FR2911253A1 - Use of peptide having phytase activity, comprising arginine, where the position of amino acids are defined in relation to phytase peptide sequence of Debaryomyces castellii, for the preparation of nutritional additive or an animal feed - Google Patents

Abstract

Use of a peptide having a phytase activity, comprising an arginine in position 211 of its peptide sequence, where the position of amino acids are defined in relation to phytase peptide sequence of Debaryomyces castellii comprising SEQ ID NO: 1 (having a defined sequence of 461 amino acid, given in the specification), for the preparation of a nutritional additive or an animal feed, is claimed. Independent claims are included for: (1) use of an isolated non-genetically modified strain or a transformed host organism, for producing the peptide; (2) use of a protein mixture or a fermentation wort to be obtained by an isolated non genetically modified strain or by host organism, for producing the peptide; and (3) use of a peptide mixture having a phytase activity comprising the peptide.

Description

The invention relates to the use of a phytase comprising an arginine

  in its active site for the manufacture of a nutritional additive or animal feed. Phytases are enzymes that catalyze the hydrolysis of phytic acid (myo-inositol hexakisphosphate, InsP6) to inorganic monophosphate, myo-inositol phosphate of lower phosphorylation degree (InsP5 to InsPI) and free myo-inositol in some case. According to the official nomenclature on enzymes of the Committee of the International Union of Eoiochemistry (IUPAC-IUB, 1977), phytases are part of hydrolases capable of hydrolyzing phosphomonoester bonds (EC 3.1.3) and are classified as 3-phytase (EC 3.1.3.8), 6-phytase (EC 3.1.3.26) and 5-phytase (EC 3.1.3.72) as a function of the first phosphate group of hydrolyzed phytic acid (the 2-position of the ring is attributed to the carbon bearing the axial connection, phytic acid having five of its phosphate groups in equatorial position and the last in axial position). Phytic acid and phytates are the major form of phosphorus storage in cereals, legumes and oleaginous plants. They are the main source of phosphorus in plant-based animal feeds, including monogastric animals (poultry and pigs).

  However, monogastric animals do not have enough intestinal enzymes to degrade phytic acid and phytates to provide the amounts of inorganic phosphate they need. In addition, phytic acid is an antinutritional factor, which forms complexes with proteins and ions (Fe3 +, Cal +, Zn2 +, Mg2 +) and thus decreases the availability of proteins and ions. Food rations for monogastric animals must therefore be supplemented with inorganic phosphate, while phosphorus contained in phytic acid and phytates is excreted and contributes to the eutrophication of surface water in areas of intensive livestock farming of monogastric animals. . Supplementation of monogastric animal diets with enzymes is a solution currently used to improve the bioavailability of phosphorus in phytic acid and phytates, and to decrease food supplementation with inorganic phosphorus and thus reduce phosphorus excretion. intensive farming areas.

  The phytases that are used as an additive in animal feed, allow on the one hand to increase the availability of phytic phosphorus, on the other hand to improve the digestibility of food. In addition, the release of phytic phosphate considerably reduces the costs due to phosphate supplementation, as well as the pollution caused by an excess of excreted phosphates. Phytases are particularly present in microorganisms, in particular fungi, bacteria and yeasts. Among the fungi, mention may be made of the genera Aspergillus (Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus), Penicillium, Mucor and Rhizopus. All extracellular phytase producing fungi are filamentous fungi. Filamentous fungi such as Peniophora lycii, Agrocybe pediades or Trametes pubescens also produce phytases. Among the bacteria, mention may be made of Pseudomonas sp., Klebsiella sp., Escherichia coli, Enterobacter sp., Bacillus subtilis, Aerobacter aerogenes, Bacillus amyloliquefaciens. The genera Bacillus and Enterobacter produce an extracellular phytase. Among the yeasts, mention may be made of Saccharomyces cerevisiae, Candida tropicalis, Torulopsis candida, Debaryomyces castellii, Debaryomyces occidentalis, Kluyveromyces fragilis, Schwanniomyces occidentalis and Schwanniomyces castellii. According to the new classification (Nakase T., Suzuki M., Phaff HJ and Kurtzman CP, 1998 p157-167 in CP Kurtzman and JW Fell (ed), The Yeasts, A taxonomic study, 4th Ed. Elsevier Sci. Publication Amsterdam), Schwanniomyces castellii Capriotti is synonym of Schwanniomyces occidentalis Kloker and Debaryomyces occidentalis Klocker var. occidentalis. Many phytases of microorganisms have been studied. Nevertheless, most of the phytases described so far only partially hydrolyze phytic acid and phytates and some with very slow kinetics.

  To date, among yeasts, only the yeast phytase Schwanniomyces occidentalis has been described as hydrolysing all phytate phosphate groups (EP 0 699 762, Segueilha L., Lambrechts C., Boze H., Moulin G., Galzy P., (1992) Purification and properties of a phytase from Schwanniomyces castelliii, J. Ferm Bioeng., 74, 7-11). According to EP 0 699 762, the Schwanniomyces occidentalis phytase is sensitive to cations (ZnCl 2 and CuCl 2 in particular), which is not favorable for use in animal feed because these cations are present in the food bolus (Segueilha et al. , 1992). The inventors of the present invention have previously studied and characterized the yeast phytase Debaryomyces castellii (patent application PCT / FR06 / 001652). This phytase is capable of hydrolyzing all the phosphate groups of phytic acid or phytate until liberation of myo-inositol.

