -23-374, -23-397, 1-353, 1-374, 1-397, 177-353, 177-374 or 177-397 SEQ ID No.11; or

allelic variants or fragments of the sequences (i) and (ii)having by activity.

A fragment of the amino acid-178-177, -159-177 or +1-177 SEQ ID No.2 or amino acid-23-353, -23-374, -23-397, 1-353, 1-374, 1-397, 177-353, 177-374 or 177-397 SEQ ID No.11 is a polypeptide in which one or more amino acids deleted from the amino and/or carboxyl end of the specified amino acid sequence. In one embodiment of the invention, the fragment contains at least 75 amino acid residues, or at least 100 amino acid residues, or at least 125 amino acid residues, or at least 150 amino acid residues, at least 160 amino acid residues, or at least a 165 amino acid residues, or at least 170 amino acid residues, or at least 175 amino acid residues.

Allelic variant is one of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variant is formed by mutation and can lead to polymorphism in populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides containing the modified and inoculate sequence. Allelic variant of a polypeptide is a polypeptide encoded by allelic variant of a gene.

The present invention relates also to a selected polypeptide, which have by activity, which is encoded by the sequences of nucleic acids, hybridizers in conditions of very low, low, medium, medium-high, high, or very high stringency with a probe nucleic acid, which it hybridises under the same conditions (a) nucleotides 25-1089, 1-1089, 1-1344, 25-1344, 559-1344 or preferably nucleotides 559-1089 SEQ ID No.1, (b) cDNA sequence, located in the nucleotide 559-1089 SEQ ID No.1, (C) suppositionally sequence (a) or (b), (d) with complementary chain sequences (a), (b) or (C) (J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York). In one specific embodiment of the invention, the probe nucleic acid selected from the above-mentioned nucleic acids sequences (a), (b), (c) or (d).

The sequence of nucleotides 559-1089, 25-1089, 1-1089, 1-1344, 25-1344 or 559-1344 SEQ ID No.1 can contain at least 100 nucleotides, or in another embodiment of the invention, at least 200 nucleotides. Furthermore, this sequence may encode a polypeptide fragment having by activity.

Sequence nucleotide sequence that is new acid, containing nucleotides 559-1089, 25-1089, 1-1089, 1-1344, 25-1344 or 559-1344 SEQ ID No.1 or suppositionally and amino acid sequences containing amino acids-178-177, -159-177 or +1-177 SEQ ID No.2 or a fragment, can be used to create a probe nucleic acid to identify and clone DNA encoding polypeptides having by activity from strains of other genera or species by methods well known in the field. In particular, such probes can be used for hybridization with the genomic or cDNA of interest genus or species by standard methods southern blot to identify and isolate the corresponding gene. Such probes can be considerably shorter than the entire sequence, but should contain at least 15, preferably at least 25 and more preferably at least 35 nucleotides. You can also use longer probes. You can use DNA and RNA probes. Probes usually mark for detecting the corresponding gene (for example,32R3H,35S, Biotin or Avidya). Such probes are included in the scope of the present invention.

Thus, it is possible to perform the screening of libraries of genomic DNA or cDNA obtained from other similar organisms for screening DNA hybridises with the above-described probes and coding on pepcid, possessing by activity. Genomic or other DNA from other organisms can be distinguished by electrophoresis in agarose or polyacrylamide gel, or other methods of selection. DNA from the libraries or selected DNA can be transferred or mobilitat on the nitrocellulose or other acceptable media. In order to identify a clone or DNA which is homologous to SEQ ID No.1 or suppositionally, the media used in the analysis by the method of southern blotting. In accordance with the purposes of the present invention, hybridization indicates that the nucleic acid sequence it hybridises with the labeled probe nucleic acid, the corresponding nucleic acid sequence presented in SEQ ID No.1, its complementary chain or suppositionally, in conditions from very low to very high stringency. Molecules with which it hybridises probe nucleic acid under these conditions, can be detected using x-ray film.

In a specific embodiment of the invention, the probe nucleic acid is a nucleic acid sequence encoding amino acids-178-177, -159-177 or +1-177 SEQ ID No.2, or suppositionally. In another embodiment of the invention, the probe nucleic acid contains a nucleotide 25-1089, 1-1089, 1-1344, 25-1344, 559-1344 librepository nucleotides 559-1089 SEQ ID No.1 (encoding a Mature polypeptide region of SEQ ID No.1). In another preferred embodiment of the invention, the probe nucleic acid is a sequence of nucleic acids or preferably its region encoding the Mature polypeptide, which is located in the plasmid in Escherichia coli DSM 14652, the nucleic acid sequence encodes a polypeptide having by activity.

For long probes containing at least 100 nucleotides conditions from very low to very high stringency is defined as prehybridization and hybridization at 42°With a 5-fold volume of SSPE, 0.3% of SDS, 200 μg/ml fragmented and denatured DNA salmon sperm and 25% formamide for very low and low severity, 35% formamide for medium duty and medium-high severity, or 50% formamide for high and very high severity in accordance with standard methods southern blot.

For long probes containing at least 100 nucleotides, media washed three times for 15 minutes using 2-fold volume of SSC, and 0.2% SDS preferably at least at 45°C (very low stringency), more preferably at least at 50°C (low stringency), more preferably at least at 55°With (medium stringency), more preferably n is at least 60° With (medium-high stringency), more preferably at least at 65°With (conditions of high stringency), and most preferably at least at 70°C (very high stringency).

To obtain short probes ranging in length from about 15 nucleotides to about 70 nucleotides stringent conditions are defined as prehybridization, hybridization and washing after hybridization at a temperature of 5°C-10°C below the calculated temperature Tmthe computation of Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, And 0.09 M Tris-HCl c pH of 7.6, 6 mm EDTA, 0,5% NP-40, 1 volume of a solution of Denhardt, 1 mm sodium pyrophosphate, 1 mm monobasic phosphate, 0.1 mm ATP, and 0.2 mg of yeast RNA per ml in accordance with standard methods southern blot.

To obtain short probes ranging in length from about 15 nucleotides to about 70 nucleotides media once washed 6 times the volume of SCC with 0.1% SDS for 15 minutes and washed twice 6-fold volume of SSC for 15 minutes at a temperature of 5°C-10°C below the calculated temperature Tm.

The present invention also pertains to variants of the polypeptide containing the amino acid sequence consisting of amino acids 1-177, -159-177 or 178-177 SEQ ID No.2, which includes the replacement, deletion and/or insertion of one or more amino acids.

Aminokislot the e sequence variant polypeptides may differ from the amino acid sequence, consisting of amino acids 1-177, -159-177 or 178-177 SEQ ID No.2, due to insertions or deletions of one or more amino acid residues and/or substitution of one or more amino acid residues with other amino acid residues. Replacement amino acid should be preferably small, for example, conservative amino acid substitutions that do not have a significant impact on the laying of the chain and/or activity of the protein, small deletions, typically of one to about 30 amino acids, small extension fragments from the amino - or carboxyl end, such as aminobenzoic a methionine residue, a small linker peptide containing about 20-25 residues; or a small extension fragment, easy-to-clean due to the change of the total charge or another function, such as polyhistidine tail, antigenic epitope or binding domain.

Examples of conservative substitutions belong to the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid or aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and amino acids with short-chain (glycine, alanine, serine, threonine and methionine). Replacement of amino acids that do not alter the specific activity of the, known in this field and are described, for example, in the publication of H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. The most common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly, and these replacement in reverse order.

The present invention relates also to a selected polypeptide, by having an activity selected from the group consisting of:

(f) polypeptides having

(i) the calculated molecular weight from about 17 to about 22 kDa, preferably from about 18 to about 21 kDa, or from about 19 to about 20 kDa; or experimentally established molecular mass (SDS-PAGE) from about 19 to about 27 kDa, preferably from about 20 to about 26 kDa, from about 21 to about 25 kDa, or from about 22 to about 24 kDa;

(ii) pI from about pH 7 to about pH 10, preferably from about pH 7.5 to about pH 9.5 or from about pH 8.0 to about pH 9,0;

(iii) optimum pH from about pH 5.5 to about pH 8.0, preferably from about pH 5.5 to about pH 7.0, or from about pH 5.5 to about pH 6.5 as determined by analysis using AZCL-casein at 45°and a buffer system with succinic acid; and/or

(iv) an optimum temperature of about 45°to 90°C, preferably from about 50°C to 85°From or from about 60°C to about 80°With that determined the th through analysis using AZCL-casein at rn;

(g) polypeptides, without inhibiting

(v) SSI (inhibitor of Streptomyces subtilisin) and/or

(vi) EDTA

when determining by analysis using AZCL-casein at pH 9 and 45°C;

(h) polypeptides that are resistant in the range from about pH 3.5 to about pH to 10.5, preferably in the range of from about pH 3.5 to about pH of 9.0 or in the range of from about pH 4.0 to pH 5.0 after incubation for 2 hours at 37°in the buffer system with succinic acid, while the residual activity is measured by analysis using AZCL-casein at rn and 45°C;

(k) polypeptides synthesized using N-terminal of propeptide; and

(l) polypeptides having the motive NEHIN.

The stability of the polypeptide according to (h) means that the residual activity is equal to at least 40%, 50%, 60%, 70% or 80% compared with the control sample which was not subjected to pre-incubation for 2 hours. Polypeptides that are sustainable in accordance with this definition, can be defined as acid polypeptides.

The polypeptide of the present invention may be a bacterial polypeptide. For example, such a polypeptide can be a polypeptide gram-positive bacteria, such as the polypeptide of Bacillus or Streptomyces polypeptide; or a polypeptide gram-negative bacteria, for example the polypeptide of E. coli or Pseudomonas sp.

The polypeptide of the present invention may be a fungal polypeptide, and more preferably a yeast polypeptide such as a polypeptide Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia; or more preferably a polypeptide filamentous fungus, such as a polypeptide Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma.

In another embodiment of the invention the polypeptide is a polypeptide Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium frost, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Thermoascus aurantiacus, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

In another embodiment of the invention the polypeptide is a polypeptide isolated from a filamentous fungus type Ascomycota, preferably class Pezizomycotina, more preferably squad Eurotiomycetes, more preferably subfamily Eurotiales and most preferably of the family Trichocomaceae.

In yet another embodiment of the invention the polypeptide allocate the C of a fungus of the genus Thermoascus, for example type Thermoascus aurantiacus, such as strain CGMCC No. 0670 Thermoascus aurantiacus, for example, the polypeptide with the amino acid sequence containing amino acids-178-177, -159-177 or +1-177 SEQ ID No.2.

It is obvious that the scope of the present invention includes the above-mentioned species in the perfect and imperfect States, and other taxonomic equivalents, such as anamorphic, regardless of the form under which they are known. Specialists in this field can easily establish the identity of the relevant equivalents.

Strains of these species can be obtained in a number of culture collections, such as the American type culture collection (ATSS), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

Further, such polypeptides can be identified and obtained from other sources including microorganisms isolated from natural sources (e.g., soil, compost, water, etc) using the above probes. Methods for isolation of microorganisms from natural habitats is well known in this field. The sequence of the nucleic acid can be isolated from the same screening libraries of genomic DNA or cDNA of another microorganism. After detection with a probe (probes) sequence nekleenov the th acid, encodes a polypeptide, the sequence can be distinguished or clone methods known to experts in this field (see, for example, Sambrook et al., 1989, cited above).

As specified in this description of the invention, "isolated" polypeptide is a polypeptide, which essentially contains no other napoleanic polypeptides, for example, the polypeptide is pure, at least about 20%, preferably at least about 40%, more preferably about 60%, more preferably about 80%, most preferably about 90% and especially preferably approximately 95% when determining the method of SDS-PAGE.

The polypeptides encoded by the sequences of nucleic acids of the present invention also include fused polypeptides or split fused polypeptides in which another polypeptide is fused to the N-end or C-end of the polypeptide or its fragment. Fused polypeptide is produced by fusion nucleic acid sequence (or portion thereof)that encodes a different polypeptide sequence (nucleic acid or part thereof) of the present invention. Methods of obtaining the fused polypeptides known in the field and include ligation of sequences encoding polypeptides in which they are framed sitiveni and expression of the fused polypeptide is controlled by the same promoter (promoter and terminator.

The nucleic acid sequences

The present invention relates also to selected sequences of nucleic acids that encode the polypeptide of the present invention. A typical sequence of nucleic acids according to this invention contain nucleotides 25-1089, 1-1089, 1-1344, 25-1344, 559-1344 and, in particular, nucleotides 559-1089 SEQ ID No.1, and last nucleotides correspond to the region that encodes a Mature polypeptide. Another typical sequence of nucleic acids according to this invention is a sequence, preferably its region encoding the Mature polypeptide, which is incorporated into the plasmid introduced into the deposited microorganism Escherichia coli DSM 14652. In the scope of the present invention also includes nucleic acid sequences that encode a polypeptide containing an amino acid sequence comprising amino acids 1-177, -159-177 or 178-177 SEQ ID No.2, which differ from the corresponding parts of SEQ ID No.1 due to the degeneracy of the genetic code. The present invention relates also to suppositionally SEQ ID No.1, which encode fragments of SEQ ID No.2 with by activity.

The present invention relates also to selected sequences of nucleic acids that encode the polypeptide of the present invention. Type CNAME sequences of nucleic acids according to this invention are SEQ ID No.10 or nucleotides 2-1129, 2-1195, 2-1267, 71-1129, 71-1195, 71-1267, 599-1129, 599-1195 or 599-1267, nucleotides 71-1129 SEQ ID No.10, corresponding to the region that encodes a Mature polypeptide.

Suppositionally SEQ ID No.1 is a sequence of nucleic acids included in SEQ ID No.1, except for deletions of one or more nucleotides at the 5'and/or 3'-end. Suppositionally preferably contains at least 225 nucleotides, more preferably at least 300 nucleotides, more preferably at least 375, 450, 500, 531, 600, 700, 800, 900, 1000, 1100, 1200 or 1300 nucleotides.

The present invention relates to nucleotide sequences with a degree of identity to nucleotides 25-1089, 1-1089, 1-1344, 25-1344, 559-1344 or preferably the nucleotides 559-1089 SEQ ID No.1, equal to at least 69%, 70%, 72%, 74%, 76% or 80%. In a specific embodiment of the invention, the degree of identity is equal to at least 85%, at least 90%, at least 95% or at least 97%. To determine the degree of identity of the nucleotides can be used in the program "align".

