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WO2006063588A1 - Polypeptides présentant une activité phosphatase acide et polynucléotides codant pour lesdits polypeptides - Google Patents

Polypeptides présentant une activité phosphatase acide et polynucléotides codant pour lesdits polypeptides Download PDF

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Publication number
WO2006063588A1
WO2006063588A1 PCT/DK2005/000787 DK2005000787W WO2006063588A1 WO 2006063588 A1 WO2006063588 A1 WO 2006063588A1 DK 2005000787 W DK2005000787 W DK 2005000787W WO 2006063588 A1 WO2006063588 A1 WO 2006063588A1
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Prior art keywords
polypeptide
seq
phytase
amino acids
phosphatase
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PCT/DK2005/000787
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English (en)
Inventor
Frank Hatzack
Vibe GLITSØ
Henrik Frisner
Mikako Sasa
Søren Flensted LASSEN
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Novozymes A/S
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Publication of WO2006063588A1 publication Critical patent/WO2006063588A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes

Definitions

  • the present invention comprises a sequence listing.
  • the strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C. F. R. ⁇ 1.14 and 35 U. S. C. ⁇ 122.
  • the deposit represents a substantially pure culture of the deposited strain.
  • the deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
  • the present invention relates to isolated polypeptides having phosphatase activity related to a phosphatase from Eupenicillium pinetorum CBS 116641 , as well as to isolated polynucleotides encoding the polypeptides.
  • the invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides, in particular the use in animal feed, in particular for boosting the activity of phytases.
  • Phytases catalyze the degradation of myo-inositol hexakisphosphate (phytate, "IP6") to myo-inositol-pentakisphosphate ("IP5") and phosphate ("P"). While certain phytases may, to a much lower extent, however, catalyze the further degradation of IP5 to "IP4", "IP3” or even lower myo-inositol phosphates such as “IP2" or “IPT, for sure, this is no general feature of phytases, or the speed of these reactions are for practical purposes too low.
  • sequences of highest homology to the phosphatase sequences of the invention are the cDNA encoding a phytase derived from Penicillium sp. CBS 109899, as well as the corresponding amino acid sequence, both of which are disclosed in WO 03/054199 as SEQ ID NO: 2, and SEQ ID NO: 3, respectively.
  • phytase in animal feed is disclosed in, e.g., WO 98/28408 A1.
  • the combination of phytase with acid phosphatase (EC 3.1.3.2) with a view to improving the degradation of phytate is disclosed in, e.g., WO 9403072 A1 and EP 619369 A1. Ullah and
  • SEQ ID NO: 1 is genomic DNA encoding a phosphatase from Eupenicillium pinetorum
  • SEQ ID NO: 2 is the deduced amino acid sequence.
  • SEQ ID NO: 3 is the cDNA corresponding to SEQ ID NO: 1
  • SEQ ID NO: 4 the amino acid sequence deduced from SEQ ID NO: 3.
  • SEQ ID NO: 2 and SEQ ID NO: 4 are identical.
  • the present invention relates to an isolated polypeptide having phosphatase activity, selected from the group consisting of: (a) a polypeptide having an amino acid sequence which has at least 65% identity with amino acids 8 to 523 of SEQ ID NO: 4, (b) a polypeptide which is encoded by a polynucleotide which has at least 65% identity with nucleotides 73 to 1620 of SEQ ID NO: 3, (c) a polypeptide which is encoded by a polynucleotide which hybridizes under at least medium-high stringency conditions with (i) nucleotides 73 to 1620 of SEQ ID NO: 3, or (ii) a complementary strand of (i), (d) a variant comprising a conservative substitution, deletion, and/or insertion of no more than fifty amino acids of amino acids 1 to 523 of SEQ ID NO: 4, and (e) a fragment of amino acids 1 to 523 of SEQ ID NO: 4 which contains at least 400 amino acid residues.
  • the present invention also relates to an isolated polynucleotide encoding a polypeptide having phosphatase activity, selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having an amino acid sequence which has at least 65% identity with amino acids 8 to 523 of SEQ ID NO: 4, (b) a polynucleotide having at least 65% identity with nucleotides 73 to 1620 of SEQ ID NO: 3, (c) a polynucleotide which hybridizes under at least medium-high stringency conditions with nucleotides 73 to 1620 of SEQ ID NO: 3, and (d) a subsequence of nucleotides 52 to 1620 of SEQ ID NO: 3 which contains at least 1200 nucleotides.
  • the present invention also relates to nucleic acid constructs, recombinant expression vectors, and recombinant host cells comprising the polynucleotides.
  • the present invention also relates to methods for producing such polypeptides having phosphatase activity comprising (a) cultivating a recombinant host cell comprising a nucleic acid construct comprising a polynucleotide encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
  • the present invention also relates to methods of using the polypeptides of the invention, preferably together with a phytase, i) in animal feed; and/or ii) for degradation of phytate and/or inositol phosphates in vivo or in vitro.
  • Phosphatase and phytase activity are defined herein as an enzyme activity which catalyzes the hydrolysis of p-Nitrophenyl phosphate di-sodium hexahydrate (pNP-phosphate).
  • pNP-phosphate p-Nitrophenyl phosphate di-sodium hexahydrate
  • a suitable phosphatase activity assay is described in Example 3 herein (pH 5.5, temperature 37°C, substrate pNP-phosphate in a concentration of 1.35 mM).
  • the term “phytase activity” is defined herein as an enzyme activity which catalyzes the hydrolysis of myo-inositol-hexakisphosphate (phytate) to 1 D-myo-inositol-pentakisphosphate and phosphate.
  • phosphatase (or polypeptide) of the invention refers to the phosphatase or polypeptide as such, as well as any use thereof as described herein.
  • the phosphatase of the invention is preferably an acid phosphatase.
  • Acid phosphatases catalyze the hydrolysis of a phosphate monoester to the corresponding alcohol plus phosphate.
  • Acid phosphatase may also be designated phosphate-monoester phospho- hydrolase (acid optimum) - which is the systematical name - or acid phosphomonoesterase; phosphomonoesterase; glycerophosphatase; acid monophosphatase; acid phosphohydrolase; acid phosphomonoester hydrolase; uteroferrin; acid nucleoside diphosphate phosphatase; or orthophosphoric-monoester phosphohydrolase (acid optimum).
  • the pH optimum of the acid phosphatase is below 7.
  • the phosphatase of the invention has a basic pH optimum (pH optimum above 7), or a neutral pH optimum (pH optimum in the range of 6-8).
  • the pH optimum may suitably be determined using the assay of Example 3 (temperature 37°C, substrate pNP-phosphate in a concentration of 1.35 mM), using appropriate buffers of various pH-values, as is known in the art. According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) acid phosphatase is classified EC 3.1.3.2.
  • the phosphatase of the invention is preferably an EC 3.1.3.2 acid phosphatase.
  • 3-phytase myo-inositol hexaphosphate 3-phosphohydrolase, EC 3.1.3.8
  • 4-phytase alternatively designated 6-phytase, based on 1 L- numbering system and not ID-numbering; myo-inositol hexaphosphate 6-phosphohydrolase, EC 3.1.3.26
  • 5-phytase myo-inositol-hexakisphosphate 5-phosphohydrolase
  • the polypeptide of the invention also has phytase activity.
  • the phosphatase of the invention is also a phytase.
  • it is a 3-phytase (EC 3.1.3.8), a 4-phytase (EC 3.1.3.26), or a 5- phytase (EC 3.1.3.72).
  • the polypeptide of the invention catalyzes the degradation of a) myo-inositol-pentakisphosphate (IP5) to myo-inositol-tetrakisphosphate (IP4) and phosphate (P); b) myo-inositol-tetrakisphosphate (IP4) to myo-inositol-trikisphosphate (IP3) and phosphate (P); c) myo-inositol-trikisphosphate (IP3) to myo-inositol-dikisphosphate (IP2) and phosphate (P); d) myo-inositol-dikisphosphate (IP2) to myo-inositol- monokisphosphate (IP1) and phosphate (P); and/or e) myo-inositol monokisphosphate (IP1) to myo-inositol (I) and phosphate (P).
  • IP5 myo-inositol-pent
  • the phytase activity is determined in the unit of FYT, one FYT being the amount of enzyme that liberates 1 micro-mol inorganic ortho- phosphate per min. under the following conditions: pH 5.5; temperature 37 0 C; substrate: sodium phytate (C 6 H 6 O 24 PeNa I2 ) in a concentration of 0.0050 mol/l.
  • Suitable phytase assays are the FYT and FTU assays described in Example 1 of WO 00/20569. FTU is for determining phytase activity in feed and premix.
  • the activity of the polypeptide of the invention can also be determined in assays identical to these FYT and FTU assays, except that the substrate phytate is replaced by IP5, IP4, IP3, IP2, and/or IP1 , as defined above.
  • another pH value may be selected in the FYT and FTU assays, e.g. a pH of 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5.
  • the polypeptides of the present invention have at least 20%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% of the specific activity, measured in the phosphatase units "U" as defined in Example 3, and/or using the phytase units of FYT, of the polypeptide consisting of the amino acid sequence shown as amino acids 1 to 523, 7 to 523, or 8 to 523 of either of SEQ ID NO: 2 or 4.
  • Isolated polypeptide refers to a polypeptide which is at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, most preferably at least 90% pure, and even most preferably at least 95% pure, as determined by SDS-PAGE.
  • substantially pure polypeptide denotes herein a polypeptide preparation which contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which it is natively associated.
  • the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99%, most preferably at least 99.5% pure, and even most preferably 100% pure by weight of the total polypeptide material present in the preparation.
  • polypeptides of the present invention are preferably in a substantially pure form.
  • the polypeptides are in "essentially pure form", i.e., that the polypeptide preparation is essentially free of other polypeptide material with which it is natively associated. This can be accomplished, for example, by preparing the polypeptide by means of well-known recombinant methods or by classical purification methods.
  • Identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity”.
  • identity is determined from an alignment thereof: The two amino acid sequences are aligned using the Needle program from the EMBOSS package (http://emboss.org) version 2.8.0.
  • the Needle program implements the global alignment algorithm described in Needleman, S. B. and Wunsch, C. D. (1970) J. MoI. Biol. 48, 443-453.
  • the substitution matrix used is BLOSUM62, gap opening penalty is 10, and gap extension penalty is 0.5.
  • invention sequence The degree of identity between an amino acid sequence of the present invention
  • the degree of identity between two amino acid sequences, as well as the degree of identity between two nucleotide sequences, may also be, or is, respectively, determined by the program align which is a Needleman-Wunsch alignment (i.e. global alignment), useful for both protein and DNA alignments.
  • the default scoring matrix BLOSUM50 is used for protein alignments, and the default identity matrix is used for DNA alignments.