  The inventors have demonstrated the origin of this activity, that is to say the hydrolysis of phytic acid and phytates until release of myo-inositol. This activity lies in the existence of an amino acid determined in position 211, in this case an arginine. The present invention provides peptides having a phytase activity, hydrolyzing all phospate groups of phytate and whose activity depends little on the conditions of implementation of the enzyme. This peptide comprises an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1.

Description of the sequences

  SEQ ID NO: 1: Debaseomyces castellii phytase sequence CBS 2923. SEQ ID NO: 2: Genomic sequence of Debaryomyces castellii phytase gene CBS 2923. SEQ ID NO: 3: Consensus sequence of acid phosphatases. SEQ ID NO: 4: Pattern of the Debaryomyces castellii phytase corresponding to the consensus sequence of acid phosphatases of SEQ ID NO: 3. SEQ ID NO: 5: Schwanniomyces occidentalis phytase sequence (NCBI accession number: CAB70441) SEQ ID NO: 6: Saccharomyces cerevisiae phytase sequence (NCBI accession number: NP_009434) SEQ ID NO: 7 Saccharomyces cerevisiae phytase sequence (accession number NCBI: NP_009650) SEQ ID NO: 8: Saccharomyces cerevisiae phytase sequence (accession number NCBI: NP_009651) SEQ ID NO: 9: Sequence of phytase of Saccharomyces cerevisiae (accession number NCBI: NP_012087) SEQ ID NO: 10: Pichia pastoris Pa phytase sequence (accession number NCBI: P52291) SEQ ID NO: 11: Sequence of Aspergillus piger phytase (phyt A) (accession number NCBI: AAS00648) SEQ ID NO: 12: Sequence of the phytase of Peniophora lycii (accession number NCBI: CAC48195) SEQ ID NO: 13: Sequence of the phytase of Aspergillus piger (phyt B ) (accession number NCBI: P34754) SEQ ID NO: 14: Sequence of the Aspergillus fumigatus phytase ( accession number NCBI: XP 751017) SEQ ID NO: 15: Escherichia coli phytase sequence / (NCBI accession number: AAN28334) Description of the invention The invention relates to the use of a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the Debaryomyces castelli phytase / according to SEQ ID NO: 1, for the preparation of a nutritional additive or feed. The present invention also relates to the use of a genetically unmodified isolated strain or a transformed host organism, producing a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence, the position of the amino acid being defined with respect to the peptide sequence of the Debaryomyces castelli phytase / according to SEQ ID NO: 1, for the preparation of a nutritional additive or an animal feed. The invention also relates to the use of a genetically unmodified isolated strain or a transformed host organism, comprising a polynucleotide encoding a peptide having a phytase activity comprising an arginine at position 211 of its sequence peptide, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1, for the preparation of a nutritional additive or an animal feed. The invention also relates to the use of a transformed host organism, comprising an expression cassette consisting of, in the direction of transcription: a functional promoter in a host organism, a polynucleotide encoding a peptide having an activity phytase comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the Debaryomyces castellii phytase according to SEQ ID NO: 1, and a terminator sequence in the same host organism, for the preparation of a nutritional additive or animal feed. The invention also relates to the use of a transformed host organism, comprising a vector comprising a polynucleotide encoding a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1, for the preparation of a nutritional additive or an animal feed.

  Another object of the present invention relates to the use of a protein mixture or a fermentation mash obtainable by an isolated strain not genetically modified or by a host organism producing a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1, for the preparation of a nutritional additive or d 'an animal feed. Still another subject of the present invention relates to the use of a mixture of peptides having a phytase activity comprising at least one peptide having a phytase activity having an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the Debaryomyces castellii phytase according to SEQ ID NO: 1, for the preparation of a nutritional additive or a feed. According to one embodiment of the present invention, the peptide having phytase activity further comprises an aspartic acid at position 338 of its peptide sequence. According to one embodiment of the present invention, the peptide having a phytase activity comprises the peptide sequence SEQ ID NO: 3. According to one embodiment of the present invention, the aforementioned uses are carried out for the hydrolysis of phytic acid to inorganic monophosphate and free myo-inositol, or for the hydrolysis of myo-inositol 2-monophosphate in inorganic monophosphate and free myo-inositol.

  Another object of the present invention relates to the use of a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1, for the hydrolysis of phytic acid to inorganic monophosphate and free myo-inositol.

  The present invention thus relates to the use of a phytase having an arginine in its active site. According to the present invention, the position of the amino acids is defined with respect to the peptide sequence of the Debaryomyces castellii phytase according to SEQ ID NO: 1. The D. castellii phytase sequence is therefore the reference sequence. All particular amino acid position indications are made with respect to the primary sequence of this phytase.

  Figure 1 shows an alignment of sequences described in the state of the art, aligned with the sequence of D. castellii (SEQ ID NO: 1) as a reference. The D. castellii phytase sequence is shown at the bottom of each page of Figure 1. Some amino acids in the sequence are underlined and a number is indicated below this amino acid. This number corresponds to the numbering of the amino acid in the sequence SEQ ID NO: 1. For example, in the first page of Figure 1, a methionine residue is underlined and the number 1 appears below, which means that said methionine is the amino acid located in the first position of the sequence SEQ ID NO: 1. Also, on page 5 of Figure 1, an arginine residue is underlined and the number 211 is below, which means that this arginine residue is in position 211 in SEQ ID NO: 1.