A particular variant embodiment of the invention relates to nucleotide sequences with a degree of identity to nucleotides 25-1089, 1-1089, 1-1344 or 25-1344 SEQ ID No.1, equal to at least 55%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76% or 80%. In a specific embodiment of the invention, the degree of identity equal to Raina least 85%, at least 90%, at least 95% or at least 97%. To determine the degree of identity of the nucleotides can be used in the program "align".

The present invention relates to mutant sequences of nucleic acids containing at least one mutation in the nucleotide 25-1089, 1-1089, 1-1344, 25-1344, 559-1344 or 559-1089 SEQ ID No.1, where the mutant nucleic acid sequence encodes a polypeptide which (i) consists of amino acids-178-177, -159-177 or +1-177 SEQ ID No.2, (ii) is a variant of any sequence (i), which comprises a substitution, a deletion and/or insertion of one or more amino acids, (iii) is an allelic variant of any sequence (i) or (iv) is a fragment of any sequence (i).

The methods used for selection or cloning nucleic acid sequence that encodes the polypeptide, are known in this area and include isolation from genomic DNA, from cDNA, or a combination of these methods. The nucleic acid sequences of the present invention can be cloned from a genomic DNA, for example, using the well known polymerase chain reaction (PCR) or serological screening of libraries of expressed sequences to detect fragments of cloned DNA with related structural recognized the AMI. See, for example, Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. You can use other methods of nucleic acid amplification, such as ligase chain reaction (LCR), legirovannye activated transcription (LAT) and amplification-based nucleic acid sequence (first NASBA). The sequence of the nucleic acid can be cloned from a strain of Thermoascus, another or related organism and thus, for example, may be an allelic or species variant encodes a polypeptide region nucleic acid sequence.

The term "isolated nucleic acid sequence" used herein is meant a sequence of nucleic acid, which essentially contains no other sequences of nucleic acids, for example, is pure, at least about 20%, preferably at least about 40%, more preferably at least about 60%, more preferably at least about 80% and most preferably at least about 90% in the determination by agarose gel electrophoresis. For example, the selected nucleic acid sequence can be obtained by standard cloning methods used in genetic engineering to move the nucleic acid sequence of the natural the military situation on another site, where it will be reproduced. Cloning techniques may include cutting and isolation of the desired fragment of the nucleic acid comprising the sequence of a nucleic acid encoding the polypeptide, insertion of the fragment into the vector molecule and the introduction of the recombinant vector into the cell host, which will be replicated multiple copies or clones of a given nucleic acid sequence. On the origin of the nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic or can be a combination of these two sequences.

The present invention relates also to sequences of nucleic acids coding for active polypeptide, which have a degree of homology with the coding of the Mature polypeptide sequence of SEQ ID No.1 (i.e. nucleotides 559-1089)equal to at least about 70%, preferably about 75%, preferably about 80%, more preferably about 90%, more preferably about 95% and most preferably about 97%. In accordance with the purposes of the present invention, the degree of homology between two sequences of nucleic acids can be determined using the above program "align".

Modification of the nucleic acid sequence that encodes a polypeptide according to astasia the invention, it may be necessary for the synthesis of polypeptides is essentially similar to a given polypeptide. The term "essentially similar" in relation to the polypeptide does not mean a naturally occurring form of the polypeptide. When designing such polypeptides may differ from the polypeptide isolated from a natural source, for example, they may represent variants that differ in specific activity, thermostability, the optimal pH or similar parameters. The variant sequence may be constructed on the basis of nucleic acid sequence, which is specified coding the polypeptide part of SEQ ID No.1, for example its suppositionally, and/or by introduction of nucleotide substitutions which do not form another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but correspond to the codon of the host organism used for the production of protease, or by introduction of nucleotide substitutions which may form another amino acid sequence. For a General introduction to the replacement of nucleotides, see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107.

Professionals in this field should be obvious that such substitutions can be made outside the regions with significant Zn is necessary for the functioning of the molecule, while allowing to obtain an active polypeptide. Amino acid residues important for activity of the polypeptide encoded allocated by the sequence of nucleic acid according to this invention, and therefore preferably not subject to substitution, may be identified by methods known in this field, such as siteprovides mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). When using the latter method, mutations are introduced at every positively charged residue in the molecule and the resulting mutant molecules are tested on protease activity to identify amino acid residues that are essential for the activity of the molecule. The sites of interaction of substrate-protease can also be determined by analysis of three-dimensional structure by methods such as nuclear magnetic resonance, crystallography or photoaffinity tagging (see, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The present invention relates also to selected sequences of the nucleic acid coding for the polypeptide of the present invention, which it hybridises under very low stringency, preferably under conditions of low stringency, more preferably in the middle row the spine, more preferably under conditions of medium to high stringency, more preferably under conditions of high stringency, and most preferably under very high stringency with a probe nucleic acid, which it hybridises under the same conditions with the nucleic acid sequence SEQ ID No.1 or its complementary chain, or allelic variants and suppositionally (Sambrook et al., 1989, see above), as defined here.

The present invention relates also to selected sequences of nucleic acids obtained (a) by hybridization of DNA under very low, low, medium, medium-high, high, or very high stringency with (i) nucleotides 559-1089, 25-1089, 1-1089, 1-1344, 25-1344 or 559-1344 SEQ ID No.1, (ii) the cDNA sequence contained in nucleotides 559-1089, 25-1089, 1-1089, 1-1344, 25-1344 or 559-1344 SEQ ID No.1, (iii) suppositionally sequence (i) or (ii) or (iv) a complementary chain sequence (i), (ii) or (iii); and (b) by separating the nucleic acid sequence. Suppositionally preferably is a sequence containing at least 100 nucleotides such as a sequence encoding a polypeptide fragment having by activity.

Methods of obtaining mutant sequences of nucleic acids

This izobreteny the further relates to methods for producing a mutant nucleic acid sequence, which include the introduction of at least one mutation in the coding for the Mature polypeptide sequence of SEQ ID No.1 or her suppositionally, and the mutant nucleic acid sequence encodes a polypeptide consisting of amino acids 1-177, -159-177 or 178-177 SEQ ID No.2, or its fragment having by activity.

The introduction of mutations in the nucleic acid sequence for the replacement of one nucleotide with another nucleotide may be produced by siteprovides mutagenesis using any of the methods known in this field. Particularly suitable is a method that uses vector-based super helical conformation was, double-stranded DNA of interest insert and two synthetic seed containing the desired mutation. Oligonucleotide priming, complementary to the opposite chains of the vector, are extended during cyclic temperature changes using a DNA polymerase Pfu. When embedding the seed is formed mutated plasmid containing irregularly distributed single-stranded breaks. After the cyclic change in temperature, the product is treated with Dpnl, which specifically affects methylated and paleometeorology DNA, splitting the parent matrix DNA and selecting synthesized DNA containing the mutation. It is possible that the use of other methods, known in this field.

Design on the basis of nucleic acids

The present invention relates also to the structures on the basis of nucleic acids that contain the nucleic acid sequence of the present invention, functionally associated with one or more regulatory sequences directing expression of the coding sequence in an appropriate cell host under conditions comparable to regulatory sequences. It is clear that the expression includes any stage, implemented with the production of the polypeptide, which include, but are not limited to, transcription, post transcriptional modification, translation, post-translational modification and secretion.

The term "design-based nucleic acid" used herein is meant a molecule of single-stranded or double-stranded nucleic acid that is isolated from a natural gene or modified with the inclusion of segments of nucleic acids, combined and arranged in the order in which they never occur in nature. The term "design-based nucleic acid" is synonymous with the term "expressing cluster", when construction on the basis of the nucleic acid contains all of the regulatory sequences necessary for expression of the coding for the overall sequence of the present invention. The term "coding sequence" used herein is meant a nucleic acid sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence is usually determined by the binding site of the ribosome (prokaryotes) or the initiating codon ATG (eukaryotes), located above the open reading frame at the 5'-end of mRNA, and the sequence termination of transcription below the open reading frame at the 3'-end of mRNA. The coding sequence can include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

The sequence of the nucleic acid encoding the polypeptide of the present invention can be modified in different ways to achieve expression of the polypeptide. Changing the sequence of the nucleic acid before insertion into a vector may be desirable or necessary depending on the expressing vector. Methods of modification of sequences of nucleic acids using methods of recombinant DNA are well known in this field.

The term "regulatory sequence" is used here in the meaning means all components which are necessary or advantageous for expression of the polypeptide present Adamu invention. Each regulatory sequence may be native or foreign to the nucleic acid sequence that encodes a polypeptide. Such regulatory sequences include, but are not limited to, leader sequence, a polyadenylation sequence, propeptide sequence, the promoter sequence of the signal peptide and the terminator of transcription. Regulatory sequences include at least a promoter and signals termination of transcription and translation. The regulatory sequence can have linkers for introducing specific restriction sites facilitating ligation of regulatory sequences with the coding region of the nucleic acid sequence that encodes a polypeptide. The term "functionally linked" is used here, the value applies to the configuration in which the regulatory sequence is in the correct position relative to the coding DNA sequence, allowing regulatory sequences to direct the expression of the polypeptide.

The regulatory sequence may be acceptable promotor sequence representing the sequence of nucleic acids that is recognized by the host-cell for the expression of nucleic acid sequence. P is motorna sequence contains the sequence regulating transcription, which mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence which shows transcriptional activity in the selected cell host, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous cell-master.

Examples of acceptable promoter used to direct transcription of structures on the basis of the nucleic acids of the present invention in the cell host, representing the filamentous fungus, are the promoters obtained from the genes TAKA-amylase of Aspergillus oryzae, aspartic proteases Rhizomucor miehei, neutral alpha-amylase, Aspergillus niger acid alpha-amylase of Aspergillus niger, glucoamylase Aspergillus niger (glaA) or Aspergillus awamori, lipase Rhizomucor miehei, alkaline protease of Aspergillus oryzae, triosephosphate Aspergillus oryzae, acetamidate Aspergillus nidulans and trypsin-like Fusarium oxysporum protease (application WO 96/00787), and the promoter NA2-tpi (a hybrid of the promoters from the genes of neutral alpha-amylase of Aspergillus niger and triosephosphate Aspergillus oryzae), and mutant, truncated, and hybrid promoters.

The regulatory sequence may also be acceptable sequence termination of transcription, sequence, resposnive the second cell host for termination of transcription. Termination sequence functionally linked to the 3'end of the nucleic acid sequence that encodes a polypeptide. In the present invention can use any terminator which is functional in the selected cell host.

Preferred terminators for host cells, representing filamentous fungi, derived from genes TAKA-amylase of Aspergillus oryzae, glucoamylase Aspergillus niger, anthranilate synthase of Aspergillus nidulans, alpha-glucosidase from Aspergillus niger and trypsin-like Fusarium oxysporum protease.

The regulatory sequence may also be acceptable leader sequence, untranslated region of mRNA that is important for translation of the host-cell. Leader sequence functionally linked to the 5'-end of nucleic acid sequence that encodes a polypeptide. In the present invention can use any leader sequence that is functional in the selected cell host.

Preferred leader sequences for host cells filamentous fungi derived from genes TAKA-amylase of Aspergillus oryzae and triosephosphate Aspergillus nidulans.

The regulatory sequence may also be a polyadenylation sequence, operatively linked to the 3'-end sequence of the nucleic KIS is the notes, which is in the process of translation is recognized by the host-cell as a signal about adding residues polyadenine to transcribed mRNA. In the present invention can use any polyadenylation sequence which is functional in the selected cell host.

The preferred sequence for polyadenylation of master cells of filamentous fungi derived from genes TAKA-amylase of Aspergillus oryzae, glucoamylase Aspergillus niger, anthranilate synthase of Aspergillus nidulans, trypsin-like Fusarium oxysporum protease and alpha-glucosidase from Aspergillus niger.

The regulatory sequence can be also encodes a signal peptide region, which encodes the amino acid sequence associated with aminocom.com polypeptide and directs the encoded polypeptide into the secretory pathway cells. 5'-end of the coding sequence, which is part of the nucleic acid sequence may contain encoding the signal peptide region naturally linked in reading frame broadcast to the segment of the coding region, which encodes a secretory polypeptide. Alternatively, the 5'-end of the coding sequence may contain encoding the signal peptide region, which is alien coding sequence. Alien region encoding a signal pepti is, may be necessary in the case where the coding sequence does not contain the natural state of the region encoding the signal peptide. Alternatively, alien region encoding a signal peptide, may simply replace the natural region encoding a signal peptide, to enhance secretion of the polypeptide. However, in the present invention can use any region encoding a signal peptide, which directs the expressed polypeptide into the secretory path selected host cell.

Effective regions encoding the signal peptide, cell host filamentous fungi are encoding the signal peptide region derived from genes TAKA-amylase of Aspergillus oryzae neutral amylase, Aspergillus niger, glucoamylase Aspergillus niger, aspartic proteases Rhizomucor miehei, cellulase Humicola insolens and Humicola lipase lanuginosa.

In a preferred embodiment of the invention, the region encoding the signal peptide comprises the nucleotide 25-81 SEQ ID No.1, which encode amino acids 1-19 SEQ ID No.2.

The regulatory sequence can also be the coding propeptide region, which encodes the amino acid sequence located at linekona polypeptide. The obtained polypeptide known as properties or propolypeptide (or zymogen in some cases). Propolypeptide about what commonly is inactive and can be converted into the Mature active polypeptide in the catalytic or autocatalytical off propeptide from propolypeptide. The region encoding propeptide, can be obtained from the genes for alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Bacillus subtilis alpha-factor and Saccharomyces cerevisiae, aspartic proteases Rhizomucor miehei and laccase Myceliophthora thermophila (application WO 95/33836).

In a preferred embodiment of the invention, the region encoding propeptide, includes nucleotides 82-558 SEQ ID No.1, which encode amino acids 20-178 SEQ ID No.2.

When the field signal peptide and propeptide are aminobenzo polypeptide, the area propeptide is located next to aminocom.com polypeptide and a region of the signal peptide is located next to aminocom.com area propeptide.