  • the penalty for the first residue in a gap is -12 for proteins and -16 for DNA, while the penalty for additional residues in a gap is -2 for proteins and -4 for DNA. Align is from the fasta package version v20u6 (see W. R. Pearson and D. J.
  • the percentage of identity of an amino acid sequence of a polypeptide with, or to, amino acids 8 to 523 of SEQ ID NO: 4 is determined by i) aligning the two amino acid sequences using the Needle program, with the BLOSUM62 substitution matrix, a gap opening penalty of 10, and a gap extension penalty of 0.5; ii) counting the number of exact matches in the alignment; iii) dividing the number of exact matches by the length of the shortest of the two amino acid sequences, and iv) converting the result of the division of iii) into percentage.
  • the percentage of identity to, or with, other sequences of the invention such as amino acids 1-523 of SEQ ID NO: 2 are calculated in an analogous way.
  • the percentage of identity between two amino acid sequences, and/or two nucleotide sequences is calculated by the program align, using the BLOSUM50 scoring matrix for protein alignments, and the default identity matrix for DNA alignments.
  • the penalty for the first residue in a gap is -12 for proteins and -16 for DNA, while the penalty for additional residues in a gap is -2 for proteins and -4 for DNA.
  • polypeptide fragment is defined herein as a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of SEQ ID NO: 2, SEQ ID NO: 4, or a homologous sequence thereof, wherein the fragment has phosphatase activity.
  • a fragment contains at least 350 amino acid residues, e.g., amino acids 10 to 360 of SEQ ID NO: 2, more preferably at least 400 amino acid residues, e.g., amino acids 10 to 410 of SEQ ID NO: 2, and most preferably at least 450 amino acid residues, e.g., amino acids 10 to 460 of SEQ ID NO: 2.
  • a fragment contains at least 500 amino acid residues, e.g., amino acids 5 to 505 of SEQ ID NO: 4, more preferably at least 505 amino acid residues, e.g., amino acids 5 to 510 of SEQ ID NO: 4, and most preferably at least 510 amino acid residues, e.g., amino acids 8 to 518 of SEQ ID NO: 4.
  • Subsequence is defined herein as a nucleotide sequence having one or more nucleotides deleted from the 5 1 and/or 3 1 end of SEQ ID NO: 1 , SEQ ID NO: 3, or a homologous sequence thereof.
  • the subsequence contains at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length.
  • the subsequence may also be at least 100, 200, 300, 400, or at least 500 nucleotides in length. Or it may be even longer, e.g., at least 600, 700, 800, or at least 900 nucleotides in length.
  • the subsequence contains at least 100 contiguous nucleotides or more preferably at least 200 contiguous nucleotides.
  • the subsequence contains at least 1050 nucleotides, more preferably at least 1200 nucleotides, and most preferably at least 1350 nucleotides.
  • the subsequence contains at least 1500 nucleotides, more preferably at 1515 nucleotides, and most preferably at least 1530 nucleotides, e.g. those corresponding to the amino acid fragments exemplified above.
  • the subsequence encodes a polypeptide fragment having phosphatase activity.
  • allelic variant denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences.
  • An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
  • substantially pure polynucleotide refers to a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered protein production systems.
  • a substantially pure polynucleotide contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1 %, and even most preferably at most 0.5% by weight of other polynucleotide material with which it is natively associated.
  • a substantially pure polynucleotide may, however, include naturally occurring 5 1 and 3' untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 90% pure, preferably at least 92% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, even more preferably at least 98% pure, most preferably at least 99%, and even most preferably at least 99.5% pure by weight.
  • the polynucleotides of the present invention are preferably in a substantially pure form.
  • the polynucleotides disclosed herein are in "essentially pure form", i.e., that the polynucleotide preparation is essentially free of other polynucleotide material with which it is natively associated.
  • substantially pure polynucleotide is synonymous with the terms “isolated polynucleotide” and “polynucleotide in isolated form.”
  • the polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
  • cDNA is defined herein as a DNA molecule which can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that are usually present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA which is processed through a series of steps before appearing as mature spliced mRNA. These steps include the removal of intron sequences by a process called splicing. cDNA derived from mRNA lacks, therefore, any intron sequences.
  • nucleic acid construct refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature.
  • nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.
  • control sequences is defined herein to include all components, which are necessary or advantageous for the expression of a polynucleotide encoding a polypeptide of the present invention.
  • Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide.
  • control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.
  • the control sequences include a promoter, and transcriptional and translational stop signals.
  • the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.
  • operably linked denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.
  • Coding sequence means a nucleotide sequence, which directly specifies the amino acid sequence of its protein product.
  • the boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG.
  • the coding sequence may a DNA, cDNA, or recombinant nucleotide sequence.
  • expression includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • Expression vector is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the invention, and which is operably linked to additional nucleotides that provide for its expression.
  • host cell includes any cell type which is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct comprising a polynucleotide of the present invention.
  • Modification means herein any chemical modification of the polypeptide consisting of (i) amino acids 1 to 523 of SEQ ID NO: 2, (ii) amino acids 7 to 523 of
  • the modification(s) can be substitution(s), deletion(s) and/or insertions(s) of the amino acid(s) as well as replacement(s) of amino acid side chain(s). Similar modifications can also be applied to other parts, e.g., the signal peptide or propeptide parts, of SEQ ID NO: 2 and 4, to SEQ ID NO: 2 and 4 as such, as well as to the corresponding DNA sequences.
  • artificial variant means a polypeptide having phosphatase activity produced by an organism expressing a modified nucleotide sequence of SEQ ID NO: 1 , or SEQ ID NO: 3.
  • the modified nucleotide sequence is obtained through human intervention by modification of the nucleotide sequence disclosed in SEQ ID NO: 1 , or SEQ ID NO: 3, respectively.
  • in vitro refers to a method of using the polypeptide of the invention outside the animal body, for example in processes for the treatment, or pre-treatment, of animal feed (including human food), or in any process for degrading phytate and/or the various myo-inositol phosphates.
  • in vivo refers to a method of using the polypeptide of the invention inside an animal body, including humans.
  • the polypeptide is catalytically active inside the intestinal tract of the animal, e.g. in the mouth, the stomach, and/or the intestines, such as the upper intestine.
  • the present invention relates to isolated polypeptides having an amino acid sequence which has a degree of identity to amino acids 8 to 523 of SEQ ID NO: 4 (i.e., the mature polypeptide) of at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 97%, which have phosphatase and/or phytase activity (hereinafter "homologous polypeptides").
  • the degree of identity to amino acids 8 to 523 of SEQ ID NO: 4 is at least 63%, 64%, 66%, 67%, 68%, or at least 69%.
  • each of the abovementioned degrees of identity is to amino acids 1 to 523 of SEQ ID NO: 2, amino acids 7 to 523 of SEQ ID NO: 2, amino acids 8 to 523 of SEQ ID NO: 2, amino acids 8 to 523 of SEQ ID NO: 4, amino acids 7 to 523 of SEQ ID NO: 4, and/or amino acids 1 to 523 of SEQ ID NO: 4.
  • the homologous polypeptides have an amino acid sequence which differs by no more than fifty amino acids, preferably by no more than forty amino acids, more preferably by no more than thirty amino acids, even more preferably by no more than twenty amino acids, most preferably by no more than fifteen amino acids, and even most preferably by no more than ten amino acids from amino acids 1 to 523 of SEQ ID NO: 4.
  • the homologous polypeptides have an amino acid sequence which differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from amino acids 1 to 523, 7 to 523, or 8 to 523 of SEQ ID NO: 4.
  • a polypeptide of the present invention preferably i) comprises, or ii) consists of: a) the amino acid sequence of SEQ ID NO: 4 (or SEQ ID NO: 2), b) amino acids 1 to 523 of SEQ ID NO: 4 (or SEQ ID NO: 2), c) amino acids 8 to 523 of SEQ ID NO: 4 (or SEQ ID NO: 2); d) amino acids 7 to 523 of SEQ ID NO: 4 (or SEQ ID NO: 2); or an allelic variant of any one of a), b), c), or d), or a fragment of any one of a), b), c), or d) that has phosphatase activity.
  • the present invention relates to isolated polypeptides having phosphatase and/or phytase activity and being encoded by a polynucleotide which has at least 65% identity with (i) nucleotides 73 to 1620 of SEQ ID NO: 3, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 97%.
  • the degree of identity to nucleotides 73 to 1620 of SEQ ID NO: 3 is at least 63%, 64%, 66%, 67%, 68%, or at least 69%.
  • each of the abovementioned degrees of identity is to (ii) nucleotides 52 to 1620 of SEQ ID NO: 3, (Ni) the cDNA sequence contained in nucleotides 68 to 1758 of SEQ ID NO: 1 , and/or (iv) the cDNA sequence contained in nucleotides 89 to 1758 of SEQ ID NO: 1.
  • the present invention relates to isolated polypeptides having phosphatase and/or phytase activity which are encoded by polynucleotides which hybridize under medium-high stringency conditions, more preferably high stringency conditions, and most preferably very high stringency conditions with (i) nucleotides 73 to 1620 of SEQ ID NO: 3 (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).
  • the hybridization is under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions.
  • the abovementioned hybridizations are to (ii) nucleotides 52 to 1620 of SEQ ID NO: 3, (iii) the cDNA sequence contained in nucleotides 68 to 1758 of SEQ ID NO: 1 , (iv) the cDNA sequence contained in nucleotides 89 to 1758 of SEQ ID NO: 1 , and/or (v) a complementary strand of any one of (i), (ii), (iii), or (iv).
  • nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or a subsequence of any of these, as well as the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or a fragment of any of these may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having phosphatase activity from strains of different genera or species according to methods well known in the art.
  • probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein.
  • nucleic acid probes can be considerably shorter than the entire sequence, but should be at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length. It is, however, preferred that the nucleic acid probe is at least 100 nucleotides in length.
  • the nucleic acid probe may be at least 200 nucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides in length.
  • nucleic acid probes which are at least 600 nucleotides, at least preferably at least 700 nucleotides, more preferably at least 800 nucleotides, or most preferably at least 900 nucleotides in length. Both DNA and RNA probes can be used.
  • the probes are typically labeled for detecting the corresponding gene (for example, with 32 P, 3 H, 35 S, biotin, or avidin). Such probes are encompassed by the present invention.
  • a genomic DNA or cDNA library prepared from such other organisms may, therefore, be screened for DNA which hybridizes with the probes described above and which encodes a polypeptide having phosphatase activity.
  • Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques.
  • DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material.
  • the carrier material is used in a Southern blot.
  • hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to the nucleotide sequence shown in SEQ ID NO: 1 , SEQ ID NO: 3, their complementary strands, or subsequences of any of these, under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using X-ray film.
  • the nucleic acid probe is polynucleotides (i)-(v) mentioned above.