  By convention in this application, the numbering of amino acids of other phytases is compared to the sequence of D. castellii. The alignment of the sequences is for example realized by means of Vector NTi 9.1.0, AlignX alignment program (Clustal W algorithm) (Invitrogen INFORMAX, http://www.invitrogen.com) or by using the tool CLUSTAW ( http://www.ebi.ac.uk/clustalw/). On the basis of such an alignment, those skilled in the art can identify the nature and position of an amino acid in another corresponding phytase sequence.

  The following abbreviations were used: [) C Debaryomyces castellii CBS 2923 (accession number NCBI EF121003) [) O Schwanniomyces occidentalis (NCBI accession number: CAB70441) SC-1 Saccharomyces cerevisiae (NCBI accession number: NP_009434) SC-2 Saccharomyces cerevisiae (accession number NCBI: NP_009650) SC-3 Saccharomyces cerevisiae (accession number NCBI: NP_009651) SC-4 Saccharomyces cerevisiae (accession number NCBI: NP_012087) PP Pichia pastoris (accession number NCBI: P52291) AN-A Aspergillus piger (phyt A) (accession number NCBI: AAS00648) PL Peniophora lycii (accession number NCBI: CAC48195) AN-B Aspergillus piger (phyt B) (NCBI accession number: P34754) AF Aspergillus fumigatus (accession number NCBI: XP_751017) EC Escherichia coli (NCBI accession number: AAN28334)

  The phytase of Debaryomyces castellii CBS 2923 is shown in the sequence listing in SEQ ID NO: 1. By "phytase" is meant a polypeptide or an enzyme which catalyzes the hydrolysis of phytic acid (myo-inositol hexakisphosphate, InsP6) to inorganic monophosphate, myo-inositol phosphate of lower phosphorylation degree (InsP5 to InsP1) and free myo-inositol in some cases. According to the official nomenclature on enzymes of the committee of the International Union of Biochemistry (IUPAC-MB, 1977), phytases belong to the class of hydrolases, under the class of ester hydrolases and under subclass phospho monoester hydrolases among which we find phosphatases and phytases. Not all phosphatases hydrolyze phytic acid and phytates. To date, some enzymes have been identified as phosphatases, but it is not known if these enzymes are also phytases. The inventors have demonstrated an arginine residue at position 211 of the peptide sequence of Debaryomyces castellii (SEQ ID NO: 1) as a residue capable of binding myo-inositol 2-monophosphate, which stabilizes the enzyme-substrate and hydrolysis complex. phytic acid and phytates until release of myo-inositol. The inventors have furthermore demonstrated that other peptides having a phytase activity and having an arginine at position 211 of their peptide sequence (position defined by alignment with SEQ ID NO: 1), are also capable of hydrolyzing phytic acid and phytates until release of myo-inositol. The present invention therefore relates to the use of a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO : 1, for the preparation of a nutritional additive or feed. According to another embodiment, the present invention relates to the use of a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1, for the hydrolysis of myo-inositol hexakisphosphate to inorganic monophosphate and free myo-inositol.

  The phytase of SEQ ID NO: 1 comprises the RHGERYP or Arg His Gly Glu Arg Tyr Pro motif (SEQ ID NO: 4, amino acids 72 to 78 of SEQ ID NO: 1) corresponding to the consensus sequence RHGXRXP (SEQ ID NO : 3). This consensus sequence according to SEQ ID NO: 3 is present in the active site of numerous acid phosphatases. The phytase of SEQ ID NO: 1 also has the HD motif (or His Asp) present in many phytases. This motif is found in the C-terminal portion at amino acids 335 and 336 of SEQ ID NO: 1.

  In one embodiment of the invention, the peptides according to the invention have the RHGERYP motif (SEQ ID NO: 4) or another motif corresponding to the consensus sequence RHGXRXP (SEQ ID NO: 3). Preferably, the peptides of the invention also have an HD pattern. Thus, according to this embodiment, the subject of the present invention is the use of a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the Debaryomyces castellii phytase according to SEQ ID NO: 1, said phytase comprising the motif RHGXRXP (or peptide sequence SEQ ID NO: 3) or the RHGERYP motif (or peptide sequence SEQ ID NO: 4), for the preparation of a nutritional additive or an animal feed.

  The inventors have furthermore demonstrated an aspartic acid residue at position 338 of the peptide sequence of Debaryomyces castellii (SEQ ID NO: 1) as a residue capable of stabilizing arginine at position 211. The inventors have further verified that the other peptides having a phytase activity, having an arginine at position 211 of their peptide sequence, and capable of hydrolyzing phytic acid and phytates until the myo-inositol is released, also have an aspartic acid residue in position 338 of their peptide sequence (position defined by alignment with SEQ ID NO: 1). Thus, according to another embodiment, the subject of the invention is the use of a peptide having a phytase activity comprising: an arginine at position 211 of its peptide sequence; an aspartic acid at position 338 of its peptide sequence , the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1, for the preparation of a nutritional additive or an animal feed.

  In another embodiment of the present invention, peptides having phytase activity are glycosylated. In addition, in yet another embodiment of the invention, peptides having phytase activity carry at least one disulfide bridge.