In addition, it may be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are such systems that include or switch off expression of a gene under the action of a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac and trp-operator system. In the yeast system can be used ADH2 or GAL1. In filamentous fungi as regulatory sequences can be used TAKA alpha-amylase promoter, glucoamylase promoter, Aspergillus niger and glucoamylase the promoter of Aspergillus oryzae. Other examples of regulatory sequences include those sequences that provide amplification of the gene. In eukaryotic systems, these sequences include gene dihydrofolate-reductase, which is amplified in the presence of methotrexate, and genes metallothionein, which are amplified by heavy metals. In such cases, the nucleic acid sequence encoding the polypeptide must be functionally linked to a regulatory sequence.

Expressing the vectors

The present invention relates to recombinant expressing the vectors containing the nucleic acid sequence of the present invention, the promoter and signals termination of transcription and translation. The various above-described nucleic acid sequences and regulatory sequences can be linked to each other to form expressing recombinant vector which may include one or more conventional sites of restriction, providing the insertion or replacement nucleic acid sequence that encodes a polypeptide at such sites. Alternatively, the nucleic acid sequence of the present invention can be expressed by introducing a nucleic acid sequence or design is based on the nucleic acid, comprising the sequence into an appropriate vector for expression. When creating expressing vector encoding the sequence of features in the vector so that the coding sequence was functionally linked to appropriate regulatory sequences to ensure the expression.

Recombinant expressing the vector may be any vector (e.g., plasmid or virus), standard variable methods of recombinant DNA and causing expression of the nucleic acid sequence. The choice of vector is usually dependent on the compatibility of the vector with the host-cell in which you want to enter a vector. The vectors may be linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e. a vector which exists in the form of extrachromosomal element, replication which does not depend on the replication of chromosomes, such as, for example, a plasmid, an extrachromosomal element, minichromosome or artificial chromosome. The vector can contain any elements that provide autoreplication. Alternatively, the vector may be a vector which, when introduced into the cell host is integrated into the genome and replicates along with the chromosome in which it was introduced. In addition, you can use a single vector or plasmid or two or the more vectors or plasmids which together contain the total DNA intended for introduction into the genome of the host cell, or a transposon.

The vectors of the present invention preferably contain one or more breeding markers, which allow quick selection of transformed cells. Breeding marker is a gene the product of which has a biocide or viral resistance, resistance to heavy metals, prototrophic to auxotrophy and similar properties. Examples of bacterial breeding markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, indicating resistance to antibiotics, such as ampicillin, kanamycin, chloramphenicol or tetracycline. Acceptable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Breeding markers suitable for use in the cell-host filamentous fungus, include, but are not limited to, amdS (acetamides), argB (emitintermediate), bar (phosphinotricin-acetyltransferase), hygB (hygromycin-phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate-decarboxylase), sC (sulfate-adenanthera), trpC (anthranilate-synthase), and their equivalents. In the Aspergillus cell, it is desirable to use the genes of the amdS and pyrG from Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

The vectors of the present invention predpochtitel what about the containing element(s), which provides a stable integration of the vector into the genome of the host cell or Autonomous replication of the vector in the cell independent of the genome.

For integration into the genome of the host cell, the vector may use the sequence of the nucleic acid encoding the polypeptide or any other element of the vector, providing a stable integration of the vector into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional nucleic acid sequences directing integration into the genome of the host cell by homologous recombination. Additional nucleic acid sequences enable the vector to integrate into the genome of the host cell in the exact place on the chromosome. To increase the likelihood of integration at a precisely defined location, integrating elements should preferably contain a sufficient number of nucleic acids, for example 100-1500 base pairs, preferably 400-1500 base pairs and most preferably 800-1500 base pairs, which are highly homologous to the corresponding target sequence to enhance the probability of homologous recombination. Inline elements can be any sequence that is homologous to a target sequence in the genome of the host cell. In addition, integrating elements can b the th non-coding or coding sequences of nucleic acids. On the other hand, the vector may be integrated into the genome of the host cell by homologous recombination.

For Autonomous replication, the vector may further include a replication origin allowing the vector to replicate autonomously in the cell host. Examples of bacterial start points of replication are the starting point of replication of plasmids pBR322, pUC19, pACYC177, and rsus that replicates in E. coli, and pUB110, re, pTA1060, and rumβ1, provides replication in Bacillus. Examples of start points of replication for use in a yeast cell host are dumitrana the replication origin ARS1, ARS4, the combination of ARS1 and CEN3 and combination ARS4 and CEN6. The replication origin may have a mutation that causes it to function depending on the temperature in the cell-the host (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433).

In the cell-host, you can enter more than one copies of the nucleic acid sequence of the present invention to increase the production of the gene product. The number of copies of the nucleic acid sequence can be increased by embedding at least one additional copy of the sequence into the genome of the host cell or the introduction amplifierarava breeding gene marker in a nucleic acid sequence, after che is about cells, containing amplificatoare copies of breeding gene marker, and hence additional copies of the nucleic acid sequence can be selected by culturing cells in the presence of an appropriate breeding agent.

The methods used for ligating the above elements with the creation of expressing recombinant vectors of the present invention, well-known specialists in this field (see, for example, Sambrook et al., 1989, cited above).

The protease according to this invention can be also co-expressed with at least one other enzyme used in animal feed, such as phytase, xylanase, galactans and/or beta-glucanase. Enzymes can be co-expressed from different vectors, one vector or by using a combination of both methods. When using different vectors, these vectors can have different breeding markers and different points start replication. When using only one vector genes can be expressed from one or more promoters. When cloning with regulation by a single promoter (di - or multicentre) on the order in which the cloning of genes can influence the levels of protein expression. The protease can also be expressed in the form of a fused protein that is a gene encoding the protease, fused in reading frame with the gene coding for another protein. This protein can be another enzyme or a functional domain of another enzyme.

Cell owners

The present invention relates to a recombinant cell host containing a nucleic acid sequence according to this invention, which are successfully used in the recombinant production of polypeptides. A vector containing the nucleic acid sequence of the present invention, is introduced into a cell of the host so that the vector was integrated into the chromosome or was a self-replicated extrachromosomal vector, described above. The term "a host cell" means any progeny of the parent cell, which neodenticula parent cell due to mutations that occur during replication. The choice of host cell largely depends on the gene encoding the polypeptide, and the source of its receipt.

A host cell may be a unicellular microorganism, such as a prokaryotic or multicellular organism, such as eukaryotes.

Suitable cells of unicellular microorganisms are bacterial cells such as gram positive bacteria, which include, but are not limited to, a Bacillus cell, a Streptomyces cell, the cells of lactic acid bacteria the bacilli; or gram-negative bacteria, such as E. coli and Pseudomonas sp. Sour milk bacteria include, but are not limited to, species of the genera Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, Pediococcus and Enterococcus.

The vector can be introduced in a bacterial cell-host by transformation of protoplasts (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168:111-115), using competent cells (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, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, for example, Koehier and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).

A host cell may be eukaryotic, such as animal cell, non-human, an insect cell, a plant cell or a fungal cell.

In one specific embodiment of the invention, a host cell is a fungal cell. The term "fungi" used here is the mean species such as Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as described in the Handbook Hawksworth et al., In Ainsworth and Bisby''s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK), and Oomycota (as described in the Handbook Hawksworth et al., 1995, supra, page 171) and all microsporidia fungi (Hawksworth et al., 1995, see above).

In another specific embodiment of the invention Gribkova a host cell is a yeast cell. The term "yeast" is used here value means ascos the orogenic yeast (Endomycetales), basidiospores yeast, and yeast belonging to the species Fungi Deuteromycota (Blastomycetes). Because in the future, the classification of yeast may change in the purposes of this invention, yeast shall be classified in accordance with the definition described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M. and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

Yeast a host cell may be a cell of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia.

Fungal a host cell may be a cell of a filamentous fungus. In the definition of the term "filamentous fungi" include all filamentous forms of subtypes Eumycota and Oomycota (as described in the above reference Hawksworth et al., 1995). Filamentous fungi are characterized by the walls of the mycelium composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth of such fungi occurs by elongation of hyphae, with dissimilation of carbon must be aerobic. In contrast, vegetative growth of yeast, such as Saccharomyces cerevisiae occurs by budding unicellular thallus, with dissimilation of carbon may be enzymatic.

Examples of host cells of filamentous fungi are the cells of the following types, which include, but are not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma.

Gribko the es cells can be transformed by the way, including the formation of protoplasts, transformation of the protoplasts, and regeneration of the cell membrane known methods. Acceptable methods for transforming host cells of Aspergillus described in EP No. 238023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81:1470-1474. Acceptable methods for transforming Fusarium species are described in the article Malardier et al., 1989, Gene 78:147-156, and in the application WO 96/00787. Yeast may be transformed using the methods described in the following documents Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153:163; and Hinnen et al., 1978, Proceedings on the National Academy of Sciences USA 75:1920.

Ways to get

The present invention relates also to methods of producing the polypeptide according to this invention, which comprise (a) cultivating a strain, which is capable of producing the polypeptide in the wild, to obtain the supernatant containing the polypeptide; and (b) isolation of the polypeptide. In one embodiment of the invention the specified strain belongs to the genus Thermoascus, for example to mind Thermoascus aurantiacus, such as a strain of Thermoascus aurantiacus CGMCC No. 0670.

The present invention relates also to methods of producing the polypeptide according to this invention, which comprise (a) cultivating the host cell under conditions suitable for production of the polypeptide; and (b) isolation of the polypeptide.

Us is Aasee the invention relates also to methods of producing the polypeptide according to this invention, which include (a) culturing the host cell under conditions suitable for production of the polypeptide, with a host cell comprises a mutant nucleic acid sequence containing at least one mutation in the nucleotide 25-1089, 1-1089, 1-1344, 25-1344, 559-1344 or 559-1089 SEQ ID No.1 and encoding a polypeptide which (i) consists of amino acids-178-177, -159-177 or +1-177 SEQ ID No.2, (ii) is a variant of any sequence (i), this variant includes a substitution, a deletion and/or insertion of one or more amino acids, (iii) is an allelic variant of any sequence (i) or (iv) is a fragment of any sequence (i).

The present invention relates also to methods of producing the polypeptide according to this invention, which comprise (a) cultivating the host cell under conditions suitable for production of the polypeptide, with a host cell contains a mutant nucleic acid sequence having at least one mutation in nucleotides of SEQ ID No.10 or nucleotides 2-1129, 2-1195, 2-1267, 71-1129, 71-1195, 71-1267, 599-1129, 599-1195 or 599-1267 sequence or encoding a polypeptide which (i) consists of SEQ ID No.11 or amino acid-23-353, -23-374, -23-397, 1-353, 1-374, 1-397, 177-353, 177-374 or 177-397 sequence.

In the implementation of the methods of obtaining of the present invention, the cells are cultivated in the pit is positive environment suitable for production of the polypeptide using methods known in this field. For example, cells can be grown in a shake flask or by small-scale or large-scale fermentation (including continuous, batch, batch, or solid state fermentation) in laboratory or industrial fermenters in an acceptable environment and in conditions that ensure the expression and/or secretion of the polypeptide. The cultivation is carried out in an acceptable nutrient medium containing sources of carbon and nitrogen and inorganic salts, using methods known in this field. Acceptable environment can be purchased from commercial suppliers or receive using the published recipe (e.g., in catalogues of the American type culture collection). Polypeptide secreted into the nutrient medium, you can select directly from the environment. Secretively polypeptide can be isolated from cell lysates.

The polypeptides can be detected by methods known in this field that are specific for the polypeptides. Such detection methods may include use of specific antibodies, formation proteasome product or destruction of proteasome substrate. For example, analysis of the protease can be used to determine the activity described polypeptide.

The resulting polypeptide can is about to allocate methods known in this field. For example, the polypeptide can be isolated from the nutrient medium by conventional methods, which include, but are not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

The polypeptides of the present invention can be cleaned by different methods known in this field, which include, but are not limited to, chromatography (e.g. ion exchange, affinity, hydrophobic, chromatofocusing and pressure), electrophoresis (e.g., preparative isoelectric focusing), differential solubilization (for example, precipitation with ammonium sulfate), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

Plants

The present invention relates to a transgenic plant, plant part or plant cell, which have been transformed by a nucleic acid sequence that encodes a polypeptide having by activity, according to the present invention for the expression and production of the polypeptide in the allocated quantities. The polypeptide can be isolated from plants or parts of plants. Alternatively, the plant or plant part containing the recombinant polypeptide can be used as is, to improve the quality of food or feed, for example, delapouite nutritional value, taste and rheological properties, or to destroy inedible factor.

In a specific embodiment of the invention the polypeptide accentuated in vacuoles of the seed that contains the endosperm. This result is achieved by the synthesis of this polypeptide as a precursor with an acceptable signal peptide, as described in article Horvath et al., in PNAS, Feb. 15, 2000, vol. 97, no. 4, p. 1914-1919.

The transgenic plant can be dicots or monocots. Examples of monocotyledonous plants are grasses, such as meadow grass (blue grass, PoA), forage grass such as festuca, lolium, bent grass, such as Agrostis, and cereals, such as wheat, oats, rye, barley, rice, sorghum and maize (corn). Examples of dicotyledonous plants include tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape and closely related plant Arabidopsis thaliana. Plants with low content of phytate, which is described in U.S. patent No. 5689054 and 6111168, are examples of genetically engineered plants.

Examples of parts of plants are stem, callus, leaves, roots, fruits, seeds and tubers. Parts of a plant are also specific plant tissue, such as the chloroplast, apoplast, mitochondrion vacuole, peroxisomes and cytoplasm. In addition, any plant cell, regardless of the origin of the fabric is the plant part.

In the scope of the present invention also includes progeny of such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing the polypeptide of the present invention, may be created by methods known in this field. In short, the plant or plant cell obtained by introducing into the genome of the host plant expressing one or more constructs encoding the polypeptide of the present invention, and reproduction obtained from modified plants or plant cells with obtaining transgenic plants or plant cells.

Expressing design is usually design based on nucleic acid containing a nucleic acid sequence encoding the polypeptide of the present invention, which is functionally connected with appropriate regulatory sequences required for expression of the nucleic acid sequence in the selected plant or plant part. In addition, expressing the design may include breeding marker, suitable for identification of master cells, which have built-in expressing this structure and DNA sequences necessary to introduce the construct into the plant (the latter condition depends on the method used for introducing DNA).