  • the nucleic acid probe is a polynucleotide sequence which encodes the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or a subsequence thereof.
  • the nucleic acid probe is SEQ ID NO: 1 , or SEQ ID NO: 3.
  • the nucleic acid probe is the mature polypeptide coding region of SEQ ID NO: 1 , or SEQ ID NO: 3.
  • nucleic acid probes are the complementary strands of i) the first 500 nucleotides of SEQ ID NO: 3, ii) the last 500 nucleotides of SEQ ID NO: 3, and iii) the nucleotides inbetween i) and ii).
  • very low to very high stringency conditions are defined as prehybridization and hybridization at 42 0 C in 5X SSPE, 0.3% SDS, 200 ⁇ g/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.
  • the carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS preferably at least at 45°C (very low stringency), more preferably at least at 50 0 C (low stringency), more preferably at least at 55 0 C (medium stringency), more preferably at least at 60°C (medium-high stringency), even more preferably at least at 65°C (high stringency), and most preferably at least at 70 0 C (very high stringency).
  • 2X SSC 0.2% SDS preferably at least at 45°C (very low stringency), more preferably at least at 50 0 C (low stringency), more preferably at least at 55 0 C (medium stringency), more preferably at least at 60°C (medium-high stringency), even more preferably at least at 65°C (high stringency), and most preferably at least at 70 0 C (very high stringency).
  • the wash is conducted using 0.2X SSC, 0.2% SDS preferably at least at 45 0 C (very low stringency), more preferably at least at 50 0 C (low stringency), more preferably at least at 55°C (medium stringency), more preferably at least at 60 0 C (medium-high stringency), even more preferably at least at 65°C (high stringency), and most preferably at least at 70°C (very high stringency).
  • the wash is conducted using 0.1 X SSC, 0.2% SDS preferably at least at 45°C (very low stringency), more preferably at least at 50 0 C (low stringency), more preferably at least at 55°C (medium stringency), more preferably at least at 60 0 C (medium-high stringency), even more preferably at least at 65°C (high stringency), and most preferably at least at 7O 0 C (very high stringency).
  • the wash is conducted using 0.02X SSC, 0.2% SDS preferably at least at 45 0 C (very low stringency), more preferably at least at 50°C (low stringency), more preferably at least at 55°C (medium stringency), more preferably at least at 60 0 C (medium-high stringency), even more preferably at least at 65°C (high stringency), and most preferably at least at 7O 0 C (very high stringency).
  • stringency conditions are defined as prehybridization, hybridization, and washing post- hybridization at about 5°C to about 10 0 C below the calculated T m using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCI, 0.09 M Tris-HCI pH 7.6, 6 mM EDTA, 0.5% NP-40, 1X Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures.
  • the carrier material is washed once in 6X SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6X SSC at 5°C to 10°C below the calculated T m .
  • the present invention relates to artificial variants comprising a conservative substitution, deletion, and/or insertion of one or more amino acids of amino acids 1 to 523 of SEQ ID NO: 4.
  • the artificial variants comprise a conservative substitution, deletion, and/or insertion of one or more amino acids in the sequence of SEQ ID NO: 2, amino acids 1 to 523 of SEQ ID NO: 2, amino acids 7 to 523 of SEQ ID NO: 2, amino acids 8 to 523 of SEQ ID NO: 2, SEQ ID NO: 4, amino acids 7 to 523 of SEQ ID NO: 4, or amino acids 8 to 523 of SEQ ID NO: 4.
  • amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl- terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
  • conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine).
  • basic amino acids arginine, lysine and histidine
  • acidic amino acids glutmic acid and aspartic acid
  • polar amino acids glutamine and asparagine
  • hydrophobic amino acids leucine, isoleucine and valine
  • aromatic amino acids phenylalanine, tryptophan and tyrosine
  • small amino acids glycine, alanine, serine, threonine and methionine.
  • non-standard amino acids such as 4- hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues.
  • "Unnatural amino acids” have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids.
  • Unnatural amino acids can be chemically synthesized, and preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.
  • amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered.
  • amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
  • Essential amino acids in the parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (i.e., phosphatase activity) to identify amino acid residues that are critical to the activity of the molecule.
  • biological activity i.e., phosphatase activity
  • the active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. MoI. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64.
  • the identities of essential amino acids can also be inferred from analysis of identities with polypeptides which are related to a polypeptide according to the invention.
  • Single or multiple amino acid substitutions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625.
  • Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochem. 30:10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).
  • Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells.
  • Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
  • the total number of amino acid substitutions, deletions and/or insertions of amino acids 1 to 523 of SEQ ID NO: 4 or 2 is 10, preferably no more than fifty, more preferably no more than forty, more preferably no more than thirty, more preferably at most thirty-five, more preferably at most thirty, more preferably at most twenty-five, even more preferably at most twenty, most preferably at most fifteen, and even most preferably at most 12.
  • the total number of amino acid substitutions, deletions and/or insertions of amino acids 8 to 523 of SEQ ID NO: 4 or 2 is no more than 10, preferably no more than 9, more preferably no more than 8, more preferably no more than 7, more preferably at most 6, more preferably at most 5, more preferably at most 4, even more preferably at most 3, most preferably at most 2, and even most preferably 1.
  • a polypeptide of the present invention may be obtained from microorganisms of any genus.
  • the term "obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a nucleotide sequence is produced by the source or by a strain in which the nucleotide sequence from the source has been inserted.
  • the polypeptide obtained from a given source is secreted extracellularly.
  • a polypeptide of the present invention may be a bacterial polypeptide.
  • the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus polypeptide; or a Streptomyces polypeptide; or a gram negative bacterial polypeptide, e.g., an E. coli or a Pseudomonas sp. polypeptide.
  • a polypeptide of the present invention may also be a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or most preferably a filamentous fungal polypeptide such as an Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Eupenicillium, Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma polypeptide.
  • yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or
  • the polypeptide derives from a filamentous fungus of the phylum of Ascomycota, of the Ascomycetes division, of the sub-division Eurotiomycetidae, of the order Eurotiales, of the family Trichocomaceae; or of the genus Eupenicillium, such as polypeptides derived from Eupenicillium abidjanum, Eupenicillium alutaceum, Eupenicillium anatolicum, Eupenicillium baarnense, Eupenicillium bovifimosum, Eupenicillium brefeldianum, Eupenicillium catenatum, Eupenicillium cinnamopurpureum, Eupenicillium crustaceum, Eupenicillium egyptiacum Eupenicillium ehrlichii, Eupenicillium erubescens, Eupenicillium hirayamae, Eupenicillium inusitatum, Eupenicillium
  • Eupenicillium katangense Eupenicillium lapidosum
  • Eupenicillium secretion
  • Eupenicillium secretion
  • Eupenicillium levitum Eupenicillium meridianum
  • Eupenicillium molle Eupenicillium ochrosalmoneum
  • Eupenicillium osmophilum Eupenicillium parvum
  • Eupenicillium pinetorum Eupenicillium reticulisporum
  • Eupenicillium rubidurum Eupenicillium senticosum
  • Eupenicillium shearii Eupenicillium stolkiae, Eupenicillium terrenum, Eupenicillium tropicum, Eupenicillium tularense, Eupenicillium zonatum, or Eupenicillium sp.
  • the polypeptide is an Eupenicillium pinetorum polypeptide, and most preferably an Eupenicillium pinetorum CBS 116641 polypeptide, e.g., the polypeptide of amino acids -17 to 523, 1 to 523, 7 to 523, or 8 to 523 of either of SEQ ID NOs: 2 or 4.
  • ATCC American Type Culture Collection
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art.
  • the polynucleotide may then be obtained by similarly screening a genomic or cDNA library of another microorganism. Once a polynucleotide sequence encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques which are well known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
  • Polypeptides of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof.
  • a fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide to a nucleotide sequence (or a portion thereof) of the present invention.
  • Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.
  • the present invention also relates to isolated polynucleotides having a nucleotide sequence which encodes a polypeptide of the present invention.
  • Preferred nucleotide sequences are set forth in SEQ ID NO: 1 and SEQ ID NO: 3.
  • the nucleotide sequence is obtainable from Eupenicillium pinetorum CBS 116641 by use of the primers specified in example 3 (SEQ ID NO: 6 and SEQ ID NO: 7).
  • the nucleotide sequence is the mature polypeptide coding region of SEQ ID NO: 1 or SEQ ID NO: 3.
  • the present invention also encompasses nucleotide sequences which encode a polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or the mature polypeptides of any of these, which differ from SEQ ID NO: 1 or SEQ ID NO: 3 by virtue of the degeneracy of the genetic code.
  • the present invention also relates to subsequences of SEQ ID NO: 1 and SEQ ID NO: 3 which encode fragments of SEQ ID NO: 2 or SEQ ID NO: 4 that have phosphatase activity.
  • the present invention also relates to mutant polynucleotides comprising at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, in which the mutant nucleotide sequence encodes a polypeptide which consists of amino acids 1 to 523, 7 to 523, or 8 to 523, of SEQ ID NO: 2, or SEQ ID NO: 4.
  • the techniques used to isolate or clone a polynucleotide encoding a polypeptide include isolation from genomic DNA, preparation from cDNA, or a combination thereof.
  • the cloning of the polynucleotides of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., lnnis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York.
  • nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used.
  • LCR ligase chain reaction
  • LAT ligated activated transcription
  • NASBA nucleotide sequence-based amplification
  • the polynucleotides may be cloned from a strain of Eupenicilium, or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleotide sequence.
  • the present invention also relates to polynucleotides having nucleotide sequences which have a degree of identity to (i) nucleotides 73 to 1620 of SEQ ID NO: 3 (i.e., the mature polypeptide coding part) of at least 65%, preferably at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97% identity, which encode an active polypeptide.
  • the degree of identity to nucleotides 73 to 1620 of SEQ ID NO: 3 is at least 63%, 64%, 66%, 67%, 68%, or at least 69%.
  • each of the abovementioned degrees of identity is to (ii) nucleotides 52 to 1620 of SEQ ID NO: 3, (iii) the cDNA sequence contained in nucleotides 68 to 1758 of SEQ ID NO: 1 , or (iv) the cDNA sequence contained in nucleotides 89 to 1758 of SEQ ID NO: 1.
  • Modification of a nucleotide sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide.
  • the term "substantially similar" to the polypeptide refers to non-naturally occurring forms of the polypeptide.
  • These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., artificial variants that differ in specific activity, thermostability, pH optimum, or the like.
  • the variant sequence may be constructed on the basis of the nucleotide sequence presented as the polypeptide encoding region of SEQ ID NO: 1 , e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleotide sequence, but which correspond to the codon usage of the host organism intended for production of the phosphatase, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence.
  • nucleotide substitution see, e.g., Ford et al., 1991 , Protein Expression and Purification 2: 95-107.