  The phytase of Debaryomyces castellii is an enzyme secreted by this yeast in its extracellular environment. For expression and secretion by a recombinant host organism, peptides having phytase activity can be fused at their N-terminus with a signal peptide recognized by that host organism.

  The phytases of the invention can be isolated or purified from their natural environment. They can be prepared by different methods. These methods include purification from natural sources such as cells naturally expressing these peptides, production of recombinant peptides by appropriate host cells and their subsequent purification, production by chemical synthesis or, finally, a combination of these different approaches. . These different production processes are known to those skilled in the art. Thus, the phytases of the present invention can be isolated from any phytase producing microorganism. The phytases of the present invention can also be isolated from recombinant host organisms expressing the peptide having this phytase activity. The invention also relates to fusion proteins, recombinant proteins or chimeric proteins comprising the peptide according to the invention.

  The peptides according to the present invention have a phytase activity and possess the ability to hydrolyze all the phosphate bonds of phytic acid.

  According to one embodiment of the present invention, a peptide having an activity, as described above, for the preparation of a nutritional additive or feed for the hydrolysis of myo-inositol is used. hexakisphosphate to inorganic monophosphate and free myo-inositol, or for the hydrolysis of myo-inositol 2-monophosphate to inorganic monophosphate and free myo-inositol.

  According to one embodiment of the present invention, a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence is used, the position of the amino acids being defined with respect to the peptide sequence of the Debaryomyces caste / Ili phytase. according to SEQ ID NO: 1, for the preparation of a nutritional additive or a feed, except the peptide of SEQ ID NO: 1 and / or the peptide of SEQ ID NO: 5.

  Isolated strain or host organism The present invention also relates to the use of a genetically unmodified isolated strain or a transformed host organism producing a phytase comprising an arginine at position 211 of its peptide sequence. , the position of the amino acids being defined with respect to the peptide sequence of the Debaryomyces castellase phytase according to SEQ ID NO: 1, for the preparation of a nutritional additive or an animal feed. The present invention also relates to the use of this strain or host organism for the hydrolysis of myo-inositol hexakisphosphate to inorganic monophosphate and free myo-inositol.

  The present invention also relates to the use of a genetically unmodified isolated strain or a transformed host organism, comprising a polynucleotide encoding a phytase comprising an arginine at position 211 of its peptide sequence, the position amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1, for the preparation of a nutritional additive or an animal feed. The present invention further relates to the use of a transformed host organism comprising an expression cassette consisting of, in the sense of transcription: a functional promoter in a host organism, a polynucleotide encoding a phytase comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the Debaryomyces castellii phytase according to SEQ ID NO: 1, and a terminator sequence in the same host organism, for the preparation of a nutritional additive or animal feed. The present invention also relates to the use of a transformed host organism, comprising a vector comprising a polynucleotide encoding a phytase comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1, for the preparation of a nutritional additive or an animal feed. It is quite possible according to the present invention to transform a host organism by integration into said host organism of at least one polynucleotide, an expression cassette or a vector. The polynucleotide can be integrated into the genome of the host organism or replicate stably in the host organism. Transformation methods of host organisms are well known to those skilled in the art and widely described in the literature. By "host organism" is meant in particular according to the invention any monocellular or multicellular organism, lower or higher, in particular selected from bacteria, yeasts, fungi and plants. By "host organism" is meant a non-human organism. As an indication, the yeasts are chosen from Pichia pastoris, Saccharomyces cerevisaeae, Yarrowia lipolytica, Schwanniomyces occidentalis, Schwanniomyces castellii and Schizosaccharomyces pombe. As an indication, the fungi are chosen from Aspergillus and Penicilliums, preferentially from Penicillium funiculosum, Trichoderma reesei, Aspergillus piger, Aspergillus awamori, Aspergillus kawachii and Trichoderma koningii. In one embodiment, the host organism is a Penicillium funiculosum strain in which a peptide having a phytase activity according to the invention is expressed. In another embodiment, the host organism is a Debaryomyces castellii strain in which a peptide having a phytase activity according to the invention is expressed or over-expressed. In another embodiment of the invention, the host organism is a strain of Pichia pastoris or Schizosaccharomyces pombe of which is expressed a peptide having a phytase activity according to the invention. The plants are for example chosen from rice (Oryza satvia L.), tobacco, soya and wheat.

  The techniques of vector construction, host-organ transformation, and expression of heterologous proteins in these organisms are widely described in the literature (Ausubel FM et al., "Current Protocols in Molecular Biology" Volumes 1 and 2, Greene Publishing Associates and Wiley -Interscience, 1989, T.Maniatis, EFFritsch, J.Sambrook, Molecular Cloning: A laboratory Handbook, 1982).