The choice of regulatory sequences, such as promoter and termination sequence, and optionally signal or transit sequences is determined based on when, where and how the polypeptide must be expressed. For example, expression of the gene encoding the polypeptide of the present invention may be constitutive, inducible or specific developmental stage or tissue, and gene product can directly affect a specific tissue or plant part such as seeds or leaves. Regulatory sequences are described, for example, Tague et al., 1988, Plant discrimination 86:506.

For constitutive expression, you can use the promoter of 35S-CaMV (Franck et al., 1980, Cell 21:285-294). Organspecific promoter can be, for example, the promoter of storing tissues, such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or metabolic tissues, such as meristem (Ito et al., 1994, Plant Mol. Biol. 24:863-878), semispecific promoter such as the promoter of rice, glutelin, prolamin, globulin, or albumin (Wu et al., 1998, Plant and Cell discrimination 39:885-889), a promoter of the Vicia faba from legumin B4 and the unknown gene seed protein from Vicia faba (Conrad et al., 1998, Journal of Plant Physioogy 152:708-711), the promoter of the protein oil calf seed (Chen et al., 1998, Plant and Cell discrimination 39:935-941), the promoter napA stored protein from Brassica napus, or any other semispecific promoter known in this field, for example, as described in the application WO 91/14772. In addition, the promoter may be misspecification promoter such as the rbcs promoter of rice or tomato (Kyozuka et al., 1993, Plant discrimination 102:991-1000), the promoter of the gene adenylatecyclase Chlorella virus (Mitra and Higgins, 1994, Plant Molecular Biology 26:85-93), the promoter of the gene aldP rice (Kagaya et al., 1995, Molecular and General Genetics 248:668-674), or induced by damage to the promoter, such as the pin2 promoter of potato (Xu et al., 1993, Plant Molecular Biology 22:573-588).

You can also use the enhancer-promoter element to achieve higher expression of the protease in the plant. For example, the enhancer element in the promoter may be an intron, which is injected between the promoter and the nucleotide sequence that encodes a polypeptide of the present invention. In the above article Xu et al., 1993, describes the use of the first intron of the gene actin 1 rice to increase expression.

In addition, to improve the expression can be optimized using codon for this species (see above article Horvath et al.).

Breeding gene marker and any other part of expressing designs can be selected from this about the region.

Design based on the nucleic acid is inserted into the genome of a plant by conventional methods known in this field, including Agrobacterium-mediated transformation, viruspositive transformation, microinjection, particle bombardment, biolistics transformation, and electroporation (Gasser et al., 1990, Science 244:1293; Potrykus, 1990, Bio/Technology 8:535; Shimamoto et al., 1989, Nature 338:274).

Currently, the most frequently used method of generating a transgenic dicotyledonous plant is gene transfer mediated Agrobacterium tumefaciens (for information on this method, see Hooykas and Schilperoort, 1992, Plant Molecular Biology 19:15-38). However, this method can also be used for transformation of monocotyledonous plants, although such plants usually use other methods of transformation. Currently, the most frequently used method of generating a transgenic monocotyledonous plants is particle bombardment (microscopic particles of gold or tungsten coated with the transforming DNA) of embryonic calluses or developing embryos (Christou, 1992, Plant Journal 2:275-281; Shimamoto, 1994, Current Opinion Biotechnology 5:158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocotyledonous plants are the transformation of protoplast described in article Omirulleh et al., 1993, Plant Molecular Biology 21:415-428.

The resulting transformation of the transformants, the soda is expressing containing design, select and regenerate from them whole plants by methods well known in this field.

The present invention relates also to methods of producing the polypeptide according to this invention, which comprise (a) cultivating a transgenic plant or plant cell containing a nucleic acid sequence encoding a polypeptide having by activity, according to the present invention under conditions suitable for production of the polypeptide; and (b) isolation of the polypeptide.

Animals

The present invention also relates to transgenic animals than humans and their products, or elements, examples of which include body fluids, such as milk and blood, organs, flesh and animal cells. Methods protein expression, for example, in mammalian cells are known in the art; see, for example, the reference Protein Expression: A Practical Approach, Higgins and Hames (eds.), Oxford University Press (1999) and three other book in this series related to gene transcription, RNA processing and post-translational processing. In General, to obtain a transgenic animal is selected cells of the selected animal transforming a nucleic acid sequence that encodes a polypeptide having by activity, according to the present invention so as to Express and produce poly is epted. The polypeptide can be obtained from an animal, for example selected from the milk of the female animal, or the polypeptide can be expressed for the benefit of the animal, for example to improve digestion of the animal. Examples of animals are presented below in the section entitled "pet Food".

To obtain a transgenic animal with the aim of identifying protease from the milk of the animal, the gene encoding the protease can be typed into a fertilized egg of the investigated animal, for example, using a vector expressing the transgene, which contains an acceptable milk protein promoter and the gene encoding the protease. The vector expressing the transgene, microinjection in a fertilized egg and preferably stably inserted into the chromosome. Once the egg begins to grow and divide, the potential embryo implanted in a surrogate mother and identify animals carrying the transgene. Received the animal can then be reproduced by conventional methods of breeding. The polypeptide can be purified from the milk of the animal by methods described, for example, in the publication Meade, H.M. et al. (1999): Expression of recombinant proteins in the milk of transgenic animals, Gene expression systems: Using nature for the art of expression, J. M. Fernandez and J. P. Hoeffler (eds.). Academic Press.

Alternatively, obtain a transgenic animal besides man, carrying in the genome of somatic and/or germ to etoc sequence of nucleic acid, including heterologous transgenic construct containing a transgene encoding a protease, the transgene can be functionally associated with a first regulatory sequence for a specific expression of the protease in the salivary gland, as described in the application WO 2000064247.

Composition

The present invention relates also to compositions containing the polypeptide of the present invention.

Polypeptide compositions can be obtained by methods known in the field, in the form of liquid or a dry composition. For example, the polypeptide composition may be in the form of granules or microgranules. Polypeptide introduced into such a composition may be stabilized by the methods known in this field.

In a specific embodiment of the invention the polypeptide compositions of this invention can be protected from decomposition under the action of acid, for example, for a better conservation of the polypeptide when passing through the stomach of the animal. Protection can provide acid-resistant coating, examples of which can be found in the pharmaceutical references.

Examples of preferable applications of the polypeptides or polypeptide compositions according to this invention.

Pet food

The present invention also relates to methods of using the polypeptides having p is teasei activity in animal feed, and feed compositions and feed additives containing polypeptides according to this invention.

The term "animal" means any animal, including humans. Examples of animals are ruminant animals and ruminants, such as cows, sheep and horses. In a specific embodiment of the invention the animal is a ruminant animal. Ruminant animals include animals mono-gastric, such as pigs or swine (including, but not limited to, newborn piglets, growing pigs and sows, poultry, such as turkeys and chickens (including, but not limited to, broiler chickens, laying hens, calves and fish (including salmon, but not limited to them).

The term "food" or "food composition" means any compound, drug, mixture or composition, suitable or intended for animal feeding.

In accordance with the purposes of this invention, the protease can be fed to the animal before, after or simultaneously with the feed. The latter is preferred.

In a specific embodiment of the invention the protease in the form in which it is added to the feed or feed additive must have a certain structure. The term "specific structure" means that proteiny the drug should be clean at least 50% of the survey is the ATA pressure chromatography (see example 12 application WO 01/58275). In other specific embodiments of the invention proteiny the drug should be clean at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94% or 95% when determining the above-mentioned method.

Proteiny the preparation of a particular composition is preferred. For example, it is much easier to accurately measure the protease introduced into the feed, which is essentially not contaminated by other proteases. The term "exact dosing" means, in particular, obtaining comparable and consistent results and to optimize the dose given to achieve the desired effect.

However, for use in animal feed this protease does not have to be completely clean; it may include other enzymes, representing in this case proteiny the drug.

Proteiny the drug can (a) be added directly to the feed (or used directly in the processing of vegetable proteins) or (b) be used to obtain one or more intermediate compositions such as feed additives or premixes are then added to feed (or used in the process). The above degree of purity refers to the purity of the original proteasome drug used in accordance with paragraphs (a) or (b).

Protease preparations with a purity specified order amount is s get in particular, methods of recombinant DNA, because these drugs are harder to get and they are characterized by a higher discrepancy between the parties in obtaining traditional methods of fermentation.

This proteiny the drug can be mixed with other enzymes.

In another specific embodiment of the invention, the protease used in accordance with this invention, may:

to increase the number of split protein degree of hydrolysis (DH) and/or the number solubilizing protein in the full model of digestion with single-chamber stomach in vitro, described in the following example 6;

to increase the number of split protein, DH and/or the number solubilizing protein in the intestinal Department models digestion with single-chamber stomach in vitro, described in the following example 6; and/or

to increase the solubility of the protein, redeployment protein and/or DH in model digestive aquaculture in vitro, described in the following example 7.

The term "vegetable protein" used herein is meant any compound, composition, preparation or mixture that include at least one protein isolated or derived from plants, including modified proteins and protein derivatives. In specific embodiments of the invention the vegetable protein composition of AET at least 10%, 20%, 30%, 40%, 50% or 60% (wt./wt.).

Plant proteins can be obtained from such sources of vegetable protein, like legumes and cereals, such as plants of the families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae and Poaceae, including soy, luminova and rapeseed flour.

In a specific embodiment of the invention a source of vegetable protein are plants of the family Fabaceae, such as soybean, lupine, peas or beans.

In another specific embodiment of the invention a source of vegetable protein are one or more plants of the family Chenopodiaceae, such as beet, sugar beet, spinach or quinoa.

Other examples of vegetable protein sources are rapeseed seed and cabbage.

Soy is the preferred source of vegetable protein.

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

Treatment of vegetable proteins, at least one protease according to this invention increases the solubilization of plant proteins.

Below are examples of the number solubilizing protein percentage, obtained using protease according to this invention: at least 100,0%, 100,1%, 100,2%, 100,4%, 100,5%, 100,6%, 100,7%, 100,8%, 100,9%, 101,0%, 101,1%, 101,2%, 101,3% or 101.4% compared to the control and the reference sample when using the years of the full model of digestion with single-chamber stomach in vitro in example 6.

Below are examples of the amount of degradable protein in percent, obtained by using the protease according to this invention: at least 100% or 101%, 102%, 103% or 104% compared with the control and the reference sample using the full model of digestion with single-chamber stomach in vitro in example 6.

Examples i) % solubilizing and ii) % of split of protein, obtained with the use of protease according to this invention: at least 100%, 101%, 102% or 103% compared to the control and the reference sample when using intestinal Department models digestion with single-chamber stomach in vitro in example 6.

Below are examples of degrees of hydrolysis (DH) in percent, obtained by using the protease according to this invention: at least 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111% or 112% compared to the control and the reference sample by using a model of the digestive aquaculture in vitro in example 7.

Examples i) % solubilizing and ii) % of split of protein, obtained with the use of protease according to this invention: at least 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112% or 113% compared to the control and the reference sample by using a model of the digestive aquaculture in vitro in example 7.

The term "solubilization of proteins refers to the transfer the proteins in solution. Such solubilization can occur due mediated by protease release of protein from other components of the complex nature of the compositions, such as food. The solubilization can be measured by the amount of solubilizing proteins compared with the control samples treated only with pepsin and Pancreatin (see examples 6 and 7).

The term "cleavage of proteins refers to proteolytic hydrolysis solubilizing proteins under the action of proteases, which are formed of low molecular weight peptides and amino acids. Splitting solubilizing proteins can be measured by the amount of solubilizing peptides and amino acids with a molecular mass equal to 1500 daltons or less, compared with the control samples treated only with pepsin and Pancreatin (see examples 6 and 7).

In a particular embodiment, a method of handling this protease(s) affects (or has solubilizers action) on vegetable proteins or protein sources. To achieve such actions vegetable protein or protein source is usually suspended in a solvent, for example an aqueous solvent such as water, and the pH and the temperature regulating characteristics of the studied enzyme. For example, processing may be performed when the so is m pH-value, in which activity actual protease equal to at least 50%, 60%, 70%, 80% or 90%. Similarly, for example, the above processing can be performed at a temperature at which activity actual protease equal to at least 50%, 60%, 70%, 80% or 90%. The above percentages activity related to the maximum activity. The enzymatic reaction continues until reaching the desired result, then it can be terminated or not terminated by inactivation of the enzyme, for example, in the execution stage of heat treatment.

In another specific embodiment, the method of processing according to this invention the action of the protease may be delayed, resulting in, for example, the protease is added to vegetable proteins or protein sources, but it has solubilizers action, or, in other words, start later when creating favorable conditions for solubilization, or inactivation of inhibitors of the enzyme, or by using any other means slowing down the action of the enzyme.

In one embodiment of the invention, the processing is advanced processing of feed for animal or vegetable protein for use in animal feed, i.e. proteins solubilizer to their absorption.

The term "improving hideway value of the feed for animals" means better availability of proteins, resulting in increased extraction of protein, is a higher protein yield and/or improving the utilization of proteins. This improves the nutritional value of feed and improved growth rate, weight gain and/or feed conversion (i.e. the ratio of the mass of consumed feed to weight gain) of the animal.

The protease can be added to feed in any form, for example in the form of relatively pure protease or in a mixture with other components that are added to animal feed, i.e. in the form of additives in animal feed, such as a so-called premix animal feed.

Another object of the present invention are compositions intended for use in animal feed and additives to feed for animals, such as premixes.

In addition to the protease according to this invention an additive to feed for animals according to this invention contain at least one fat-soluble vitamin, at least one water-soluble vitamin, and/or at least one micromineral, and/or at least one macromineral.

In addition, an optional ingredients added to food, as are the colors, flavors, stabilizers, antimicrobial peptides and/or at least one other enzyme selected from of enzymes such as phytase EC 3.1.3.8 or 3.1.3.26; xylanase EC 3.2.1.8; galactans EU 32.1.89 and/or beta-glucanase EC 3.2.1.4.

In a specific embodiment of the invention these other enzymes are well-known enzymes (described above for protease drugs).

Examples of antimicrobial peptides (AMP) are SAR, leucocin And, triptycene, protegrin-1, Tinatin, defensin and viperin, such as novispirin (Robert Lehrer, 2000), plectin and statins, including compounds and polypeptides described in PCT/DK02/00781 and PCT/DK02/00812, as well as variants or fragments of the above substances with antimicrobial activity.

Examples of antifungal proteins (AFP) are peptides Aspergillus giganteus and Aspergillus niger, and variants and fragments possessing antifungal activity described in the application WO 94/01459 and WO 02/090384.