  • amino acid residues essential to the activity of the polypeptide encoded by an isolated polynucleotide of the invention may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for phosophatase activity to identify amino acid residues that are critical to the activity of the molecule.
  • Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see, e.g., 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 also relates to isolated polynucleotides encoding a polypeptide of the present invention, which hybridize under medium-high stringency conditions, more preferably high stringency conditions, and most preferably very high stringency conditions with (i) nucleotides 73 to 1620 of SEQ ID NO: 3; or allelic variants and subsequences thereof (Sambrook et al., 1989, supra), as defined herein.
  • the hybridization is under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions.
  • the abovementioned hybridizations are to (ii) nucleotides 52 to 1620 of SEQ ID NO: 3, (iii) the cDNA sequence contained in nucleotides 68 to 1758 of SEQ ID NO: 1 , (iv) the cDNA sequence contained in nucleotides 89 to 1758 of SEQ ID NO: 1 , and/or (v) a complementary strand of any one of (i), (ii), (iii), or (iv).
  • the present invention also relates to nucleic acid constructs comprising an isolated polynucleotide of the present invention operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
  • An isolated polynucleotide encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide's sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well known in the art.
  • the control sequence may be an appropriate promoter sequence, a nucleotide sequence which is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention.
  • the promoter sequence contains transcriptional control sequences which mediate the expression of the polypeptide.
  • the promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venen
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.
  • Preferred terminators for filamentous fungal host cells are obtained from the genes for
  • Aspergillus oryzae TAKA amylase Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin- like protease.
  • the control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell.
  • the leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention.
  • Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans those phosphate isomerase.
  • the control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the nucleotide sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenbsine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.
  • Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase.
  • the control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway.
  • the 5' end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide.
  • the 5' end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence.
  • the foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region.
  • the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide.
  • any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.
  • Effective signal peptide coding regions for filamentous fungal host cells are the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.
  • the signal peptide coding region is nucleotides 17 to 67 of SEQ ID NO: 1 which encodes amino acids -17 to -1 of SEQ ID NO: 2 (or of SEQ ID NO: 4).
  • the control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide.
  • the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
  • a propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • the propeptide coding region may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836).
  • the propeptide coding region is nucleotides 68-85, or 68-88 of SEQ ID NO: 1 which encodes amino acids 1 to 6, or 1 to 7, respectively, of SEQ ID NO: 2 (and SEQ ID NO: 4).
  • the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.
  • regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell.
  • regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences.
  • Other examples of regulatory sequences are those which allow for gene amplification.
  • these include the dihydrofolate reductase gene which is amplified in the presence of methotrexate, and the metallothionein genes which are amplified with heavy metals.
  • the nucleotide sequence encoding the polypeptide would be operably linked with the regulatory sequence.
  • the present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals.
  • the various nucleic acids and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites.
  • a nucleotide sequence of the present invention may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression.
  • the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
  • the recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleotide sequence.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vectors may be linear or closed circular plasmids.
  • the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
  • the vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells.
  • a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.
  • Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.
  • the vectors of the present invention preferably contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
  • the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or nonhomologous recombination.
  • the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s).
  • the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity with the corresponding target sequence to enhance the probability of homologous recombination.
  • the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell.
  • the integrational elements may be non-encoding or encoding nucleotide sequences.
  • the vector may be integrated into the genome of the host cell by non-homologous recombination.
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
  • the origin of replication may be any plasmid replicator mediating autonomous replication which functions in a cell.
  • the term "origin of replication" or “plasmid replicator” is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo.
  • AMA1 and ANSI examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et al., 1991 , Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.
  • More than one copy of a polynucleotide of the present invention may be inserted into the host cell to increase production of the gene product.
  • An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
  • the present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention, which are advantageously used in the recombinant production of the polypeptides.
  • a vector comprising a polynucleotide of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier.
  • the term "host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
  • the host cell may be a unicellular microorganism, e.g., a prokaryote, or a non- unicellular microorganism, e.g., a eukaryote.
  • Useful unicellular microorganisms are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell.
  • the host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
  • the host cell is a fungal cell.
  • "Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).
  • the fungal host cell is a yeast cell.
  • yeast as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi lmperfecti (Blastomycetes).
  • the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.
  • the fungal host cell is a filamentous fungal cell.
  • “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).
  • the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
  • the filamentous fungal host cell is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell.
  • Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81 : 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N.
  • the present invention also relates to methods for producing a polypeptide of the present invention, comprising (a) cultivating a cell, which in its wild-type form is capable of producing the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
  • the cell is of the genus Eupenicillium, and more preferably Eupenicillium pinetorum, e.g., Eupenicillium pinetorum CBS 116641.
  • the present invention also relates to methods for producing a polypeptide of the present invention, comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
  • the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods well known in the art.
  • the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
  • the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
  • the polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme (phosphatase, and/or phytase) assay may be used to determine the activity of the polypeptide as described herein.
  • the resulting polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
  • polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J. -C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
  • chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelectric focusing
  • differential solubility e.g., ammonium sulfate precipitation
  • SDS-PAGE or extraction
  • the present invention also relates to a transgenic plant, plant part, or plant cell which has been transformed with a nucleic acid sequence encoding a polypeptide of the invention so as to express and produce the polypeptide.
  • the polypeptide may be recovered from the plant or plant part.
  • the plant or plant part containing the recombinant polypeptide may be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor.
  • the polypeptide is targeted to the endosperm storage vacuoles in seeds.
  • This can be obtained by synthesizing it as a precursor with a suitable signal peptide, see Horvath et al in PNAS, Feb. 15, 2000, vol. 97, no. 4, p. 1914-1919.
  • the transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot) or engineered variants thereof.
  • monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, triticale (stabilized hybrid of wheat (Triticum) and rye (Secale), and maize (corn).
  • dicot plants are tobacco, legumes, such as sunflower (Helianthus), cotton (Gossypium), lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.
  • Low-phytate plants as described e.g. in US patent no. 5,689,054 and US patent no. 6,111 ,168 are examples of engineered plants.
  • plant parts are stem, callus, leaves, root, fruits, seeds, and tubers, as well as the individual tissues comprising these parts, e.g. epidermis, mesophyll, parenchyma, vascular tissues, meristems. Also specific plant cell compartments, such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part. Likewise, plant parts such as specific tissues and cells isolated to facilitate the utilisation of the invention are also considered plant parts, e.g. embryos, endosperms, aleurone and seed coats.
  • the transgenic plant or plant cell expressing a polypeptide of the present invention may be constructed in accordance with methods known in the art. Briefly, the plant or plant cell is constructed by incorporating one or more expression constructs encoding a polypeptide of the present invention into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.
  • the expression construct is a nucleic acid construct which comprises a nucleic acid sequence encoding a polypeptide of the present invention operably linked with appropriate regulatory sequences required for expression of the nucleic acid sequence in the plant or plant part of choice.
  • the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).
  • regulatory sequences such as promoter and terminator sequences and optionally signal or transit sequences are determined, for example, on the basis of when, where, and how the polypeptide is desired to be expressed.
  • the expression of the gene encoding a polypeptide of the present invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific cell compartment, tissue or plant part such as seeds or leaves.
  • Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiology 86: 506.
  • the following promoters may be used: The 35S-CaMV promoter (Franck et al., 1980, Cell 21 : 285-294), the maize ubiquitin 1 (Christensen AH, Sharrock RA and Quail 1992. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation), or the rice actin 1 promoter (Plant Mo. Biol. 18, 675-689.; Zhang W, McElroy D. and Wu R 1991 , Analysis of rice Act1 5' region activity in transgenic rice plants. Plant Cell 3, 1155-1165).
  • Organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant MoI. Biol.
  • a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al., 1998, Journal of Plant Physiology 152: 708-711 ), a promoter from a seed oil body protein (Chen et al., 1998, Plant and Cell Physiology 39: 935-941 ), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772.
  • a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39: 885-889)
  • the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiology 102: 991-1000, the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26: 85-93), or the aldP gene promoter from rice (Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Molecular Biology 22: 573-588).
  • the promoter may be inducible by abiotic treatments such as temperature, drought or alterations in salinity or inducible by exogenously applied substances that activate the promoter, e.g. ethanol, oestrogens, plant hormones like ethylene, abscisic acid, gibberellic acid, and/or heavy metals.
  • abiotic treatments such as temperature, drought or alterations in salinity or inducible by exogenously applied substances that activate the promoter, e.g. ethanol, oestrogens, plant hormones like ethylene, abscisic acid, gibberellic acid, and/or heavy metals.
  • a promoter enhancer element may also be used to achieve higher expression of the polypeptide in the plant.
  • the promoter enhancer element may be an intron which is placed between the promoter and the nucleotide sequence encoding a polypeptide of the present invention.
  • Xu et al., 1993, supra disclose the use of the first intron of the rice actin 1 gene to enhance expression.
  • codon usage may be optimized for the plant species in question to improve expression (see Horvath et al referred to above).
  • the selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.
  • the nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).
  • Agrobacterium tumefaciens-mediated gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38), and it can also be used for transforming monocots, although other transformation methods are more often used for these plants.
  • the method of choice for generating transgenic monocots, supplementing the Agrobacterium approach is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli 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 monocots is based on protoplast transformation as described by Omirulleh et al., 1993, Plant Molecular Biology 21 : 415-428.
  • the transformants having incorporated therein the expression construct are selected and regenerated into whole plants according to methods well-known in the art.
  • the transformation procedure is designed for the selective elimination of selection genes either during regeneration or in the following generations by using e.g. co- transformation with two separate T-DNA constructs or site specific excision of the selection gene by a specific recombinase.
  • the present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a transgenic plant or a plant cell comprising a nucleic acid sequence encoding a polypeptide of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
  • the present invention also relates to a transgenic, non-human animal and products or elements thereof, examples of which are body fluids such as milk and blood, organs, flesh, and animal cells.
  • body fluids such as milk and blood, organs, flesh, and animal cells.
  • Techniques for expressing proteins, e.g. in mammalian cells are known in the art, see e.g. the handbook Protein Expression: A Practical Approach, Higgins and Hames (eds), Oxford University Press (1999), and the three other handbooks in this series relating to Gene Transcription, RNA processing, and Post-translational Processing.
  • to prepare a transgenic animal selected cells of a selected animal are transformed with a nucleic acid sequence encoding a polypeptide of the present invention so as to express and produce the polypeptide.
  • the polypeptide may be recovered from the animal, e.g. from the milk of female animals, or the polypeptide may be expressed to the benefit of the animal itself, e.g. to assist the animal's digestion. Examples of animals are mentioned below in the section headed Animal Feed.
  • a gene encoding the polypeptide may be inserted into the fertilized eggs of an animal in question, e.g. by use of a transgene expression vector which comprises a suitable milk protein promoter, and the gene encoding polypeptide.