  Expression cassettes With regard to the expression cassettes according to the invention, any type of promoter sequence may be used. The choice of the promoter will depend in particular on the host organism chosen for the expression of the gene of interest. Some promoters allow constitutive expression whereas other promoters are inducible on the contrary. Among the functional promoters in fungi, mention may be made in particular of glyceraldehyde-3-phosphate dehydrogenase from Aspergillus nidulans (Roberts et al., Current Genesis 15: 177-180, 1989). Among the functional promoters in bacteria, mention may be made in particular of the bacteriophage T7 RNA polymerase (Studier et al., Methods in Enzymology 185: 60-89, 1990). Among the functional promoters in yeasts, mention will be made especially of that of the Gall gene (Elledge et al., Proc Natl Acad Sciences, USA 88: 1731-1735, 1991) or the GAL4 and ADH promoters of S. cerevisiae. All these promoters are described in the literature and well known to those skilled in the art. For expression in Penicillium funiculosum, for example, expression cassettes comprising an H4B histone promoter, an aspartyl protease acid promoter or a csI13 promoter (WO 00/68401) will be chosen. For expressing in yeast Pichia pastoris, for example, expression cassettes comprising the methanol-inducible AOX1 promoter (Tschopp, JF, Sverlow, G., Kosson, R., Craig, W. and Grinna, L) will be selected. (1987) High-level secretion of glycosylated invertase in the methylotrophic yeast, Pichia pastoris, Biotechnology 5, 1305-1308) or the strong constitutive promoter GAP (Waterham, HR, Digan, ME, Koutz, PJ, Lair, SV and Cregg JM (1997) Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter, Gene 186, 37-44).

  For the expression in Schizosacchromyces pombe, for example, expression cassettes will be selected comprising the Thiarnine-repressed Nmtl regulatory promoter and inactivated in the absence of thiamine (Maundrell, K., (1989) Nmtl of fission yeast. The expression cassettes according to the present invention may further include any other sequence necessary for the expression of the peptides or polynucleotides, such as by example regulatory elements or signal sequences allowing the secretion of polypeptides produced by the host organism. In particular, it is possible to use any regulatory sequence making it possible to increase the level of expression of the coding sequence inserted in the expression cassette. According to the invention, it is possible in particular to use, in association with the promoter regulatory sequence, other regulatory sequences, which are located between the promoter and the coding sequence, such as enhancer enhancers. In addition, the expression cassettes according to the present invention may include any other sequence necessary for the secretion of polypeptides produced by the host organism such as signal sequences. For secretion by Pichia pastoris, it is possible, for example, to use the sequence of factor a as a secretion signal. A wide variety of terminator sequences can be used in the expression cassettes according to the invention, these sequences allow termination of the transcription and polyadenylation of the mRNA. Any functional terminator sequence in the selected host organism may be used. For expression in Penicillium funiculosum, for example, expression cassettes comprising an H4B histone terminator, an aspartyl protease acid terminator or a cs113 terminator (WO 00/68401) will be selected. The subject of the present invention is also a polynucleotide comprising an expression cassette according to the invention, advantageously the expression cassettes according to the present invention are inserted into a vector.

  Vectors The replication or expression vectors for transforming a host organism according to the present invention comprise at least one polynucleotide or an expression cassette as defined above. This vector may in particular be constituted by a plasmid, a cosmid, a bacteriophage or a virus into which is inserted a polynucleotide or an expression cassette according to the invention. The techniques for constructing these vectors and for inserting a polynucleotide of the invention into these vectors are known to those skilled in the art. In general, any vector capable of maintaining, self-replicating or propagating in a host cell and especially in order to induce the expression of a polynucleotide or a polypeptide can be used. Those skilled in the art will choose the appropriate vectors, in particular according to the host organism to be transformed and according to the transformation technique used.

  The vectors of the present invention are especially used to transform a host organism for the purpose of replicating the vector and / or the expression of a polypeptide according to the invention in the host organism.

  Food Additives and Feeds "Nutritional additive" means a substance intentionally added to a food, usually in small quantities, to improve its nutritional characteristics or digestibility. Nutritional additives for animals may for example contain vitamins, mineral salts, amino acids and enzymes.

  Typically, according to the invention, the nutritional additives for animals may comprise a polypeptide, a non-genetically modified strain, a host organism, a culture supernatant, a protein mixture, a fermentation broth of a host organism or a strain. not genetically modified according to the invention. Thus, peptides having phytase activity can be used directly for the manufacture of a nutritional additive for animals. Alternatively, a non-genetically modified strain or a host organism producing phytases according to the invention can be used directly for the manufacture of a nutritional additive for animals.

  In one embodiment of the invention, a culture supernatant, a protein mixture or a fermentation must is used for the preparation of a nutritional additive or animal feed. According to the invention, this protein mixture, culture supernatant or fermentation broth is likely to be obtained by a genetically unmodified isolated strain or by a host organism producing a peptide having a phytase activity, this peptide comprising a arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debatyomyces castellii according to SEQ ID NO: 1. This embodiment is particularly advantageous when the peptide in question is secreted in the extracellular medium by the strain or the host organism. Usually, this culture supernatant is concentrated or lyophilized for the manufacture of the nutritional additive. This culture supernatant, protein mixture or fermentation must may then be concentrated or lyophilized for the formulation of a food additive or feed. The method may comprise additional steps of purifying the peptide having phytase activity from the culture supernatant. If the host organism does not secrete the peptide into the culture medium, an additional step of breaking the cells and purifying the cell extract may be necessary. The nutritional additives of the present invention comprise at least one peptide according to the invention but may also comprise other nutritional substances such as vitamins, amino acids or mineral salts. The additives according to the invention increase the digestibility of food, thus contributing to a better nutritional value of cereal-based diets (wheat, barley, corn, oats, rye, etc.) and oilcake (soybean, sunflower, rapeseed, ...) in particular.