Fat and water - soluble vitamins and trace minerals usually form part of the so-called premix designed to add to the stern, while the major minerals added to feed separately. Any of these compositions, enriched by the protease according to this invention, is additive to feed for animals according to this invention.

In a specific embodiment of the invention an additional feed for animals according to this invention is administered (or prescribe for injection) in the diet or animal feed in an amount of from 0.01 to 10.0%, more specifically from 0.05 to 5.0%, or from 0.2 to 1.0% (% means the number is in the additives in grams per 100 g food). These amounts also apply to the premix.

Below are non-limiting examples of these components.

Examples of fat-soluble vitamins are vitamin a, vitamin D3, vitamin E, and vitamin K, such as 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 minerals are manganese, zinc, iron, copper, iodine, selenium and cobalt.

Examples of major minerals are calcium, phosphorus and sodium.

Food requirements specified components (for example, poultry and piglets/pigs)that are listed in table a application WO 01/58275. The term "nutritional requirements" means that these components must be present in the diet in the indicated concentrations.

Alternatively, it should be noted that additive to feed for animals according to this invention includes at least one of the components identified in table a application WO 01/58275. The term "at least one" means one or more, for example one, two, three, four and so on up to thirteen or fifteen individual components. More specifically, at least one separate component is introduced into the additive according to this invention in such amount that is required to DOS is to achieve concentrations in the feed is within the range, specified in the fourth, fifth or sixth column of table A.

The present invention relates also to compositions of feed for animals. Composition or feed for animals are characterized by a relatively high protein content. Diets for poultry and pigs may be similar to those shown in the table in the application WO 01/58275, columns 2-3. Rations for fish can be similar to that shown in the 4th column of the table C. in Addition, these diets for fish usually include crude fat 200-310 g/kg

The animal feed composition according to this invention contains crude protein in the amount of 50-800 g/kg, and at least one protease according to this invention.

In addition or as an alternative (the above crude protein content), the animal feed composition according to this invention is characterized by the exchange of energy equal to 10-30 MJ/kg, contains calcium in an amount of 0.1-200 g/kg available phosphorus in an amount of 0.1-200 g/kg, and/or methionine in an amount of 0.1-100 g/kg, and/or methionine to cysteine in an amount of 0.1-150 g/kg, and/or lysine in the amount of 0.5-50 g/kg

In specific embodiments of the invention the amount of metabolizable energy and crude protein, calcium, phosphorus, methionine, methionine to cysteine and/or lysine match one of the ranges 2, 3, 4 or 5, are shown in table B for the Cai WO 01/58275 (R. 2-5).

Crude protein is calculated by multiplying the amount of nitrogen (N) by a factor of 6.25, i.e. crude protein (g/kg) = N (g/kg) x 6,25. The nitrogen content determined by the Kjeldahl method (A.O.A.C., 1984, Official Methods of Analysis, 14th ed. Association of Official Analytical Chemists, Washington DC).

The magnitude of the exchange energy can be calculated on the basis of data contained in NRC publications "Nutrient Requirements in Swine", ninth revised edition 1988, subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council. National Academy Press, Washington, D.C., pp. 2-6, and the European Table of Energy Values for Poultry Feed-stuffs, Spelderholt centre for poultry research and extension, 7361 DA Beekbergen, The Netherlands, Grafisch bedrijf Ponsen & Iooijen bv, Wageningen. ISBN 90-71463-12-5.

Calcium, available phosphorus and amino acids in complete rations for animals is calculated based on the feed table, such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a specific embodiment of the invention, the animal feed composition according to this invention contains at least one of the above vegetable protein or protein source.

In other specific embodiments of the invention, the animal feed composition according to this invention contains 0-80% corn, 0-80% sorghum, 0-70% wheat, 0-70% barley, 0-30% oats, 0-40% soy flour: 0-10% fish meal and/or 0-20% whey.

Pet food can be obtained in the form of crushed feed (agranular the frame) or pelleted diets. Chopped forage is usually mixed and add sufficient amount of essential vitamins and minerals in accordance with the requirements of this species. Enzymes can be added in the form of a solid or liquid enzyme preparations. For example, the solid enzyme preparation is usually added before or during the stage of mixing; and a liquid enzyme preparation is usually added after the stage of granulation. The enzyme can also enter in feed additive or premix.

The final concentration of the enzyme in the diet is in the range from 0.01 to 200 mg protein enzyme per kg of diet, for example in the range of 5-30 mg protein enzyme per kg of feed for animals. In specific embodiments of the invention the concentration of the enzyme in the feed is 0,1-180, 0,1-160, 0,1-150, 0,2-125, 0,2-100, 1-180, 2-160, 5-150, 10-100, 10-90, 10-80, 10-70 or 10-50 mg protein enzyme per kg of feed for animals.

In specific embodiments, the application of the method according to this invention for the treatment of vegetable proteins perform the additional step of adding phytase. In other specific embodiments of the invention in addition to the joint addition of phytase and protease in the composition can be also introduced other enzymes, and these enzymes are selected from the group including other protease, phytase, lipolytic enzymes and enzymes glucosidase/carbohydra the s. Examples of such enzymes described in the application WO 95/28850.

Protease, of course, must be entered in an effective amount, i.e. in an amount necessary to improve the solubilization and/or improve the nutritional value of food. Currently, it is believed that this enzyme should be entered in one or more of the following quantities (dose ranges), equal 0,01-200, 0,01-100, 0,05-100, 0,05-50 or 0.10 to 10, expressed in mg of protein protease per kg feed (ppm).

To determine the protein concentration of the protease in mg per kg of feed protease purified from the composition of the feed and to determine the specific activity of the purified protease with an appropriate analysis (see Proteasa activity, substrates and analyses"). Protease activity feed composition is also determined using the above analysis and on the basis of these two definitions is calculated dose of protein protease in mg per kg of feed.

Similar principles apply to the determination of protein protease mg in feed supplements. If the sample protease used to retrieve the feed additive or feed, the specific activity is determined on the basis of this sample (no need to clean the protease from the composition of the feed or supplements).

The present invention will be described hereinafter with reference to the following examples, which should not be construed ka is limiting the scope of the invention.

Examples

Reagents, environment and equipment

Reagents

Except where otherwise indicated, the chemicals used are commercially produced products at least with the purity of the reagents.

AZCL-casein (Megazyme, No. I-AZCAS). For analysis of endoprotease use sewn Surinam casein obtained by dyeing and stitching highly purified casein. Specified casein comes in the form of finely ground powder, which is not soluble in the buffer solution, but quickly hydrates with the formation of the gel particles, which are easily and rapidly hydrolyzed by endoprotease, releasing soluble dye-labeled fragment.

pNA substrates:

(AAPF) Ala-Ala-Pro-Phe-pNa (Sigma S-7388)

(GGF) Gly-Gly-Phe-pNa (Sigma S-1899)

(FGL) Phe-Gly-Leu-pNA (Sigma S-6768)

Ba-pNA (Sigma B-4875)

(AAPE) Ala-Ala-Pro-Glu-pNa (BACHEM C-1710)

SSI (inhibitor of Streptomyces subtilisin): SSI was purified from the fermentation supernatant of Streptomyces albogriseolus FERM P-1205 (S-3253) using chromatography. The drug SSI had a purity of more than 95% when determining the method of SDS-PAGE. Alternative SSI can be purchased at the company Wako Japan, catalog No. 303-05201, the production company of Daiwa Kasei K.K. (see, for example:

http://search.wako-chem.co.jp/lifedb_e/lifedocs_e/44834.asp).

EDTA (Gibco BRL, catalog No. 15576-028)

IPTG (Promega, catalog No. V3951)

X-gal (Promega, catalog No. V3941)

Sodium salt of ampicillin (GIBCOL., No. katal is the 11593-019)

LMP-agarose (Promega, catalog No. V2111)

The name "trypsin" is used to denote a trypsin-like protease obtained from a strain of Fusarium oxysporum. Obtaining specified substances described in EP No. 471265, examples 1 and 2 ("protease II").

SAVINASE™ is subtilisin-protease derived from Bacillus clausii (formerly Bacillus lentus NCIB 10309), which is commercially produced by the company Novozymes A/S, Krogshoejvej, DK-2880 Bagsvaerd, Denmark. Obtaining specified substances described in U.S. patent No. 3723250.

Pepsin (Sigma P-7000; 539 u/mg solid)

Pancreatin (Sigma P-7545; 8xU.S.P (USP))

Cytochrome C (12500 Dalton, Boehringer Mannheim 103870)

Aprotinin (6500 daltons, Sigma A-1153)

Gastrin I (2126 Dalton, Sigma G-1276)

Substance P (1348 Dalton, Sigma S-6883)

Environment

The buffer system (pH 3-pH 11): 100 mm succinic acid, 100 mm HEPES, 100 mm CHES, 100 mm CABS, 1 mm CaCl2, 150 mm KCl, 0.01% of Triton® X-100 with pH brought to 2,0, 2,5, 3,0, 3,5, 4,0, 4,5, 5,0, 6,0, 7,0, 8,0, 9,0, 10,0 or 11.0 using HCl or NaOH (hereinafter briefly referred to as "buffer system with succinic acid").

Borax/NaH2PO4pH 9: the resulting mixing of 8.25 ml of 0.05 M borax from 1.75 ml of 0.1 M NaH2PO4.

1 x TAE buffer: 0.04 M Tris-acetate; 0,001Ì EDTA.

0.5 x TBE buffer: OF 0.045 M Tris-borate; 0.001 M EDTA.

1 mm pNa substrates: weigh 5 mg pNa-substrate and dissolved in 100 μl DMSO, and then diluted with 10 ml of a mixture of borax/NaH2PO4buffer with a pH of 9.0

Wednesday SVN:

Avicel25 g(NH4)2SO41.4 g
KN2RHO42 gUrea0.3 g
CaCl2·2H2O0.3 gMgSO4·7H2O0.3 g
FeSO4·7H2O5 mgMnSO4·H2O1.6 mg
Peptone1 gYeast extract10 g
Tween 801 mlGlucose5 g
H2About1000 ml

80 ml placed in a 500 ml Erlenmeyer flask and treated in an autoclave for 20 minutes at 121°C.

Liquid medium LB: to 950 ml of deionized H2About add 10 g of bacteriophora, 5 g bacto-yeast extract, 10 g NaCl. Shake to dissolve the soluble substances. Adjusted to pH 7.0 by adding 5 n NaOH solution (˜0.2 ml). The solution volume was adjusted to 1 liter by adding de-ionized H2O. the Mixture sterilizers in the autoclave for 20 minutes under a pressure of 1 ATM (15 psi) on liquid cycle.

Tablets for LB with ampicillin/IPTG/X-Gal: 15 g of agar add to one liter among the s LB. Enter ampicillin at a final concentration of 100 μg/ml, then add 0.5 mm IPTG and 80 μg/ml X-gal and poured on the tablets.

1% LMP-agarose gel: 1 g LMP-agarose is added to 100 ml single volume TAE buffer.

The original solution of IPTG (0.1 M):

Distilled water added to 1.2 g IPTG to achieve a final volume of 50 ml, sterilized through a filter and stored at 4°C.

A diet based on corn/SBM (SBM = soybean meal): the ratio of corn and SBM is 6:4 (wt./wt.), the protein content is 43% (wt./wt.) in SBM and 8.2% (wt./wt.) in corn flour. The total amount of protein in 10 g of diet based on corn/SBM is 2,21,

Superseded SBM: the protein content is 45%. The total amount of protein in 10 g of water displaced SBM is equal to 4.5,

Equipment, including different sets

Membrane 5K (Millipore BIOMAX-5, 13442AM)

The 0.45 µm filter (Nalgene 190-2545)

Column of Q Sepharose FF (Amersham Pharmacia 17-0510-01)

Superdex75 column (Amersham Pharmacia 17-1047-01)

IEF gel (Amersham Pharmacia 80-1124-80)

Thermometer Comfort (Eppendorf)

Minnebar RNeasy (QIAGEN, catalog No. 74904)

Set of 3' RACE (GIBCO, catalog No. 18373-019), which includes a priming Adapter and a mixture of dNTP AUAP (100 mm, Promega, catalog No. U1330)

System Taq polymerase (Promega, catalog No. M), including a buffer for PCR (200 mm Tris-HCl (pH of 8.4), 500 mm KCl)

Purification system DNA PCR Preps (Promega, catalog No. A)

The system of vectors pGEM-T (Promega, catalog No. A), enabling the I double volume of buffer with DNA ligase T4

High-efficiency competent cells JM109 (Promega, catalog No. L1001)

Purification system DNA Minipreps (Promega, catalog No. A)

Kit for sequencing BigDye Terminator Cycle Sequencing Ready Reaction (PE Applied Biosystems, catalog No. 4303149)

DNA sequencing machine (ABI Prism 377 (PE)

System 5' RACE (GIBCO, catalog No. 18374-058), including anchor the seed with bridge connection

System DNA polymerase pfu (Promega, catalog No. M)

Polycarbonate filters of 0.45 μm (Sartorius)

Column for gel filtration Superdex Peptide PE (7.5 x 300 mm) (Global)

Spectrophotometer DU7500 (Beckman)

System for PCR GeneAmp 9700 (PE)

Laboratory vacuum manifold Vac-Man Jr. (Promega, catalog No. A)

Example 1. Analyses of protease

Were made following the analysis activity of the protease.

Analysis using AZCL-casein

A solution of 0.2% blue substrate AZCL-casein suspended under stirring in a buffer with a mixture of borax/NaH2PO4with a pH of 9. (To achieve the profile of pH in example 4 used a buffer system with a pH 3-pH 11). Stir the solution was dispensed into the wells tiralongo microplate (100 μl in each well), add 30 ál sample of enzyme and tablets incubated in thermosensitive company Eppendorf for 30 minutes at 45°With a speed of 600 revolutions/min Denatured sample enzyme (boiling at 100°C for 20 minutes) and the use as a control sample. After incubation the reaction is stopped by transferring tetrazinni the microplate on the ice, and painted the solution is separated from the solids by centrifugation with a speed of 3000 rpm for 5 minutes at 4°C. 60 μl of the supernatant is transferred to tetrazinni the microplate and measured the optical density at 595 nm in a spectrophotometer to read the BioRad microplate.