  • the transgene expression vector is microinjected into fertilized eggs, and preferably permanently integrated into the chromosome. Once the egg begins to grow and divide, the potential embryo is implanted into a surrogate mother, and animals carrying the transgene are identified. The resulting animal can then be multiplied by conventional breeding.
  • the polypeptide may be purified from the animal's milk, see e.g. Meade, H. M.
  • the transgene in order to produce a transgenic non-human animal that carries in the genome of its somatic and/or germ cells a nucleic acid sequence including a heterologous transgene construct including a transgene encoding the polypeptide, the transgene may be operably linked to a first regulatory sequence for salivary gland specific expression of the polypeptide, as disclosed in WO 00/064247.
  • compositions and Uses in a still further aspect, relates to compositions comprising a polypeptide of the present invention.
  • polypeptide compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition.
  • the polypeptide composition may be in the form of a granulate or a microgranulate.
  • the polypeptide to be included in the composition may be stabilized in accordance with methods known in the art.
  • the present invention is directed to methods for using the polypeptides having phosphatase and/or phytase activity in animal feed, as well as to feed compositions and feed additives comprising the polypeptides of the invention.
  • animal includes all animals, including human beings. Examples of animals are non-ruminants, and ruminants. Ruminant animals include, for example, animals such as sheep, goats, horses, and cattle, e.g. beef cattle, cows, and young calves. In a particular embodiment, the animal is a non-ruminant animal. Non-ruminant animals include mono-gastric animals, e.g.
  • pigs or swine including, but not limited to, piglets, growing pigs, and sows
  • poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); young calves; and fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns).
  • feed or feed composition means any compound, preparation, mixture, or composition suitable for, or intended for intake by an animal.
  • human food or food composition means any compound, preparation, mixture, or composition suitable for, or intended for intake by a human being.
  • the polypeptide can be fed to the animal before, after, or simultaneously with the diet.
  • the latter is preferred.
  • the polypeptide, in the form in which it is added to the feed, or when being included in a feed additive is well defined.
  • Well-defined means that the polypeptide preparation is at least 50% pure as determined by Size-exclusion chromatography (see Example 12 of WO 01/58275).
  • the polypeptide preparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined by this method.
  • a well-defined polypeptide preparation is advantageous. For instance, it is much easier to dose correctly to the feed a polypeptide that is essentially free from interfering or contaminating other polypeptides.
  • dose correctly refers in particular to the objective of obtaining consistent and constant results, and the capability of optimizing dosage based upon the desired effect.
  • polypeptide For the use in animal feed, however, the polypeptide need not be that pure; it may e.g. include other enzymes, in which case it could be termed a polypeptide, or phosphatase, preparation.
  • the polypeptide preparation can be (a) added directly to the feed (or used directly in a treatment process of vegetable proteins), or (b) it can be used in the production of one or more intermediate compositions such as feed additives or premixes that is subsequently added to the feed (or used in a treatment process).
  • the degree of purity described above refers to the purity of the original polypeptide preparation, whether used according to (a) or (b) above.
  • Polypeptide preparations with purities of this order of magnitude are in particular obtainable using recombinant methods of production, whereas they are not so easily obtained and also subject to a much higher batch-to-batch variation when the polypeptide is produced by traditional fermentation methods.
  • polypeptide of the invention may be used together with other enzymes, in particular one or more phytases.
  • the polypeptide of the invention is combined with a (at least one) phytase, and/or used in combination with a (at least one) phytase.
  • the phytase may be a wildtype phytase or any mutant or variant thereof, such as those disclosed in, e.g., WO 00/43503, as well as completely synthetic phytases, such as those disclosed in, e.g., EP 897985.
  • the phytase is i) a fungal phytase, such as a basidiomycete phytase (WO 98/28409), or an ascomycete phytase, such as a phytase derived from Aspergillus (Yamada et al., 1986, Agric. Biol. Chem. 322:1275-1282; Piddington et al.,
  • a bacterial phytase such as a phytase derived from Bacillus (Paver and Jagannathan, 1982, Journal of
  • a plant or animal phytase such as wheat-bran phytase (Thomlinson et al, Biochemistry, 1 (1962), 166-171), or the IiIIy pollen phytase (Barrientos et al, Plant. Physiol., 106 (1994), 1489-1495).
  • the phytase is selected from phytases having at least 50%, preferably at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identity to either of amino acids 23-432 of SEQ ID NO: 10 (E.coli appA, SPTREMBL Q8GN88), amino acids 1-410 of SEQ ID NO: 12 (Peniophora phytase, WO 98/28408), amino acids 1-411 of SEQ ID NO: 14 (Citrobacter braakii phytase, PCT/DK2005/000632), amino acids 1-410 of SEQ ID NO: 16 (Citrobacter gillenii phytase, PCT/DK2005/000631 ), or amino acids 1-411 of SEQ ID NO: 17 (Citrobacter braakii KCCM 10427 phytase,
  • Examples thereof are the phytases having the specific sequences of SEQ ID NOs: 10, 12, 14, 16, and 17, as well as homologues, fragments, muteins, variants and/or modifications of any of these which retain phytase activity. Additional preferred phytases are those having at least 50%, preferably at least 55, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identity to the consensus phytase (SEQ ID NO: 31 of WO 00/43503), or the Aspergillus ficuum/niger phytase (amino acids 1-444 of SEQ ID NO: 32 of US Patent No. 5,436,156).
  • the term "used in combination with” or simply “together with” means that the two enzymes are both active. Their activity can be simultaneous, i.e. the two enzymes are active at the same time, or sequential, i.e. one is acting first, the second subsequently.
  • the first type may be active or non-active, when the second type excerts its effect, viz. a simultaneous and sequential, and a pure sequential action, respectively.
  • the polypeptide of the invention is preferably acting subsequently to the phytase, which is acting first.
  • the polypeptide of the invention in a dosage of no more than 1000 FYT/ml, preferably no more than 900, 800, 700, 600, 500, 400, 300, 200, 100, or no more than 50 FYT/ml, when acting at 40 0 C together with 500 FYT/ml of the Peniophora phytase, improves the degradation of phytate (2 mM sodium phytate in 50 mM Glycine buffer pH 4) by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, or at least 85%, after incubation in 5, 10, or 30 minutes, by reference to the concentration of IP6, IP5, IP4, IP3, or IP2.
  • a preferred incubation time is 5 minutes.
  • the polypeptide of the invention is dosed 134 ul/ml of the supernatant of Example 1 (the supernatant being prepared by as follows: Cultivation of E.
  • the polypeptide of the invention releases at least 10% (or at least 12%, 15%, 20%, 25%, 30%, or 35%) of all phosphorous from the phytic acid content of a substrate consisting of 30% Soy Bean Meal (SBM), 70% ground maize and including CaCI 2 to a final concentration of 5 g calcium / kg substrate; as a result of the following reaction steps:
  • step (vi) determining the concentration of phosphorous (P) bound in IP (sum of IP6, IP5, IP4 and IP3) in the reaction mixture resulting from step (v), preferably by HPIC, for example as described in Example 4; and
  • polypeptide of the invention in combination with a phytase, releases at least 40% (or at least
  • step (vi) determining the concentration of phosphorous (P) bound in IP (sum of IP6, IP5, IP4 and IP3) in the reaction mixture resulting from step (v), preferably by HPIC, for example as described in Example 4; and
  • the total amount of phosphorous released is bigger than the sum of that released by the phytase and the polypeptide of the invention when used separately. This may be due to the different substrate specificities and the consequential possibility for sequential activity, cf. Example 10 herein.
  • the activity of the polypeptide of the invention on the substrate IP6 is at most 50% (or at most 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of the activity on IP4; and/or (ii) the activity of the polypeptide of the invention on the substrate IP5 is at most 60% (or at most 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%) of the activity on IP4.
  • the phosphatase activity of the polypeptide of the invention in the pH-range of 3.0-5.0 is at least 35% (or a least 40%, 45%, 50%, 55%, or 60%) of the phosphatase activity of the same polypeptide at pH 3.5 (or at the pH-optimum), using p-Nitrophenyl phosphate di-sodium hexahydrate (pNP-phosphate) as a substrate, and at a temperature of 37°C.
  • pNP-phosphate p-Nitrophenyl phosphate di-sodium hexahydrate
  • the substrate concentration is 1.35 mM
  • the incubation time is 15 minutes
  • the activity is measured using the phosphatase microtiter assay of Example 3 herein (at a desired pH- value).
  • vegetable proteins refers to any compound, composition, preparation or mixture that includes at least one protein derived from or originating from a vegetable, including modified proteins and protein-derivatives.
  • the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60% (w/w).
  • Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example materials from plants of the families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal and rapeseed meal.
  • Fabaceae Leguminosae
  • Cruciferaceae Chenopodiaceae
  • Poaceae such as soy bean meal, lupin meal and rapeseed meal.
  • the vegetable protein source is material from one or more plants of the family Fabaceae, e.g. soybean, lupine, pea, or bean.
  • the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa.
  • Other examples of vegetable protein sources are rapeseed, sunflower seed, cotton seed, and cabbage. Soybean is a preferred vegetable protein source.
  • vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, triticale, and sorghum.
  • the treatment is a pre-treatment of animal feed or vegetable proteins for use in animal feed.
  • the polypeptide of the invention when added to animal feed leads to an improved nutritional value of the feed, in particular due to the fact that the phytate present in the animal feed is degraded more efficiently. But also, e.g. the growth rate and/or the weight gain and/or the feed conversion (i.e. the weight of ingested feed relative to weight gain) of the animal may be improved. This may be due to, in turn, one or more of the following effects:
  • the phosphate moieties of phytic acid chelates divalent and trivalent cations such as metal ions, i.a. the nutritionally essential ions of calcium, iron, zinc and magnesium as well as the trace minerals mangane, copper and molybdenum.
  • the phytic acid also to a certain extent binds proteins by electrostatic interaction.
  • pi the positively charged protein binds directly with phytate.
  • the negatively charged protein binds via metal ions to phytate.
  • Phytic acid and its salts, phytates are often not metabolized, since they are not absorbable from the gastro intestinal system, i.e. neither the phosphorous thereof, nor the chelated metal ions, nor the bound proteins are nutritionally available. Accordingly, since phosphorus is an essential element for the growth of all organisms, food and feed preparations need to be supplemented with inorganic phosphate. Quite often also the nutritionally essential ions such as iron and calcium, must be supplemented. And, besides, the nutritional value of a given diet decreases, because of the binding of proteins by phytic acid.
  • the polypeptide of the invention can be added to the feed in any form, be it as a relatively pure enzyme, or in admixture with other components intended for addition to animal feed, i.e. in the form of animal feed additives, such as the so-called pre-mixes for animal feed.
  • animal feed additives such as the so-called pre-mixes for animal feed.