  By "food" is meant anything that can be used for the food of animals. According to one embodiment of the present invention, the feed comprises a nutritional base and a nutritional additive as defined above. These foods are usually in the form of flours or granules in which the additives according to the invention are incorporated. According to one embodiment of the present invention, the feed comprises a peptide according to the invention, a non-genetically modified strain, a host organism according to the invention, a fermentation wort, a protein mixture, or a supernatant. of this strain or host organism.

  The present invention also relates to the use of a mixture of peptides having a phytase activity, said mixture comprising at least one peptide having a phytase activity, the peptide having an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castel / il according to SEQ ID NO: 1, for the preparation of a nutritional additive or an animal feed. The present invention also relates to the use of this peptide mixture for the hydrolysis of phytic acid to inorganic monophosphate and free myo-inositol.

  In one embodiment of the invention, the nutritional additives and animal feeds according to the invention comprise a combination of at least two peptides having complementary activities. These additives and these foods thus comprise at least one peptide having a phytase activity as defined above, said peptide being associated with another peptide. The peptide of the invention hydrolyzes phytic acid to inorganic monophosphate and free myosinol. A peptide having a phytase activity, for example a 3-phytase activity or an 6-phytase activity, or a peptide having a phytase activity which does not hydrolyze all the phosphate groups can be associated with it. The phytase associated with the phytase according to the invention may for example be chosen from the phytases of the following organisms: Western shrimpworm, Pichia pastoris, Escherichia coli, Aspergillus awamori (phytase A and phytase B), Aspergillus piger (phytase A and phytase B), Enicillium funiculosum, Aspergillus oryzae, Peniophora lycii, Aspergillus ficuum, Aspergillus nidulans, Talaromyces thermophilus, Aspergillus fumigatus and Aspergillus terreus. As an indication, the phytase according to the present invention is associated with the Aspergillus piger phytase (Ullah, AH J and Sethumadhavan, K., (2003) PhyA gene product of Aspergillus ficuum and Peniophora lycii produces dissimilar phytases.Biochemical and Biophysical Research Communications 303: 463-468) or a phytase of a strain of Penicillium funiculosum (WO 03/054199, WO 99/57325). For intensive animal husbandry, feed usually comprises a nutritional base and nutritional additives. By "nutritional basis" is meant, what constitutes the bulk of the animal's diet, constituted for example by a mixture of cereals, proteins and fats of animal and / or vegetable origin . The nutritional bases for animals are adapted to the diet of these animals. Typically, these nutritional bases include, for example, corn, wheat, pea and soybeans. These nutritional bases are adapted to the needs of the different animal species for which they are intended. These nutritional bases may already contain nutritional additives such as vitamins, mineral salts and amino acids. By "monogastric animals" is meant in particular poultry and pigs. Poultry includes laying hens, broilers, turkeys and ducks. Pigs include growth and finishing pigs as well as piglets.

  FIG. 1: alignment of sequences described in the state of the art, aligned with the D. castellii phytase sequence (SEQ ID NO: 1) as a reference. FIG. 2: representation of a monomer of the phytase of D. castellii Figure 3: representation of a trimer of D. castellii phytase Figure 4: representation of a tetramer of D. castellii phytase Figure 5: activity of P. pastoris phytase as a function of pH ( pH 3 to 3.5 200 mM glycine buffer, pH 3.5 to 7 200 mM sodium acetate buffer, pH 7 to 7.5 200 mM tris HCI buffer) Figure 6: P. pastoris phytase activity in function temperature, at pH 3, for 20 minutes

  The strain used is listed in the Centraal office voor Schimmelculture (Delft) under the name Debaryomyces castellii CBS 2923.

  The D. castelli phytase has been characterized biochemically and biophysically (PCT / FR2006 / 001653 patent application filed July 7, 2006). This phytase belongs to the group of high molecular weight phosphatases. It is characterized as an HAP (acid phosphatase to histidine) and is active in a wide pH range (2-6.5), with an optimum pH of activity of 4-4.5. It is stable at 66 C for 1 hour, its optimum temperature of activity is 60 C.

  Example 1: Determination of the structure of D. castellii phytase

  Crystal Analysis and Data Processing Several crystals were obtained using crystallization kits under different conditions. Preliminary X-ray diffraction tests made it possible to judge the diffracting quality of these crystals (resolution, mosaicity, etc.). The best crystals were obtained with the following crystallization conditions: 0.02M CaCl 2 0.1M Na Acetate pH 4.6 15% MPD (2-methyl-2,4-pentanediol)

  Under such conditions, the crystal can be frozen (vitrified) in liquid nitrogen directly without prior specific soaks. The dataset collected at the ESRF at 100K was indexed and integrated by the MOSFLM software (Leslie, 1992) and then scaled by the SCALA software (CCP4). The crystal belongs to the P6522 space group with a mesh of dimensions a = b = 121.65 A, c = 332.24 A and a = R = 90, y = 120. The structure was resolved by molecular replacement (PHASER software, Mac Coy, 2005) using the structure of Aspergillus piger phytase (code pclb: 1QFX, Kostrewa et al., 1999) as a model. The step of automatic construction / refinement of the structure was conducted by the software ARP 19 (Perrakis et al., 1999). REFMAC software (Murshudov et al., 1997) and TRUBO-FRODO (Roussel & Carnbillaud, 1992) were used for final manual refinement.