Analysis using pNA-substrate

50 µl containing protease sample is placed on tetrazinni the microplate and perform the analysis by adding 100 ál of 1 mm pNA substrate (5 mg dissolved in 100 μl DMSO and diluted to 10 ml buffer with a mixture of borax/NaH2PO4pH of 9.0). The protease activity determined by measuring the increase in optical density OD405 at room temperature.

Example 2. Cultivation of strain CGMCC No. 0670 Thermoascus aurantiacus

A strain of Thermoascus aurantiacus CGMCC No. 0670 were grown at 45°C for 60 hours in shake flasks with medium SUN. The culture broth was harvested by centrifugation (7000 rpm for 20 minutes at 4°). Received a total of 1500 ml of culture broth.

Example 3. Purification of protease strain of Thermoascus aurantiacus CGMCC No. 0670

1500 ml of the supernatant obtained in example 2, precipitated with ammonium sulfate (80% saturation) and re-dissolved in 40 ml of 25 mm buffer with Tris-HCl, pH 7.4. The resulting solution, for example, the Ali ultrafiltration through a membrane 5K to remove salts and replaced the buffer 25 mm Tris-HCl with pH 7.4, then the solution was filtered through a 0.45 µm filter. The final volume was 30 ml of the Solution was injected into a 20 ml column of Q Sepharose FF equilibrated 25 mm Tris-HCl, pH 7.4, and proteins were suirable linear gradient of NaCl (0-0,4 M). Faction, erwerbende from the column were analyzed in relation to the activity of the protease using AZCL-casein, pH of 9.0, with SSI or without him. Collected fractions with by activity and not inhibiting SSI. Then the collected solution was injected into the column Superdex75, balanced 25 mm Tris-HCl, pH 7.4, and was suirable proteins with the same buffer. Containing protease fractions were analyzed by the method of SDS-PAGE and pure fractions collected.

The degree of purity of the purified protease was examined by the method of SDS-PAGE and using the IEF-gel. The sample contained only one protease, which was given the name AR. Molecular mass of approximately 23 kDa, and pI corresponds to pH 8.5.

Example 4. The study of the protease, the strain of Thermoascus aurantiacus CGMCC No. 0670

Test for inhibition

Purified protease AR was tested for inhibition by various inhibitors, including SSI and EDTA. The protease was incubated with 1,12 mg/ml SSI or 0.5 M EDTA for 5 minutes at room temperature and determined the residual activity using the analysis using AZCL-casein in example 1 (pH 9, 45°). As control samples used trypsin and SAVINASE™. It is to show the results shown in figure 1, neither SSI nor EDTA, apparently, does not inhibit the activity AR.

The temperature profile

The relationship between temperature and activity of the protease was determined by analysis using AZCL-casein (20 minutes with shaking in thermosensitive). The obtained temperature profile shown in figure 2.

Protease OR is active in a wide range of temperatures from 20°to 90°C, and the optimum temperature is about 70°C. But even at 90°this activity is at least 60% compared with the activity at 55°C.

Profile of pH

The relationship between pH and activity of the protease was determined by analysis using AZCL-casein in example 1 (a buffer system with a pH 3-11). The resulting profile of pH is shown in figure 3.

Protease OR is active in a wide range of pH from 5 to 10. The optimum pH is about 6.

Resistance to pH

To measure the resistance to pH a mixture of 15 ml sample of the enzyme and 200 ml of buffer with pH 3, 4, 5, 6, 7, 8, 9, 10, 11 incubated at 37°C for 2 hours and then determined the residual enzyme activity using an analysis using AZCL-casein in example 1 (add 900 ál of AZCL-casein in buffer with a mixture of borax/NaH2PO 4pH of 9.0, and incubated the mixture at 45°With speed 1200 rpm/min for 10 minutes and measure the optical density at 595 nm). The results are shown in figure 4. Protease is stable in a very wide range of pH from 4 to 10, but optimum stability corresponds to approximately pH 4.

The specificity of pNA-substrate

The substrate specificity of the protease AR was determined using more pNA substrates, including AAPF, AAPE, GGF, BA and FGL, and SAVINASE™ and trypsin as control samples.

The results, shown in figure 5, show that the protease OR has no significant activity on any of these substrates.

Sequencing of N-terminal sequence

N-terminal amino acid sequence of the protease OR represented: QRISSCSGSRQSALTTALRN (SEQ ID No.3).

Example 5. Cloning of the gene encoding the protease, the strain of Thermoascus aurantiacus CGMCC 0670

Fungal strain and its cultivation

A strain of Thermoascus aurantiacus CGMCC No. 0670 were grown in medium SUN at 45°With the speed of 165 rpm for 3 days. The mycelium was collected by centrifugation speed 7000 rpm for 30 minutes. The harvested mycelium was stored at -80°C until used for RNA extraction.

Extraction of total RNA

Total RNA was extracted from 100 mg of mycelium with ISOE what Lovanium of minnesora RNeasy.

Grown seed

The following grown seed were created on the basis of N-terminal amino acid sequence QSALTTA:

T025-3 5' - CA(A/G) TC(T/C/A/G) GC(T/C/A/G) CT(T/C/A/G) AC(T/C) AC(A/G) GC 3' (SEQ ID No.4)

T025-12 5' - CA(A/G) AG(T/C) GC(T/C/A/G) CT(T/C/A/G) AC(A/G) AC(A/G) GC 3' (SEQ ID No.5)

Cloning of the 3'end of the protease OR

For synthesis of cDNA protease OR used a set of 3' RACE. Approximately 5 mg of total RNA was used as matrix and priming Adapter used for the synthesis of the first chain cDNA. Then cDNA protease OR amplified by PCR using the above-grown seed.

Were used the reaction system for performing PCR and reaction conditions, to the following:

10-fold volume of buffer for PCR5 ál
25 mm MgCl23 ál
10 mm dNTP mix1 ál
3'-the seed (T-3, T-12; 10 μm)1 ál
AUAP (10 μm)1 ál
Taq polymerase0,5 ál
The reaction mixture for the synthesis of cDNA2 ál
Add processed in the autoclave distilled water to50 µl

Conditions

94° 3 minutes
94°40 seconds
55°40 seconds30 cycles
72°1 minute
72°10 minutes

The analysis of the PCR product in the gel when using nucleating AR-3 and AR-12 was detected a specific band corresponding to the fragment length of about 800 BP Obtained products were isolated from a 1% LMP-agarose gel, purified by incubation in a bath with 70°With subsequent use of the system for purification of DNA PCR Preps. Concentration of the purified products was determined by measuring the optical density a and A in the spectrophotometer. Then purified fragments ligated with the vector pGEM-T using the corresponding set Promega:

2-fold volume of buffer with DNA ligase T41 ál
The vector pGEM-T (50 ng)1 ál
The PCR product40 ng
DNA T4 ligase (3 units Weiss/µl)1 ál
Distilled H20 to a final volume10 ál

The reaction mixture was incubated over night at 4°C.

2-4 µl of the ligation products were introduced which 50 μl of high-efficiency competent cells JM109 method "heat shock" (J. Sambrook, E.F., Fritsch, T. Maniatis (1989) Molecular Cloning 1.74, 1.84).

The transformed culture was placed on the tablet LB with a mixture of ampicillin/IPTG/X-Gal and incubated overnight at 37°C. the Recombinant clones were identified by color screening on indicator tablets and screening colonies by PCR as follows.

The screening system of the colonies by PCR

10-fold volume of buffer for PCR5 ál
25 mm MgCl23 ál
10 mm dNTP mix1 ál
3'-the seed (10 μm, T-3 or T-12)2 ál
AUAP (10 μm)2 ál
Taq polymerase0,5 ál
Add processed in the autoclave distilled water to50 µl

In a white colony immerse the pipette tip and pipeinput colony in the mix for PCR used as the matrix.

The PCR conditions

94°3 minutes
94°40 seconds
55°40 seconds30 cycles
72°1.5 minutes
10 minutes

Positive clones were inoculable in 3 ml of liquid LB medium and incubated with shaking (250 rpm) overnight at 37°C. the Cell precipitate was separated by centrifugation with an acceleration of 10000 x g for 5 minutes and from the resulting cell sediment was obtained plasmid samples using the purification system DNA Minipreps. And finally, plasmids sequenced using the kit for sequencing BigDye Terminator Cycle Sequencing Ready Reaction and sequencing machine AV. The sequencing reaction was performed as follows.

A mixture of Terminator Ready Reaction8 ál
Plasmid DNA1.0 to 1.5 mcg
Seed3.2 pmol
Distilled H2About to make a final volume10 ál

The sequencing results showed that the strip PCR obtained using seed AR-3 and seed AR-12, is a 3'-terminal sequence AR.

Cloning of the 5'-end AR

On the basis of 3'-terminal sequence OR created three specific seed that was used to clone the 5'-end of the sequence.

AR-5'-15' AAG GTA TAT GGC ATT CGC AT 3' (SEQ ID No.6)
AP025-5'-25' GCA GCC TGG TAG CCA TAC3' (SEQ ID No.7)
AR-5'-35' TTG ATC CTG AGC GTG ACA G3' (SEQ ID No.8)

System 5' RACE were used for the synthesis of 5'-terminal fragment AR. For the synthesis of the first chain was added to 5 μg total RNA and a dose of 5'-1. Then cDNA was purified using the system for purification of DNA (included with system 5' RACE and 3'-end of cDNA was added to the policy-terminal fragment. For synthesis of the second circuit used the seed crystal 5'-2.

Were used and PCR conditions for obtaining cDNA from dC-terminal fragment below.

10-fold volume of buffer for PCR (200 mm Tris-HCl (pH of 8.4), 500 mm KCl)5 ál
25 mm MgCl23 ál
10 mm dNTP mix1 ál
5'-seed (10 μm) (AR-5'-1/2/3))2 ál
Anchor the seed with bridge connection2 ál
Taq polymerase0,5 ál
cDNA with the dC-terminal fragment5 ál
Add processed in the autoclave distilled water to50 µl

The PCR conditions

94°2 min is you
94°40 seconds
55°40 seconds30 cycles
72°1 minute
72°10 minutes

To obtain a specific product, the authors present invention amplified the 5'-end using a nested PCR, using the seed AR-5'-3 (Frohman, M.A. (1990) PCR Protocols: A Guide to Methods and Applications (Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J., eds.) p. 28, Academic Press, San Diego.

Sterilized distilled water33,5 ál
10-fold volume of buffer for PCR5,0 ál
25 mm MgCl23,0 ál
10 mm dNTP mix1,0 ál
The seed AR-5'-3 (10 μm)1,0 ál
AUAP (10 mm, supplied in 5' RACE)1,0 ál
Dilution of the primary PCR product (1:100)5,0 ál
Taq polymerase (2-5 u/ál)0,5 ál

The PCR conditions

94°2 minutes
94°40 seconds
58°40 seconds30 cycles
72°1 minute
72°10 minutes

When using seed AR-5'-3 in the 5' RACE was obtained specific band related to the fragment length 1000 BP PCR Products were purified, ligated into the vector pGEM-T and transformed into competent JM109 cells and sequenced. The sequencing results confirmed that was cloned 5'-terminal fragment AR.

Cloning of the complete gene OR

Kind of primer for cloning full sequence was created on the basis of the above 3'- and 5'-terminal sequences.

AR-CDS-25' AAG TCT ACC CAG TAT CCT GT-3'(SEQ ID No.9)

Seed AR-CDS-2 and AUAP used to amplify the full-size gene from cDNA AR. Were used the reaction system and the PCR conditions described below.

10-fold volume of buffer for PCR5 ál
25 mm MgCl23 ál
10 mm dNTP mix1 ál
The seed AR-CDS-2 (10 μm)1 ál
AUAP (10 μm)1 m the l
DNA polymerase pfu0,5 ál
The reaction mixture for the synthesis of cDNA2 ál
Add processed in the autoclave distilled water to50 µl

Conditions

95°2 minutes
95°40 seconds
59°30 seconds30 cycles
72°3.5 minutes
72°5 minutes

As a result of the amplification was obtained specific band length of about 1.4 KBP To add the poly-terminal fragment used Taq polymerase, and incubated the reaction mixture at 72°C for 30 minutes. dA-terminal fragment was isolated from gel using the system DNA purification PCR Preps. Then the purified fragment is ligated into the vector pGEM-T and transformed into competent cells (JM109). Screening of some of the positive clones was performed using PCR for the colonies.

System perform PCR for colonies

10-fold volume of buffer for PCR5 ál
25 mm MgCl2/sub> 3 ál
10 mm dNTP mix1 ál
AR-CDS-2 (10 μm, AR-3 or AR-12)2 ál
AUAP (10 μm)2 ál
Taq polymerase0,5 ál
Add processed in the autoclave distilled water to50 µl

In a white colony immerse the pipette tip and pipeinput colony in the mix for PCR used as the matrix.

Conditions

94°3 minutes
94°40 seconds
55°40 seconds30 cycles
72°1.5 minutes
72°10 minutes

Then the plasmids were extracted from these clones using a purification system DNA Minipreps. The plasmids sequenced using the kit for sequencing BigDye Terminator Cycle Sequencing Ready Reaction, which produced a full sequence.

The sequence analysis of cDNA clone showed that the sequence contains the coding region of 1065 nucleotides. Product broadcast the specified coding region is a peptide containing 55 amino acids. Deduced amino acid sequence of this gene with the signal peptide (AA 1-19), propeptide (20-178) and a Mature peptide (AA 179-335) is shown in Fig.6 and 7.

Example 6. The action of the protease, the strain of Thermoascus aurantiacus CGMCC 0670, in the model of digestion with single-chamber stomach in vitro

Protease AR was expressed in and isolated from Aspergillus oryzae. The purified protease was characterized by the index And280equal to 1.15, and a260equal to the value of 0.52. The activity of purified protease was tested in the in vitro model simulating digestion in animals with single-chamber stomach. In particular, we tested the ability of the protease to improve the solubilization and cleavage of proteins of corn/SBM (corn/soy flour).

The in vitro system consisted of 10 flasks in which initially incubated substrate corn/SBM with HCl/pepsin, simulating digestion in the stomach, and then with Pancreatin, simulating digestion in the intestine. In the early phase of digestion in the stomach protease AR was introduced in five flasks, while the rest of the flask served as the control flasks. In the late phase of incubation, corresponding to digestion in the intestine, digested samples feed in vitro were removed and analyzed for contents of solubilizing and cleaved protein.