  • the present invention relates to compositions for use in animal feed, such as animal feed, and animal feed additives, e.g. premixes.
  • the animal feed additives of the invention contain at least one fat soluble vitamin, and/or at least one water soluble vitamin, and/or at least one trace mineral. It may also contain at least one macro mineral.
  • feed-additive ingredients are colouring agents, e.g.
  • carotenoids such as beta-carotene, astaxanthin, and lutein; aroma compounds; stabilisers; antimicrobial peptides; polyunsaturated fatty acids; reactive oxygen generating species; and/or at least one other enzyme selected from amongst acid phosphatase (EC 3.1.3.2); phytase (EC 3.1.3.8, EC 3.1.3.26, EC 3.1.3.72); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4.-.-), phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (EC 3.1.4.3); phospholipase D (EC 3.1.4.4); amylase such as, for example, alpha-amylase (EC
  • antimicrobial peptides examples include CAP18, Leucocin A, Tritrpticin,
  • Protegrin-1 Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins, and Statins, including the compounds and polypeptides disclosed in WO 03/044049 and WO 03/048148, as well as variants or fragments of the above that retain antimicrobial activity.
  • AFP's antifungal polypeptides
  • Aspergillus giganteus and Aspergillus niger peptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94/01459 and WO 02/090384.
  • polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma- linoleic acid.
  • reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.
  • the animal feed additive of the invention is intended for being included (or prescribed as having to be included) in animal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05 to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This is so in particular for premixes.
  • fat soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin K, e.g. vitamin K3.
  • water soluble vitamins are vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g. Ca-D-panthothenate.
  • trace minerals are manganese, zinc, iron, copper, iodine, selenium, and cobalt.
  • Examples of macro minerals are calcium, phosphorus and sodium.
  • the nutritional requirements of these components are listed in Table A of WO 01/58275. Nutritional requirement means that these components should be provided in the diet in the concentrations indicated.
  • the present invention also relates to animal feed compositions.
  • Animal feed compositions or diets have a relatively high content of protein.
  • Poultry and pig diets can be characterized as indicated in Table B of WO 01/58275, columns 2-3.
  • Fish diets can be characterized as indicated in column 4 of this Table B.
  • Furthermore such fish diets usually have a crude fat content of 200-310 g/kg.
  • WO 01/58275 corresponds to US 09/779334 which is hereby incorporated by reference.
  • An animal feed composition according to the invention has a crude protein content of
  • the animal feed composition of the invention has a content of metabolisable energy of 10-30 MJ/kg; and/or a content of calcium of 0.1-200 g/kg; and/or a content of available phosphorus of 0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or a content of methionine plus cysteine of 0.1-150 g/kg; and/or a content of lysine of 0.5-50 g/kg.
  • the content of metabolisable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO 01/58275 (rows 2-5).
  • Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25, i.e.
  • N (g/kg) N (g/kg) x 6.25.
  • the nitrogen content is determined by the Kjeldahl method (A.O.A.C, 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC).
  • Metabolisable energy can be calculated on the basis of the NRC publication 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 & looijen bv, Wageningen. ISBN 90-71463-12-5.
  • the dietary content of calcium, available phosphorus and amino acids in complete animal diets is calculated on the basis of feed tables 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.
  • the animal feed composition of the invention contains at least one vegetable protein or protein source as defined above.
  • the animal feed composition of the invention contains 0-80% maize; and/or 0- 80% sorghum; and/or 0-70% wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or 0-20% whey.
  • Animal diets can e.g. be manufactured as mash feed (non pelleted) or pelleted feed.
  • the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question.
  • Enzymes can be added as solid or liquid enzyme formulations.
  • a solid enzyme formulation is typically added before or during the mixing step; and a liquid enzyme preparation is typically added after the pelleting step.
  • the enzyme may also be incorporated in a feed additive or premix.
  • the final enzyme concentration in the diet is within the range of 0.01-200 mg enzyme protein per kg diet, for example in the range of 0.5-10 or 5-30 mg enzyme protein per kg animal diet.
  • the polypeptide should of course be applied in an effective amount, i.e. in an amount adequate for improving the degradation of phytate in the diet, and/or for improving the nutritional value of the feed.
  • the enzyme is administered in one or more of the following amounts (dosage ranges): 0.01-200; 0.01-100; 0.5-100; 1-50; 5- 100; 1-10; 10-100; 0.05-50; or 0.10-10 - all these ranges being in mg enzyme protein per kg feed (ppm).
  • the phosphatase is purified from the feed composition, and the specific activity of the purified phosphatase is determined using a relevant assay such as the assay of Example 3.
  • the phosphatase activity of the feed composition as such is also determined using the same assay, and on the basis of these two determinations, the dosage in mg phosphatase enzyme protein per kg feed is calculated.
  • Chemicals used as buffers and substrates were commercial products of at least reagent grade.
  • Wheat bran is commercially available from, e.g., Cerealia Mills Moellegade 12, DK- 7100 Vejle, Denmark, DK.
  • ROFEC dried corn residual is commercially available from, e.g., Rocquette Freres FF-62136 Lestrem, France.
  • SOLCAFLOC (DICACEL) cellulose powder is commercially available from Dicalite Europe nv, Scheepzatestraat 100, 9000 Gent, Belgium. Shake flasks were inoculated with the Eupenicillium pinetorum CBS 116641 growing in
  • Example 2 Purification of the phosphatase of Eupenicillium pinetorum Purification
  • Example 2 3 liter culture supernatant from Example 1 was filtered through a 0.45 ⁇ m filter and concentrated 8-fold by ultra filtration through a 10 kDa membrane. The concentrate was washed with water to a final conductivity of 2 mSi. Sample pH was adjusted to pH 4 and the resulting precipitate was removed by centrifugation at 1800xg for 10 min. Supernatant pH was readjusted to pH 7 and filtered though a 0.22 ⁇ m filter.
  • the pooled eluate fraction was adjusted to pH 4 and subjected to a primary cation exchange chromatography on a Resource S (Amersham Biosciences, Uppsala, Sweden) column equilibrated with 20 mM sodium acetate, pH 4.0. Sample loading was followed by a 30 vol. wash with the equilibration buffer. Target protein was eluted with approx. 0.25 M NaCI applied in a linear gradient from 0 til 0.5 M over 20 column volumes in 20 mM sodium acetate, pH 4.0.
  • sample was mixed 1 :1 with a standard SDS-PAGE sample buffer containing dithiothreitol (DTT) in an Eppendorf tube and heated to 95 0 C for 4 minutes. Following the heating a 20 ⁇ l aliquot of the diluted sample was applied to a precast 4-20% SDS polyacrylamide gel (Novex) in parallel with a molecular marker (Mark 12, Novex).
  • DTT dithiothreitol
  • the gel was run in a Xcell Il (Novex) gel apparatus in a Tris/glycine based electrophoresis for 90 min with initial power settings of 40 mA at maximum 135 V. Following the electrophoresis, the gel was incubated in a blotting solution consisting of 10 mM CAPS, pH 11 containing 6% methanol for 5 minutes. A membrane (ProBlott, Applied Biosystems) for electroblotting was wetted for 1 minute in pure methanol before being placed in the blotting solution for 5 minutes.
  • Electroblotting was carried out in a Semi Dry Blotter Il apparatus as follows. Six pieces of Whatman no. 1 paper wetted in blotting solution were placed on the positive electrode of the blotting apparatus followed by the membrane, the polyacrylamide gel and 6 pieces of Whatman no. 1 paper wetted in blotting solution. The blotting apparatus was assembled thereby putting the negative electrode in contact with the upper stack of Whatman no. 1 paper. A weight of 11.3 kg was placed on top. The electroblotting was done at a current of 175 mA for 180 minutes.
  • the membrane was then stained for 1 minute in 0.1 % (w/v) Coomassie Brilliant Blue R-250 dissolved in 60% methanol, 1% acetic acid, 39% H2O, destained in 40% aqueous methanol for 5 minutes, rinsed in deionized water, and air-dried.
  • N-terminal amino acid sequencing a stained membrane piece containing a band of approximately 60 kDa was cut out and placed in the blotting cartridge of an Applied Biosystems Procise protein sequencer. The N-terminal sequencing was carried out using the method run file for PVDF membrane samples (Pulsed liquid PVDF). From comparing a blank chromatogram, an amino acid standard chromatogram and 11 chromatograms corresponding to amino acid residues 1 to 11 , the following sequence could be deduced: TSTIPDYFQTS (SEQ ID NO: 5)
  • Example 3 Recombinant Eupenicillium phosphatase, its preparation, characterization and assay Reagents and media PDA plates:
  • the genomic DNA was used as template for PCR amplification of the phosphatase structural gene from Eupenicillium pinetorum CBS 116641 using below primers, primer 1893 and primer 1894, designed with appropriate restriction sites added to the primer ends (BamHI for primer 1893 and Xhol for primer 1894) to facilitate sub-cloning of the PCR product.
  • Primer 1893 5'-CTG GGG ATC CTT CAC CAT GCA TAT CCC TTC TCA GTT TCT ACT TC-
  • Primer 1894 5'- GAT CTC GAG CTA GAT CGC ACC CGT CGT GAT GTC-3' (SEQ ID NO: 7)
  • PCR amplification was performed using Phusion High-Fidelity DNA Polymerase (Finnzymes, Finland) following the manufacturer's instructions and using an annealing temperature of 56°C and an extension temperature of 72°C for 35 cycles.
  • the resulting PCR fragment was purified (GFX PCR DNA and Gel Band Purification
  • the digested and purified PCR fragment was cloned into the BamHI and Xhol digested Aspergillus expression vector pMStr57 using standard techniques.
  • the expression vector pMStr57 contains the same elements as pCaHj483 (WO 98/00529), with minor modifications made to the Aspergillus NA2 promoter as described for the vector pMT2188 in WO 01/12794, and has sequences for selection and propagation in E. coli, and selection and expression in Aspergillus. Specifically, selection in Aspergillus is facilitated by the amdS gene of Aspergillus nidulans, which allows the use of acetamide as a sole nitrogen source.
  • Aspergillus is mediated by a modified neutral amylase Il (NA2) promoter from Aspergillus niger which is fused to the 5' leader sequence of the those phosphate isomerase (tpi) encoding-gene from Aspergillus nidulans, and the terminator from the amyloglucosidase- encoding gene from Aspergillus niger.
  • NA2 neutral amylase Il
  • the Aspergillus oryzae strain BECh2 (WO 00/39322) was transformed with pEuphy using standard techniques (T. Christensen et al (1988), Bio/Technology 6:1419-1422). Transformants were cultured in YP+2%G medium shaken at 275 RPM at 3O 0 C. The supernatant was removed after 6 days of incubation and tested on SDS-PAGE for presence of a band of roughly the expected size. The best transformant as indicated by a large band of the expected size was selected.