  Analysis of the Monomer Structure The structure of a phytase monomer is composed of 457 amino acids. See SEQ ID NO: 1. The first four residues of the protein are too mobile to be resolved by X-ray diffraction.

  As shown in Figure 2, a monomer is composed of a small domain a and a broad domain a / I3. The small estate has 5 propellers a. The area a / 13 is composed of 6 sheets 3 in the center surrounded on one side by two long propellers a and on the other by four smaller propellers.

  The residues of the consensus catalytic site (72RHGERYP78 and 335HD336) are located in a deep cleft at the interface of the broad domain a / (3 and small domain a.

  Analysis of dimer structure The asymmetric unit has two monomers as suggested by the Mathews coefficient (VR, 3.46 A3.Da-1) and the percentage of solvent (63%) calculated for two molecules in the asymmetric unit. The analysis of the contact (surface and nature) between these two monomers clearly shows that they form a dimer, as shown in FIG. 3. The contact area between two monomers within the dimer is 2395 A2, which represents 12% of the total surface of a monomer. These two molecules are perfectly superimposable but have minor differences in their composition in N-acetylglucosamine (NAG) residues and in the number of disulfide bridges.

  For monomer A, the 8 cysteine residues are involved in Cys62-Cys385, Cys214-Cys435, Cys262-Cys275, Cys405-Cys413 disulfide bridges. For monomer B, the Cys62-Cys385 disulfide bridge is not formed. Monomer A contains 3 NAG residues, linked to 3 of the 9 potential N-glycosylation residues (Asn 158, 314 and 439). Monomer B contains only 2 bound to Asn 314 and 439. These residues correspond to the first residue of N-glycosyl chains left after hydrolysis by endoglycosidase H. Other NAG residues are present (proteolysis analysis). and mass) on other potential sites of glycosylation but are too mobile to be resolved.

  Analysis of the Quaternary Structure The crystalline stack shows that the phytase is a tetramer, confirming the results obtained by exclusion chromatography. This tetramer consists of two strictly identical dimers connected by an axis of symmetry in the crystal. See Figure 4. The tetramer has a bi-pyramid shape with a square base. At the level of the tetramer, the contact area for a monomer is 4900 A2, which is 24% of the total area of a monomer. In the tetrameric structure, the 4 active sites are exposed to the solvent and easily accessible to the substrate.

  The structure is accessible on the RCSB Protein Data Bank website with the following pdb code: 2GFI.

  EXAMPLE 2 Comparison with Other Histidine Acid Phosphatases The phytase of D. caste / Ili possessing a domain a and a domain a / [3, it can be classified in the structural family of histidines acid phosphatases (PAHs) according to the classification. SCOP (Structural Classification Of Protein, Murzin et al., 1995). This family contains in particular the phosphatase of A. Piger (PAAN), the phytases of A. piger (phytAn), the phytases of A. fumigatus (phytAf) and E. coli (phytEc).

  By superposition of the monomers, three families can be defined. The first comprises the phytDc and PAAn monomers which are very similar. The superposition of the phytAn or phytAf monomers with phytDc is less perfect. In contrast, the phytAn and phytAf structures are very similar and form a second family. The most notable difference between these first two families is at the N-terminus.

  For phytAn and phytAf phytases, the N-terminus is maintained contiguous to the protein by a disulfide bridge. For the phytDc and PAAn proteins, the N-terminal end (less constrained) forms an extended loop that can go to make a consequent contact with the second monomer. This interaction plays a major role in the stability of the dimer and consequently of the tetramer. This partly explains the quaternary structure for phytDc and PAAn (tetrameric) and monomeric for phytAf and phytAn

  The third family is represented by the phytEc phytase which has a structure more distant from those of the previously described phytases. The different structures of the PAH family are not absolutely superimposable. Despite this structural diversity, one fact is remarkable: the 5 residues of the catalytic site, namely RHGXRXP, are perfectly superimposable.

  Example 3 Determination of the Structure of D. Castellii's Phytase in the Presence of Phytic Acid Similarly to Example 1, a second structure was elucidated from apo phytase crystals complexed subsequently with a concentrated solution of phytic acid. Once the structure was resolved, it was found that the phytic acid had been hydrolysed and persisted in the active site only two phosphates, one in the reaction site and the other in the vicinity. A model of the complex between D. castel / il phytase and myo-inositol 2-monophosphate was carried out by placing the phosphate of the latter in the position identified in the reaction site, followed by an exploration of the accessible space. by rotation along the two axes between the phosphate and the 6-carbon ring. This made it possible to detect an arginine residue at position 211 as a residue capable of binding myo-inositol 2-monophosphate, thus stabilizing the PhytDC-myo-lnositol 2-monophosphate complex and thus facilitating the hydrolysis of the latter. In addition, it has been shown that the side chain of Arginine 211 is stabilized by an aspartic acid at position 338 (Asp 338).

  Example 4: Aliquancy of primary sequences and capacity for hydrolysis of myo-inositol 2-monophosphate Sequence alignment with other phosphatases / phytases (see FIG. 1) shows that 2 other phosphatases / phytases have an arginine at position 211, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1. These are the phosphatases / phytases of Schwanniomyces occidentalis and F'ichia pastoris. These 2 sequences also have an aspartic acid at position 338. The inventors have previously shown that D. castellii's phytase is capable of hydrolyzing all phosphate groups of phytate until the release of myo-inositol (see patent application PCT / FR2006 / 001653).