Scheme digestion in vitro

pH
Added components Pace-temperatureTimePhase simulated digestion
The substrate 10 g corn/SBM (6:4),

41 ml of HCl (0,105 M)
3,140°t=0 minMixing
5 ml of HCl (0,105 M)/ pepsin

(3000 u/g substrate), protease A (100 mg protein enzyme protease/kg diet)
3,140°t=30 minDigestion in the stomach
16 ml of N2About3,140°t=1 hourDigestion in the stomach
7 ml of NaOH (0,39 M)6,840°t=1.5 hoursDigestion in the intestine
5 ml NaHCO3(1 M)/ Pancreatin (8 mg/g diet)6,840°t=2 hoursDigestion in the intestine
The end of the incubation7,040°t=6 h

Conditions

Substrate:4 g SBM, 6 g of corn (pre-mixed)
pH:3,1 stage of digestion in the stomach/6,8-7,0 at the stage of digestion in the intestine
HCl:0,105 M for 1.5 hours (that is here pre-mixing HCl substrate for 30 minutes)
pepsin:3000 u/g diet for 1 hour
Pancreatin:8 mg/g diet for 4 hours
temperature:40°
the replicate:5

Solutions

0,39 M NaOH

0,105 M HCl

0,105 M HCl containing 6000 units of pepsin per 5 ml

1 M NaHCO3containing 16 mg of Pancreatin per ml

125 mm buffer with NaAc, pH 6.0

Determination of protein enzyme

The amount of protein enzyme protease is calculated based on the values of A and amino acid sequence (amino acid compositions) on the basis of the principles outlined in article S.C.Gill & P.H. von Hippel, Analytical Biochemistry 182, 319-326 (1989).

Experimental methods of using the model in vitro

The experimental method was similar to that described above. The pH was measured in periods of time corresponding to 1 to 2.5 and 5.5 hours. The incubation was finished after 6 hours, after which 30 ml samples were removed and placed on ice until centrifugation (10000 x g, 10 minutes, 4°). Supernatant was removed and stored at -20°C.

Analysis

All samples were analyzed in relation to the degree of hydrolysis (DH) in the interest method based on the ORA, as well as in the content solubilizing and split protein using gel filtration.

Determination of degree of hydrolysis method on the OS is ove ORA

The degree of hydrolysis (DH) of the protein in different samples was determined by the colorimetric method using semi-automated titrating microplate (Nielsen, P.M.; Petersen, D.; Dambmann, C. Improved method for determining food protein degree of hydrolysis. J. Food Sci. 2001, 66, 642-646). Reagent OPA received as follows: 7,620 g decahydrate of dynamicattribute and 200 mg of sodium dodecyl sulfate (SDS) was dissolved in 150 ml of deionized water. The reagents are completely dissolved. Then 160 mg of 97% on-phthaldialdehyde (ORA) was dissolved in 4 ml of ethanol. The solution ORA transferred quantitatively into the above solution by rinsing with deionized water. To the solution was added 176 mg, 99% dithiothreitol (DTT), which was brought to 200 ml using deionized water. Serine standard (0,9516 mEq/l) was obtained by dissolving 50 mg of serine (Merck, Germany) in 500 ml of deionized water.

The sample solution was obtained, diluting each sample until reaching an optical density (280 nm), equal to approximately 0.5. In General, supernatant diluted (100-fold) by using an unattended installation for cultivation Tecan (Mannedorf, Switzerland). All other readings of the spectrophotometer to read at 340 nm, using deionized water as a control sample. 25 μl of sample, standard and control sample were dispersible in titrations the microplate. Tetrazinni the microplate was placed in the spectrophotometer the La reading plates (Labsystems, Finland) and automatically dispersible 200 ál of PR. The tablets were shaken (2 minutes; 700 rpm) until the optical density measurement. Then calculate the degree of hydrolysis. Determination of degree of hydrolysis of all samples was performed eight times.

Definition solubilizing and split protein

Content supernatant solubilizing protein of split samples in vitro was determined by quantitative measurement of crude protein (CP) using HPLC with gel-filtration. Supernatant thawed, filtered through polycarbonate filters of 0.45 μm and diluted (1:50, V/V) H2O. Diluted samples were chromatographically using HPLC using a column of Superdex Peptide PE (7.5 x 300 mm) for gel filtration (Global). As eluent for isocratic elution was used 50 mm buffer phosphate (pH 7.0)containing 150 mm NaCl. The total volume of eluent used in one cycle, was equal to 26 ml at a flow rate of 0.4 ml/min elution Profiles were recorded at 214 nm and the total area under the profiles was determined by integration. To determine the protein content on the integrated plots constructed calibration curve (R2=0,9993) on the basis of the number of dilutions in vitro split control sample is a mixture of corn/SBM with known total protein content. Determination of protein in ocasinomorongot the sample was performed by the method of Kjeldahl (definition in % nitrogen; AOAC (1984) Official Methods of Analysis 14th ed., Washington DC).

The content of the cleaved protein was determined by integrating the area of the chromatogram corresponding to peptides and amino acids with a molecular weight of 1500 daltons or less (Savoie, L.; Gauthier, S.F. Dialysis Cell For The In-vitro Measurement of Protein Digestibility. J. Food Sci. 1986, 51, 494-498; Babinszky, L.; Van, D.M.J.M.; Boer, H.; Den, H.L.A. An In-vitro Method for Prediction of The Digestible Crude Protein Content in Pig Feeds. J. Sci. Food Agr. 1990, 50, 173-178; Boisen, S.; Eggum, B.O. Critical Evaluation of In vitro Methods for Estimating Digestibility in Simple-Stomach Animals. Nutrition Research Reviews 1991, 4, 141-162). To define the line of division corresponding to 1500 Da, a column for gel filtration were calibrated using cytochrome C, Aprotinin, gastrin I and substance P as molecular weight standards.

Results

The results given in the following tables 1 and 2 show that the protease OR significantly increased the amount of cleaved protein, the degree of hydrolysis and the amount of soluble protein was increased in the numerical ratio compared with the control sample.

When testing only part of the model, simulating digestion in the intestine, protease OR (in a similar dose of 100 mg protein enzyme per kg of substrate) significantly increased the level solubilizing and split protein (respectively 103,0 and 103.1% compared with the control sample), the degree of hydrolysis was not increased (965% compared with the control sample).

Table 1
The degree of hydrolysis (DH), the absolute and relative values
SamplenOf the total proteinCompared to control
% DHSD% DH%CV
Control526,6ab0,8100,0ab2,9
AR527,6b1,4103,5b5,0

Different letter symbols in the same column indicate significant differences (unidirectional method ANOVA, the test of Tukey-Kramer, P<0,05). SD = standard deviation. %CV = coefficient of variation = (standard deviation/average) x 100%.

Table 2
The results of determining solubilizing and split protein (SAT) by HPLC ACT
SampleNOf the total protein Compared to control
% split SBSD% solubilizing SBSD% split SB% CV% solubilizing SB% CV
Con

control
561,5and1,692,2and0,7100,0and2,6100,0and0,7
AR564,1and0,893,5ab1,0104,2b1,2101,4ab1,1

Different letter symbols in the same column indicate significant differences (unidirectional method ANOVA, the test of Tukey-Kramer, P<0,05). SD = standard deviation. %CV = coefficient of variation = (standard deviation/average) x 100%.

Example 7. The action of the protease, the strain of Thermoascus aurantiacus CGMCC 0670, in model digestive aquaculture in vitro

The drug of the protease AR, similar to that described in example 6, was tested in a model of aquaculture in vitro, simulating digestion in fish that live in cold water. Compared with the model of the digestion chamber in the stomach. is the W system aquaculture produced in several other pH (pH 3.3-V phase of digestion in the stomach and pH of 7.2-7.8 in phase of digestion in the intestine). The incubation temperature was lower (15°instead of 40° (C) and incubation periods longer (listed below). Ten flasks containing extruded SBM as a substrate, first incubated with HCl/pepsin, simulating digestion in the stomach, and then with Pancreatin, simulating digestion in the intestine. Five flasks protease AR was introduced in the early phase of digestion in the stomach, the remaining flasks were used as control samples. In the late phase of incubation, corresponding to digestion in the intestine, digested samples feed in vitro was removed and analyzed in relation to solubilizing and cleaved protein.

Study design aquaculture in vitro, simulating digestion in fish that live in cold water

5 ml NaHCO3(1M)/ Pancreatin (8 mg/g extruded SBM)
Added componentspHTemperatureTimePhase simulated digestion
10 g of extruded SBM, 46 ml of HCl (0,18 M)/pepsin (3000 u/g extruded SBM), protease A (100 mg protein enzyme protease/kg extruded SBM), 16 ml of N2About3,315°t=0 minutesDigestion in the stomach
7 ml of NaOH (1.10 M)7,215°t=6 hoursDigestion in the intestine
7,215°t=7 hoursDigestion in the intestine
The end of the incubation7,815°t=24 hours

Conditions

Substrate:10 g of extruded SBM
pH:3,3 stage of digestion in the stomach/7,2-7,8 at the stage of digestion in the intestine
HCl:0.18 M to 6 hours
pepsin:3000 u/g replaced by 6 hours
Pancreatin:8 mg/g extruded SBM 18 hours
temperature:15°
The replicate:5

Solutions

1.10 M NaOH

0.18 M HCl containing 652 units of pepsin per ml

1 M NaHCO3containing 16 mg of Pancreatin per ml

125 mm buffer with NaAc, pH 6.0

Experimental methods of using the model digestive aquaculture in vitro

The experimental method was similar to that described above. The pH was measured after 1, 5, 8 and 23 hours. The incubation was completed after 24 hours, and then selected 30 ml samples and put them on ice until centrifugation (10000 x g, 10 minutes, 4°). Supernatant udalenie kept at -20° C.

Analysis

All supernatant analyzed by a method based on the PR (degree of hydrolysis in percent), solubility and cleaved protein was investigated using Akta HPLC (see example 6).

Results

The results given in the following tables 3 and 4 show that the protease OR significantly increases the solubility of the protein, redeployment protein and the degree of hydrolysis compared with the control sample.

Table 3
Results to determine the degree of hydrolysis in the interest method based on the ORA
SamplenOf the total proteinCompared to control
% DHSD% DH%CV
Control520,41,3and100,06,4and
AR523,00,2bcof 112.81,1bc

Different superscripts in the same column indicate significant differences (unidirectional method ANOVA, the test of Tukey-Kramer, P<0,05).

Table 4
The results of the analysis of the split (% SATRossand solubilizing (% SATSol) total protein by HPLC Akta
SamplenOf the total proteinCompared to control
% split SBSD% solubilizing SBSD% split SB% CV% solubilizing SB% CV
Control545,9and2,982,7and5,6100,0and6,2100,0and6,8
AR551,8bc0,890,9bc2,4112,9bc1,6109,9bc2,6

Different superscripts indicate significant differences (ANOVA method, the criterion Tukey-Kramer, P<0,05).

Deposited biological material

The following biological material has been deposited in accordance with the provisions of the Budapest dog the thief in the collection DSMZ (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany) and CGMCC (China center collections microbiological cultures, Institute of Microbiology, Chinese Academy of Sciences, Haidian, Beijing 100080, China), where specified material were assigned the following numbers:

DepositThe access numberDate Deposit
Escherichia coliDSM 14652on November 29, 2001
Thermoascus aurantiacusCGMCC No. 0670December 27, 2001

The strain Escherichia coli includes a plasmid containing the nucleic acid sequence of the protease Thermoascus aurantiacus (i.e. SEQ ID No.1, encoding SEQ ID No.2).

These strains were deposited on the conditions that guarantee access to the cultures during the whole period of consideration of this application for patent to the person designated by the patent and trademark office in accordance with article 37 C.F.R (Code of Federal regulations U.S.), §1.14 and article 35 U.S.C. (, United States Code), §122. Deposits are essentially pure culture of the deposited strains. Deposits are provided in accordance with the requirements of the foreign patent laws in countries where it is considered that this application or subsequent application. However, it is obvious that access to de is to osita does not imply the granting of licenses for the use of this invention in patent infringement, provided by state law.

Strain No. CGMCC 0670 Thermoascus aurantiacus was isolated from soil sample received 21 July 1998 in Yunnan province, hesuan-Banna, China.

The invention presented in this description and the claims, is not limited to the specific variants of its implementation, which serve only to illustrate some of the objects of the present invention. All such embodiments of the invention are included in the scope of this invention. From the above description of the invention the experts in this field should be obvious other modifications of the invention in addition to the above. Such modifications are also included in the scope of the attached form of the invention. In the event of a conflict should refer to the description of the invention, containing definitions of terms.

All materials are fully included in the description of the invention as a reference.

1. The selected polypeptide, by having an activity selected from the group consisting of

(a) a polypeptide with the amino acid sequence, the degree of identity with amino acids from -178 to 177, -159 to 177, or +1 to 177 of the sequence SEQ ID No.2 is at least 80%; and

(b) a polypeptide encoded by a nucleic acid sequence that hybridizes in conditions of medium stringency

(i) part of the plasmid in Escherichia coli DSM 14652, which encodes the Mature protease;

(ii) with nucleotides 25-1089, 1-1089, 1-1344, 25-1344, 559-1344 or 559-1089 sequence SEQ ID No.l;

(iii) a complementary chain specified in (i) or (ii);

(c) allelic variants of the polypeptide (a) or (b), and

(d) a fragment of the polypeptide (a), (b) or (C)possessing by activity.

2. The polypeptide according to claim 1, characterized in that

(i) is metalloendopeptidases EC 3.4.24;

(ii) is metalloprotease family of M and/or

(iii) contains a motif HEFTH.

3. The method of producing the polypeptide according to claim 1 or 2, providing for (a) cultivating a strain of the genus Thermoascus, and (b) isolation of the polypeptide.

4. A strain of Thermoascus aurantiacus CGMCC No. 0670 to obtain the polypeptide by the method according to claim 3.

5. The method of processing vegetable proteins in order to modify their properties to increase their digestibility which the stages of adding at least one protease according to claim 1 or 2, at least one vegetable protein or protein source.

6. The method according to claim 3, characterized in that at least one of the sources of vegetable protein is soy.



 

Same patents:

FIELD: biotechnology, proteins, immunology.