  • Ion exchange was conducted on an AKTA FPLC system (Amersham, Sweden) with a 70 ml SP-Sepharose (Amersham, Sweden) column, 26 mm diameter, equilibrated with 25 mM Na-acetate, pH 3.75. Sample was loaded and washed at 8 ml/min flowrate. The target protein eluted at approx. 0.4 M NaCI under a 0 to 1 M linear NaCI gradient in 25 mM Na-acetate, pH 3.75, over 20 column volumes at 5 ml/min.
  • Phosphatase fractions were pooled based on purity in Coomassie-stained sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and phosphatase activity (see the below phosphatase assay).
  • GXXTIPDYFQ SEQ ID NO: 8
  • XX must designate "TS,” which probably escape from identification due to O-glycosylation.
  • the concentration of the purified phosphatase was estimated to 10 mg/ml (the absorbance at 280 nm, A 280 . was 20.2.
  • Amino acid analysis gave as a result 11.99 mg/ml.
  • the deviation of 20% can be explained by C-terminal truncation and/or impurities. Based on experience it is estimated that impurities are in the order of 10-20%. Together with SDS-PAGE, this leads to the final concentration estimate of 10 mg/ml).
  • SEQ ID NO: 1 is the DNA sequence encoding the phosphatase from Eupenicillium pinetorum CBS 116641 , as PCR-amplified with primer 1893 and primer 1894.
  • Nucleotides 1- 44 correspond to the primer 1893 sequence
  • nucleotides 17-19 is the start codon
  • nucleotides 17-1758 is the genomic copy of the phosphatase structural gene
  • nucleotides 138-202 is a first intron
  • nucleotides 360-416 a second intron
  • nucleotides 1759-1761 the stop codon.
  • Nucleotides 1738-1770 correspond to the reverse and complement sequence of primer 1894.
  • SEQ ID NO: 3 is the phosphatase CDS of Eupenicillium pinetorum CBS 116641 (consisting of joined CDS's of SEQ ID NO: 1 (17..137, 203..359, 417..1758). Please note, that the numbers indicated in this paragraph are not according to the sequence listing, but counts from the very first nucleotide of SEQ ID NO: 1 , C, as no. 1.
  • SEQ ID NO: 2 and SEQ ID NO: 4 are the phosphatase amino acid sequences encoded by SEQ ID NO: 1 and SEQ ID NO: 3, respectively.
  • Amino acids 1-17 is the predicted signal peptide.
  • an active enzyme fragment starts at amino acid no. 25, viz. amino acid no. 8 of SEQ ID NO: 2 and 4 (Thr), whereas according to the present Example an active enzyme fragment starts at amino acid no. 7 of SEQ ID NO: 2 and 4 (GIy). Accordingly, amino acids 1-6, or 1-7, of SEQ ID NO: 2 and 4 probably constitute a kind of a propeptide.
  • This assay is based on enzymatic hydrolysis of the substrate p-Nitrophenyl phosphate di-sodium hexahydrate (pNP-phosphate), C 6 H 4 NNa 2 O 6 P ⁇ H 2 O, being hydrolysed to phosphate and p-nitrophenol, which is yellow at neutral-basic pH and can be measured spectrophotometrically at 405 nm.
  • the reaction conditions are: pH 5.5, temperature 37 0 C, substrate concentration 1.35 mM, wavelength 405 nm, incubation time 15 min, format 96-well
  • MTP micro titer plates, e.g. Nunc 269620.
  • One phosphatase unit (U) is defined as the enzyme activity releasing 1 ⁇ mol phosphate/min under the reaction conditions given above. Buffers and reagents
  • di-sodiumtetraborate 38.1 g di-sodiumtetraborate-10 H 2 O (Merck 106308, Mw 381.37 g/mol) is dissolved in 1000 mL mQ H 2 O
  • 75 ⁇ l/well enzyme solution (or buffer blind) is dispensed in a microtiter plate.
  • 75 ⁇ l substrate is added and the plate is sealed with a piece of adhesive plate sealer.
  • the plate is quickly placed in an Eppendorff Thermomixer equiped with an MTP-holder and shaken with 750 rpm at 37°C for 15 min.
  • 75 ⁇ l stop reagent is added.
  • Absorbance at 405 nm is measured in a MTP spectrophotometer, e.g. a Molecular Devices Spectramax.
  • Example 4 Use of the Eupenicillium phosphatase for boosting the phytase-catalyzed degradation of sodium phytate
  • Example 1 The supernatant of Example 1 was tested in combination with the phytase from Peniophora (amino acids 1-410 of SEQ ID NO: 12) prepared as described in Examples 1 and
  • Inositol Phosphates in an incubation system at pH 4 using sodium phytate as a substrate and taking samples after different time intervals.
  • phytase activity is given in the units of FYT as defined above.
  • the buffer is 5OmM Glycine (pH 4), viz. 5OmM glycine in deionized water, pH 4 with HCI.
  • the incubations were performed in Eppendorph tubes by mixing the substrate, 500 uL 4 mM sodium phytate in 50 mM Glycine (pH 4), with one of two enzyme solutions: 1 ) 500 uL Peniophora phytase diluted in 50 mM Glycine (pH 4) to 1.087 FYT/ml;
  • the incubations were performed at 40 0 C and samples were removed for immediate inactivation after 0, 5, 10, and 30 minutes of incubation.
  • the inactivation was done by adding 500 uL of 1.5 M HCI giving a final concentration of 0.5 M HCI, which subsequently acted to extract the inositol phosphates.
  • the inositol phosphates (IP6 and isomers of IP5, IP4, IP3 and IP2) were separated by HPIC (High Performance Ion Chromatography; Dionex Systems; Carbo-Pac PA 100 column; 0.5 M HCL as eluent) and detected by UV absorption following post-column derivatisation with 0.1% Fe(NO 3 ) 3 x 9H 2 O, 2% HCIO 4 .
  • the quantification was made based on an IP6 standard curve, using correction factors for the lower inositol phosphates (Skoglund, E.; Carlsson, N.-G. and Sandberg, A.S. (1997): Determination of isomers of inositol mono- to hexaphosphates in selected foods and intestinal contents using high-performance ion chromatography. Journal of Agricultural and food chemistry, 45 (2), pp. 431-436). The retention times were determined using a phytate hydrolysate (made by autoclaving of sodium phytate).
  • Table 1 shows the concentration of IP6 and the various degradation products (IP5, IP4, IP3, IP2) per gram sodium phytate after different time intervals (0, 5, 10 and 30 min). More specifically, the figures in Table 1 represent the phosphorus-content (in mg) of each respective inositol phosphate, relative to the starting amount of sodium phytate (in g), viz. "mg IP-P/g phytate”.
  • Example 5 Use of the Eupenicillium phosphatase for boosting phytase activity in an in vitro stomach assay
  • Example 1 The supernatant of Example 1 was tested in combination with the phytase from Peniophora prepared as described in Examples 1 and 2 of WO 98/28408, in an in vitro system mimicking the passage of the stomach using a model-feed (30% Soy Bean Meal (SBM) and 70% maize (ground maize) as a substrate.
  • SBM Soy Bean Meal
  • SBM Soy Bean Meal
  • the stomach is mimicked by applying pepsin and controlling pH to 3.0 during automated incubations of the model-feed and the enzymes performed as follows:
  • Model-feed 10 g composed of 3 g SBM and 7 g maize (ground maize) including 5 g CaCI 2 /kg.
  • Pepsin 5 ml pepsin (Sigma) in 0.085 M HCI containing 0.0132 mg pepsin/ml.
  • Peniophora phytase 1 ml 125 mM sodium acetate (NaAc) buffer (pH 6.0) containing 7.5 FYT/ml of the Peniophora phytase + 5 ml water.
  • Peniophora phytase + Eupenicillium phosphatase 1 ml 125 mM NaAc-buffer (pH 6.0) containing 7.5 FYT/ml of the Peniophora phytase + 5 ml supernatant from Example 1 (Eupenicillium phosphatase).
  • the Peniophora phytase Compared to the control without addition of phytase and phosphatase, the Peniophora phytase efficiently degraded IP6 as present in soluble (supernatant) as well as insoluble form (residue).
  • the combination of the Peniophora phytase with the Eupenicillium phosphatase was able to degrade the IP6 degradation products even further during the in vitro stomach incubation.
  • the IP5 and to a smaller extent the IP4 content was clearly reduced when the Peniophora phytase was combined with the Eupenicillium phosphatase.
  • Example 6 Use of recombinant Eupenicillium phosphatase for boosting phytase- initiated phosphate release in vitro
  • the purified recombinant enzyme from Example 3 was tested alone and in combination with different commercially or publicly available phytases, in an in vitro system mimicking the passage of the stomach using a model-feed consisting of 30% Soy Bean Meal (SBM), 70% ground maize and including CaCI 2 to a final concentration of 5 g Calcium / kg feed, as substrate.
  • Feed samples were prepared and pre-incubated at 40 0 C and pH 3.0 (using HCI for pH adjustment) for 30 minutes followed by addition of pepsin (3000 U/g feed) and enzymes as indicated for the individual treatments in table 3. A blank with no added enzyme was also included. The samples were incubated at 40 0 C and pH 3.0 for 60 minutes at which point HCI was added to increase the pH to 4.0 and incubation continued a further 30 minutes.
  • IP-P inositol-phosphate bound phosphorous
  • Example 7 Dose-response of recombinant Eupenicillium phosphatase in combination with Peniophora phytase in vitro
  • IP-P inositol-phosphate bound phosphorous
  • Example 8 Dose-response of Peniophora phytase in combination with recombinant
  • Eupenicillium phosphatase in vitro Three dosages of Peniophora phytase (amino acids 1-410 of SEQ ID NO: 12) were tested in combination with a supernatant containing recombinant Eupenicillium phosphatase from Example 3, in an in vitro system mimicking the passage of the stomach. In vitro digestions, inositol-phosphate extraction and quantification were performed as described in Example 6. The 68.75 ml supernatant is estimated to correspond to 2.75 mg/kg.
  • IP-P inositol-phosphate bound phosphorous
  • Table 5 Phosphorous in inositol-phosphate after digestion in vitro with the indicated enzymatic treatments.
  • Eupenicillium phosphatase permits reduction of phytase dosage without concomitant reduction of P liberation. Even at the lowest phytase dosage tested (250 FYT/kg feed) combination with Eupenicillium phosphatase results in a larger P release than with the highest dosage of phytase alone (750 FYT/kg feed).
  • a series of phytases viz. those listed in row 1 , 2, 4, 5 and 6 in Table 3 of Example 6, were tested for their ability to degrade phytic acid, alone and in combination with the Eupenicillium phosphatase.
  • the reactions were performed in NMR-tubes and 31 P spectra recorded with 10 min time intervals to follow the phosphate hydrolysis.