  The inventors have also investigated whether the phytases / phosphatases of occidentallis and P. pastoris hydrolyze or not phytic acid and / or myo-inositol 2-monophosphate.

  The kinetics of phosphate release are carried out in 250 mM sodium acetate buffer, pH4 or pH3 (P. pastoris) in the presence of 0.08 U / ml of phytase. The measurements are carried out at 37 ° C: after 30 minutes of hydrolysis of Ins (2) P1, and after 240 minutes of hydrolysis of phytic acid.

  The results are shown in the table below:% Ins (n) pH hydrolyzed Phytic acid Ins (2) Pi (9 mM) (1 mM) D. castellii 100 84 S. occidentalis 100 88 P. pastoris 100 55 A. piger (PhytA) 69 2 Table: Hydrolysis of phytic acid and ins2P1 by the phytases of D. castellii, S. occidentalis, P. pastoris, A. piger These results show that the phytases / phosphatases of D. castellii S. occidentallis and P. pastoris hydrolyze phytic acid and myo-inositol 2-mono-phosphate.

  P. pastoris phytase completely hydrolyzes phytic acid to inositol and inorganic phosphate. It is able to hydrolyze ins (2) P1 like the phytases of D. castellii and S. occidentalis and contrary to A phytase A. to draw. These data constitute an important element supporting the essential role of arginine in the catalytic site in the hydrolysis of Ins (2) P1, phytic acid and phytates.

  In addition, phytases belonging to the class of histidine acid phosphatases (histidine acid phosphatase, PAH), that is to say the phytases of S. cerevisiae, P. Iycii, A. piger A (phytAn), A Niger B (PAAn), A. Fumigatus (phytAf), E. Coli (phytEc), which do not have arginine at position 211 (position determined by alignment with SEQ ID NO: 1, see FIG. not completely hydrolyze phytic acid (see table above for A. piger phytase). Myo-inositol 2-monophosphate (Ins (2) PI) is the final product. These data are further elements in the direction of the essential role of arginine in the catalytic site in the total hydrolysis of phytic acid and phytates and in the hydrolysis of Ins (2) P1.

  Example 5: Enzymatic properties of P. pastoris phytase

  5.1. Effect of pH The effect of pH on the phytase is determined by measuring the enzymatic activity at 37 C, in the presence of different buffers: for pHs between 2 and 3.5 (glycine buffer), for pH 3 5-7 (sodium acetate buffer) and for pH 7-7.5 (tris HCI buffer). The phytase is active between pH values of 2.3 to 6, with an optimum at pH 3 (see Figure 5).

  5.2. Effect of temperature The optimal temperature is determined on the native phytase, by measuring the phytase activity at different temperatures: from 10 to 80 ° C at pH 3. The optimal temperature of the phytase is 50 ° C (see Figure 6). ). 5.3. Temperature stability study A study of thermal denaturation at various temperatures shows that the enzyme is stable for one hour at 60 ° C. when it is in 125 mM acetate buffer, pH 3. It is denatured above 65 ° C. C, with a loss of activity of 30% after 20 min at 65 C.

Claims (12)

  1. Use of a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1, for the preparation of a nutritional additive or animal feed.   2. Use of a genetically unmodified isolated strain or a transformed host organism, producing a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1 for the preparation of a nutritional additive or an animal feed.   3. Use of a genetically unmodified isolated strain or a transformed host organism, comprising a polynucleotide encoding a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the Debaryomyces castellii phytase according to SEQ ID NO: 1, for the preparation of a nutritional additive or an animal feed.   4. Use of a transformed host organism, comprising an expression cassette consisting of, in the direction of transcription: a functional promoter in a host organism; a polynucleotide encoding a peptide having a phytase activity comprising an arginine; position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1, and a terminator sequence in the same host organism, for the preparation of a nutritional additive or feed.   5. Use of a transformed host organism, comprising a vector comprising a polynucleotide encoding a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1, for the preparation of a nutritional additive or an animal feed.   6. Use of a protein mixture or a fermentation mash obtainable by a genetically unmodified isolated strain or by a host organism producing a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1, for the preparation of a nutritional additive or an animal feed.   7. Use of a mixture of peptides having a phytase activity comprising at least one peptide having a phytase activity having an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the phytase of Debaryomyces castellii according to SEQ ID NO: 1, for the preparation of a nutritional additive or an animal feed.   The use according to any of the preceding claims, wherein the peptide having phytase activity further comprises an aspartic acid at position 338 of its peptide sequence.   9. Use according to any one of the preceding claims, wherein the peptide having a phytase activity comprises the peptide sequence SEQ ID NO: 3.   10. Use according to any one of the preceding claims for the hydrolysis of phytic acid to inorganic monophosphate and free myoinositol.   11. Use according to any one of claims 1 to 9 for the hydrolysis of myo-inositol 2-monophosphate to inorganic monophosphate and free myo-inositol.   12. Use of a peptide having a phytase activity comprising an arginine at position 211 of its peptide sequence, the position of the amino acids being defined with respect to the peptide sequence of the Debaryomyces castellii phytase according to SEQ ID NO: 1, for hydrolysis of phytic acid to inorganic monophosphate and free myo-inositol.