SUBSTANCE: invention proposes fragment of antibody MR1-1 that retains capacity for recognition and binding EGFRvIII and nucleic acid encoding its. Polypeptide is prepared from the known antibody MR1 by mutation of CDR3 VH- and VL-chains. Also, invention describes using a polypeptide for preparing immunotoxin possessing the cytotoxicity property to cells carrying antigen of epidermal growth factor EGFRvIII receptors. Also, invention disclosed immunotoxin based on this polypeptide. Also, invention proposes a method for preparing polypeptide comprising amino acid substitution of at least one amino acid in hypervariable region CDR with amino acid encoded by a codon comprising nucleotide belonging to a hot point motif. The hot point motif is chosen from AGY or RGYW wherein R represents A or G; Y represents C or T, and W represents A or T. Prepared polypeptide is characterized by value Kd for EGFRvIII 7 nM or less. Also, invention describes a method for cell killing that expresses antigen EGFRvIII using a polypeptide. Using this invention provides preparing antibodies showing enhanced cytotoxicity and improved binding EGFRvIII as compared with the parent antibody MR1 that can be used for target-directed delivery of immunotoxins in treatment of malignant neoplasms.

EFFECT: valuable medicinal properties of polypeptide.

34 cl, 7 tbl, 6 dwg, 8 ex

FIELD: biotechnology, microbiology, peptides.

SUBSTANCE: invention relates to a method for preparing dipeptide from L-amino acid ester and L-amino acid using culture of microorganisms possessing ability for producing dipeptide from L-amino acid ester and L-amino acid. Method involves using cells of microorganisms of genus Pseudomonas or Corynebacterium isolated from culture or disrupted cells of these microorganisms, or lyophilized cells of such microorganisms. By other variant method involves using a protein possessing proline iminopeptidase activity instead microorganism culture. Invention provides preparing dipeptides with high effectiveness degree using inexpensive parent substance by industrially economy and simple way.

EFFECT: improved preparing method.

13 cl, 1 dwg, 4 tbl, 14 ex

FIELD: biotechnology, peptides.

SUBSTANCE: invention relates to a method for preparing peptide arenicin possessing the antimicrobial activity. Method involves culturing the recombinant strain-producer Escherichia coli BL21(DE3)/pE-His8-TrxL-Ar2 in nutrient medium followed by disruption of bacterial cells, isolation of inclusion bodies comprising fusion protein His8-TrxL-Ar2. Then this fusion protein is purified by the method of metal-chelating chromatography, subjected for solubilization and the following splitting with cyanogen bromide by methionine residue in acid medium. Then reaction products are separated and arenicin is subjected for final purification by reversed-phase HPLC method. Using the invention provides expanding assortment of antimicrobial preparations of broad spectrum of effect and to carry out the high-technological effectiveness of production of arenicin. Prepared peptide is similar to peptide isolated from annelid worm Arenicola marina and can be used in medicinal and veterinary practice as antibiotic of the broad spectrum of effect.

EFFECT: improved preparing method of peptide.

1 tbl

FIELD: biotechnology, microbiology, biochemistry, enzymes.

SUBSTANCE: method involves culturing corynebacterium comprising the inserted gene encoding neutral metalloprotease from Streptoverticillium and prostructural moiety of protransglutaminase is split off by neutral metalloprotease produced by this microorganism. Features of neutral metalloprotease are given in the invention text. Also, invention relates to nucleic acid molecule encoding neutral metalloprotease. Invention provides preparing protease that can be used for producing transglutaminase that splits prostructural moiety of protransglutaminase selectively.

EFFECT: improved producing method of enzyme.

8 cl, 12 dwg, 2 ex

FIELD: biotechnology, gene engineering, medicine.

SUBSTANCE: claimed composition contains inert carrier, diluent, and antigen component in effective amount. As antigen component it contains hybrid proteins such as PEPHER2/neu.1-HSP70, PEPHER2/neu.2-HSP70, PEPHER2/neu.3-HSP70 or PERErbB1vIII-HSP70, or mixture thereof. Also disclosed are recombinant plasmid DNA encoding biosynthesis of hybrid proteins comprising in vaccine composition and method for production thereof by their gene expression in composition of recombinant proteins in E.coli followed by purification of target product.

EFFECT: new anti-tumor preparation.

6 cl, 1 dwg, 9 ex

FIELD: medicine, biotechnology.

SUBSTANCE: invention proposes variants of antibodies showing specificity to peptide domain located by both side of hinged site R76S77 in pro-BNP(1-108). Indicated antibodies recognize specifically also circulating pro-BNP(1-108) in human serum or plasma samples but they don't recognize practically peptides BNP(1-76) or BNP(77-108). Also, invention describes variants of peptides used in preparing antibodies. Amino acid sequence is given in the invention description. Also, invention discloses methods for preparing indicated antibodies and among of them by using indicated peptides. Also, invention describes methods for preparing antibody-secreting hybridoma, and hybridoma is disclosed prepared by indicated method. Also, invention describes a monoclonal antibody secreted by hybridoma 3D4 and deposited at number CNCM I-3073. Also, invention discloses variants for diagnosis of cardiac insufficiency in vitro and by using antibodies proposed by the invention. Also, invention describes a set used for detecting pro-BNP(1-108) in a biological sample. Using this invention simplifies detection of pro-BNP(1-108) circulating in human serum or plasma samples and provides specific detection of pro-BNP(1-108) that can be used in early diagnosis of human cardiac insufficiency.

EFFECT: valuable medicinal properties of antibodies.

24 cl, 16 dwg, 5 tbl, 20 ex

FIELD: biochemistry, in particular, methods and devices for producing coloring substances, possible use in food and cosmetic industry, and also during various biological research.

SUBSTANCE: phycoerythrin protein pigment is produced by extraction from seaweed. It is extracted from seaweed, selected from a group including Galaxaura oblongata, Halymenia ceylanica, Helminthocladia australis and Porphyra dentate.

EFFECT: phycoerythrin has high optical density.

2 cl, 27 dwg, 2 tbl

FIELD: equipment for growing plant tissues.

SUBSTANCE: in accordance to the invention, unit for accelerating growth of plant tissues contains a set of boards, forming matrices of holes. Each hole contains a tissue sample. Support for boards is provided by a rack which contains a set of vertically stacked shelves, containing one or more holding recesses, which forcedly move boards to given positions. Light for tissue samples is provided by a set of matrices of light diodes, mounted on mounting plates. Light diodes emit white light. Each mounting plate is supported by corresponding end comber-type rack connector, so that light diodes are close to boards, supported by shelves, positioned lower. Matrix of light diodes preferably matches matrix of holes, supported by a lower positioned shelf in fixed position, so that each light diode is centered above a corresponding hole.

EFFECT: creation of high capacity system for processing samples of tissues which require light for supporting cell reproduction.

7 cl, 7 dwg

FIELD: microbiology, veterinary science, biotechnology.

SUBSTANCE: invention relates to diagnostic immunobiological preparations comprising biologically active components of cells Bacillus anthracis. Method involves submerged culturing the strain B. anthracis, the strain CTI-1 in apparatus BIOP-1,0 in liquid nutrient medium based on hydrochloric hydrolyzate of fish flour for 8-12 h followed by washing out, centrifugation and devitalization of cells. Then alkali-soluble components are extracted from prepared bacterial mass. Borate buffer is added to the substance and dry allergen is prepared in sublimating device LZ-45. Drying temperature regimen is regulated by heat supply to material from -32°C to +28°C. The total duration of lyophilization process is 20-24 h. One dose of the prepared dry allergencomprises 20 mcg of protein. Biological activity of dry anthrax allergen exceeds the activity of commercial analog by two-fold, it is stable in prolonged storage and effective in estimation of anti-anthrax immunity expression and in diagnosis of anthrax infection.

EFFECT: enhanced and valuable properties of allergen.

4 tbl, 2 ex

FIELD: biotechnology, immunology.

SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.

EFFECT: valuable biological and medicinal properties of antibodies.

53 cl, 4 tbl, 10 dwg, 2 ex

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to development of a novel strain used for preparing enzyme representing a complex of acid subacid proteases. Strain is prepared by selection from the known strain Aspergillus oryzae (VKPM F-683) by multistep selection using effective methods of mutagenesis. Strain is stored as lyophilic dried culture and on slants with wort-agar in the biotechnology section of enzyme preparations in the food processing department of the State Scientific Institute VNII of food processing technology in Moscow. Invention provides preparing the strain possessing the high level of synthesis of acid and subacid proteases and high total activity of enzyme in cultural fluid exceeding activity of analogue by 2.0-2.6-fold and in reducing culturing process time.

EFFECT: improved and valuable properties of strain.

2 tbl, 5 ex

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to the strain Aspergillus oryzae-12 providing the high level of synthesis of acid proteases and xylanase. The strain is obtained by multistep selection of the strain Aspergillus oryzae-387 (VKPM F-683) using effective methods of mutagenesis. The strain is deposited in collection VKPM at № F-932. Invention provides preparing the enzyme complex showing the high proteolytic and xylanolytic activity in cultural fluid wherein their level exceeds activity in analogue by 2.5-2.8-fold.

EFFECT: valuable properties of strain.

2 tbl, 3 ex

FIELD: biotechnological methods.

SUBSTANCE: invention concerns new polynucleotides, which encode polypeptide having tripeptidylpentidase activity. To produce this polypeptide, host cell is transformed with polynucleotide or polynucleotide-containing vector and then cultured under suitable polynucleotide expression conditions.

EFFECT: enabled production of new protease from thread fungi.

14 cl, 3 tbl, 11 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to producing ethyl alcohol or fodder for monogastral animals. The polyenzyme product with glucoamylase, proteolytic and xylanase activities is prepared by fermentation of wheat bran using microorganism Aspergillus niger. Indicated glucoamylase, proteolytic and xylanase activities have the following minimal values: glucoamylase activity - at least 100 U/g of dry matter; proteolytic activity - at least 100 U/g of dry matter; xylanase activity - at least 100 U/g of dry matter under condition that glucoamylase activity has to be at least 750 U/g of dry matter and/or xylanase activity has to be at least 300 U/g of dry matter. Invention provides enhancing the soluble nitrogen content in wort after saccharification, reducing viscosity and effectiveness in using.

EFFECT: improved preparing method, valuable properties of hydrolyzed bran.

14 cl, 10 tbl, 7 ex

The invention relates to biotechnology and concerning the processing of leather and fur raw materials with the help of a new complex enzyme preparation

The invention relates to biotechnology, in particular, to obtain the strain - producer of complex hydrolytic enzymes, which consists of acidic and weakly acidic protease,- amylases and related enzymes, such as Exo -- glucanase, cicasa, xylanase, and can be applied in microbiological industry for preparation of enzyme preparations for hydrolysis of plant, animal and microbial substrates in the food industry, fermentation industry, agriculture, upon receipt of amino acid mixtures, fermentolizate yeast and other biologically active substances

The invention relates to the medical industry, in particular the microbiological synthesis of enzymes for medical purposes

FIELD: biotechnology, molecular biology.

SUBSTANCE: invention proposes a polynucleotide VEGI-192a encoding polypeptide that inhibits growth of human vascular endothelial cells. Invention describes expressing vector comprising polynucleotide and E. coli cell-host comprising vector. Invention discloses polypeptide encoded by polynucleotide and fused protein based on indicated polypeptide. Invention describes polynucleotide encoding fused protein and expressing vector based on indicated polynucleotide. Invention discloses a pharmaceutical composition used for inhibition of angiogenesis based on polypeptide-inhibitor of growth of human vascular endothelial cells and polynucleotide encoding its. Invention describes therapeutic methods for inhibition of angiogenesis and suppression of tumor growth based on this composition. Invention describes an antibody raised to polypeptide that inhibits growth of human vascular endothelial cells. Using this invention provides novel forms of inhibitor of human growth of vascular endothelial cells and can be used in medicine.

EFFECT: valuable biological and medicinal properties of inhibitor.

27 cl, 27 dwg, 13 tbl, 34 ex

FIELD: biotechnology, genetic engineering.

SUBSTANCE: invention relates to a plasmid vector able to autonomic replication in the strain Escherichia coli K-12 at any temperature. Plasmid comprises region or with mutation, gene rep from pMW119 and DNA fragment. DNA fragment can be integrated into chromosome of microorganism belonging to Escherichia coli W or Escherichia coli B as result of homologous recombination. Plasmid is fused into a microorganism belonging to Escherichia coli W or Escherichia coli B followed by preparing amino acid. Also, invention relates to strains Escherichia coli DH5α/pMTS11910 (FERM BP-6904) and Escherichia coli DH5α/pMTS11914 BP-6905) designated for storage the plasmid vector. Strains Escherichia coli WLA-131 (FERM BP-6902) and Escherichia coli WL-1133 (FERM BP-6903) are producers of amino acid leucine. Invention provides carrying out superexpression of gene and to obtain stable plasmids.

EFFECT: valuable biological properties of plasmids and strains.

13 cl, 2 dwg, 1 tbl, 5 ex

FIELD: biotechnology, immunology.

SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.

EFFECT: valuable biological and medicinal properties of antibodies.

53 cl, 4 tbl, 10 dwg, 2 ex

FIELD: biotechnology, immunology, molecular biology, pharmacy.

SUBSTANCE: invention describes variants of MCP-1-binding molecules. One of MCP-1-binding molecule comprises at least one variable region of immunoglobulin (VH) heavy chain comprising of hypervariable sites CDR1, CDR2 and CDR3 while other molecules comprises both light and heavy chains. Invention proposes DNA constructs encoding indicated MCP-1-binding molecules and expressing vector carrying at least one of these DNA constructs. Invention describes a method for preparing MCP-1-binding molecule. Invention discloses a method for treatment of disease or disorder mediated by MCP-1 or eotaxine-1 based on antibody raised to MCP-1 that binds eotaxine-1 by cross mode. Invention describes a pharmaceutical composition based on antibody raised to MCP-1 that binds eotaxine-1 by cross mode and used in treatment of disease or disorder mediated by MCP-1 or eotaxine-1 in a patient. MCP-1-binding molecules inhibit binding MCP-1 with its receptor. The full immobilized antibody is highly specific as far as it binds human recombinant MCP-1 with value KD = (43 ± 2.9) x 1012 and can be used in medicine.

EFFECT: valuable medicinal properties of antibodies, improved method of treatment.

13 cl, 5 dwg, 4 tbl, 2 ex

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