  • the conditions were: 50 mM phytic acid, 20 FYT/mL phytase in D 2 O, pD 5.0 adjusted with CD 3 COOD, 30 0 C.
  • the phosphatase was used in a dosage of 0.019 mg/mL with D 2 O.
  • Example 10 Determination of the relative activity of Eupenicillium phosphatase towards different inositol-phosphate substrates
  • the supernatant from a liquid culture of a transformant Aspergillus oryzae expressing the Eupenicillium phosphatase was prepared as described in Example 3 and used for testing the activity of the phosphatase towards different inositol-phosphate substrates.
  • Inositol- phosphate substrates were prepared from partially hydrolysated phytic acid (IP6) prepared by autoclaving (124 0 C) a solution of 10 g/l sodium phytate in 0.5 M HCI for one hour.
  • IP5 Inositol- pentaphosphate
  • IP4 inositol-tetraphosphate
  • Activity assays were performed under standard conditions for assaying phytase activity: pH 5.5, temperature 37°C and a substrate concentration of 5 mM. Enzyme concentration in the assay was adjusted to ensure conversion of less than 5% of the substrate during the 30 minutes incubation time.
  • Enzyme activity was measured by quantification of liberated phosphate using a malachite green based method as described by the manufacturer (QuantichromTM phosphate assay kit, BioAssay Systems; California, USA).
  • Table 6 the relative activities of the Eupenicillium phosphatase towards IP6, IP5 and IP4 are compared to the corresponding values obtained with Peniophora phytase. For each enzyme the highest observed activity has been normalized to 100.
  • IP6 is hydrolysed by a phytase to IP5 and/or IP4 which can be further hydrolysed by the Eupenicillium phosphatase
  • IP6 is hydrolysed by a phytase to IP5 and/or IP4 which can be further hydrolysed by the Eupenicillium phosphatase
  • IP6 is hydrolysed by a phytase to IP5 and/or IP4 which can be further hydrolysed by the Eupenicillium phosphatase
  • IP6 is hydrolysed by a phytase to IP5 and/or IP4 which can be further hydrolysed by the Eupenicillium phosphatase
  • Table 6 Relative activities of Eupenicillium phosphatase and Peniophora phvtase towards IP6, IP5 and IP4.
  • the pH profile of the purified Eupenicillium phosphatase of Example 3 was tested using the phosphatase microtiter assay of Example 3 at various pH values. The highest activity was found at pH 3.5. The activity at pH 3.5 was set to 100%, and the activity at the other pH values re-calculated into percentage, relative to this value. The results are shown in Table 7 below.
  • the Eupenicillium phosphatase has a rather broad pH-activity range with more than 60% of the maximum activity in the pH range of 3.0 to 5.0.

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Abstract

La présente invention décrit des polypeptides isolés présentant une activité phosphatase, ainsi que des polynucléotides isolés codant pour lesdits polypeptides. La structure desdits polypeptides est proche de celle d'une phosphatase dérivée de Eupenicillium pinetorum CBS 116641. La présente invention décrit également des structures d'acides nucléiques, des vecteurs, et des cellules hôtes incluant lesdits polynucléotides, ainsi que des méthodes de production et d'application desdits polypeptides, en particulier leur application à l'alimentation animale. Lesdits polypeptides peuvent plus particulièrement être employés dans l'amplification de l'activité des phytases.
PCT/DK2005/000787 2004-12-13 2005-12-13 Polypeptides présentant une activité phosphatase acide et polynucléotides codant pour lesdits polypeptides WO2006063588A1 (fr)

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WO2007112739A1 (fr) * 2006-04-04 2007-10-11 Novozymes A/S Variants de phytase
WO2010054513A1 (fr) * 2008-11-14 2010-05-20 Fujian Fuda Biotech Co. Ltd. Phytase thermotolérante d'une souche d'escherichia coli non-k12 et production de cette phytase
US7833768B2 (en) 2004-10-04 2010-11-16 Novozymes A/S Polypeptides having phytase activity
JP2010539888A (ja) * 2006-09-21 2010-12-24 ヴェレニウム コーポレイション フィターゼ、フィターゼをコードする核酸、並びにフィターゼを製造および使用する方法
US20120233717A1 (en) * 2010-01-15 2012-09-13 Institute Of Animal Science, Chinese Academy Of Agricultural Sciences Method for preparing a transgenic animal of simultaneous multiple-gene expression
WO2015035914A1 (fr) 2013-09-11 2015-03-19 Novozymes A/S Procédés de production de produits de fermentation
WO2017112540A1 (fr) 2015-12-22 2017-06-29 Novozymes A/S Procédés de production de produits de fermentation
WO2019055455A1 (fr) 2017-09-15 2019-03-21 Novozymes A/S Mélanges d'enzymes et procédés pour améliorer la qualité nutritionnelle d'aliments pour animaux
WO2019083831A1 (fr) 2017-10-23 2019-05-02 Novozymes A/S Procédés pour la réduction d'acide lactique dans un système de fermentation de biocarburant
WO2019231944A2 (fr) 2018-05-31 2019-12-05 Novozymes A/S Procédés d'amélioration de la croissance et de la productivité de levures
EP3670653A1 (fr) * 2018-12-21 2020-06-24 AB Enzymes Oy Polypeptides dotés d'une activité de phytase
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WO2021026201A1 (fr) 2019-08-05 2021-02-11 Novozymes A/S Mélanges d'enzymes et procédés de production d'un ingrédient d'alimentation animale à haute teneur en protéines à partir d'un sous-produit de type résidu de distillation entier
WO2021126966A1 (fr) 2019-12-16 2021-06-24 Novozymes A/S Procédés de production de produits de fermentation
CN114395484A (zh) * 2021-12-21 2022-04-26 上海市水产研究所（上海市水产技术推广站） 一株腐质霉菌KC0924g及其生产的菌剂和应用
WO2024137250A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Polypeptides de la famille 3 de gludice estérase (ce3) présentant une activité acétyl xylane estérase et polynucléotides codant pour ceux-ci
WO2024137252A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Procédé de réduction de la viscosité du sirop à la fin d'un processus de production d'un produit de fermentation
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WO2024137704A2 (fr) 2022-12-19 2024-06-27 Novozymes A/S Procédés de production de produits de fermentation faisant appel à des enzymes de dégradation de fibres avec levure modifiée
WO2024137246A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Polypeptides de la famille 1 d'estérase de glucide (ce1) présentant une activité d'estérase d'acide férulique et/ou d'estérase d'acétyl xylane et polynucléotides codant pour ceux-ci
WO2024258820A2 (fr) 2023-06-13 2024-12-19 Novozymes A/S Procédés de fabrication de produits de fermentation à l'aide d'une levure modifiée exprimant une bêta-xylosidase

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US7833768B2 (en) 2004-10-04 2010-11-16 Novozymes A/S Polypeptides having phytase activity
EP2272964A3 (fr) * 2004-10-04 2011-08-31 Novozymes A/S Polypeptides ayant une activite de phytase et polynucleotides codant ces polypeptides
US8076115B2 (en) 2004-10-04 2011-12-13 Novozymes A/S Polypeptides having phytase activity and polynucleotides encoding same
US8450096B2 (en) 2004-10-04 2013-05-28 Novozymes A/S Polypeptides having phytase activity and polynucleotides encoding same
US8877471B2 (en) 2006-04-04 2014-11-04 Novozymes A/S Phytase variants
WO2007112739A1 (fr) * 2006-04-04 2007-10-11 Novozymes A/S Variants de phytase
EP2365064A1 (fr) * 2006-04-04 2011-09-14 Novozymes A/S Variantes de phytase
US10041052B2 (en) 2006-04-04 2018-08-07 Novozymes A/S Phytase variants
US9451783B2 (en) 2006-04-04 2016-09-27 Novozymes A/S Phytase variants
US8460656B2 (en) 2006-04-04 2013-06-11 Novozymes A/S Phytase variants
JP2010539888A (ja) * 2006-09-21 2010-12-24 ヴェレニウム コーポレイション フィターゼ、フィターゼをコードする核酸、並びにフィターゼを製造および使用する方法
CN102209785B (zh) * 2008-11-14 2013-04-03 福建福大百特科技发展有限公司 一种耐热非-k12大肠杆菌植酸酶及其生产
US8652820B2 (en) 2008-11-14 2014-02-18 Fujian Fuda Biotech Co. Ltd. Thermotolerant Non-K12 Escherichia coli phytase and its production
WO2010054513A1 (fr) * 2008-11-14 2010-05-20 Fujian Fuda Biotech Co. Ltd. Phytase thermotolérante d'une souche d'escherichia coli non-k12 et production de cette phytase
US8742085B2 (en) * 2010-01-15 2014-06-03 Institute Of Animal Science, Chinese Academy Of Agricultural Sciences Method for preparing a transgenic animal of simultaneous multiple-gene expression
US20120233717A1 (en) * 2010-01-15 2012-09-13 Institute Of Animal Science, Chinese Academy Of Agricultural Sciences Method for preparing a transgenic animal of simultaneous multiple-gene expression
WO2015035914A1 (fr) 2013-09-11 2015-03-19 Novozymes A/S Procédés de production de produits de fermentation
EP3712274A1 (fr) 2013-09-11 2020-09-23 Novozymes A/S Procédés de production de produits de fermentation
WO2017112540A1 (fr) 2015-12-22 2017-06-29 Novozymes A/S Procédés de production de produits de fermentation
WO2019055455A1 (fr) 2017-09-15 2019-03-21 Novozymes A/S Mélanges d'enzymes et procédés pour améliorer la qualité nutritionnelle d'aliments pour animaux
WO2019083831A1 (fr) 2017-10-23 2019-05-02 Novozymes A/S Procédés pour la réduction d'acide lactique dans un système de fermentation de biocarburant
WO2019231944A2 (fr) 2018-05-31 2019-12-05 Novozymes A/S Procédés d'amélioration de la croissance et de la productivité de levures
EP3670653A1 (fr) * 2018-12-21 2020-06-24 AB Enzymes Oy Polypeptides dotés d'une activité de phytase
WO2020128152A1 (fr) * 2018-12-21 2020-06-25 Ab Enzymes Oy Polypeptides présentant une activité phytase
CN113166740A (zh) * 2018-12-21 2021-07-23 生化酶股份有限公司 具有植酸酶活性的多肽
WO2020160126A1 (fr) 2019-01-31 2020-08-06 Novozymes A/S Polypeptides ayant une activité xylanase et leur utilisation pour améliorer la qualité nutritionnelle d'aliments pour animaux
WO2021026201A1 (fr) 2019-08-05 2021-02-11 Novozymes A/S Mélanges d'enzymes et procédés de production d'un ingrédient d'alimentation animale à haute teneur en protéines à partir d'un sous-produit de type résidu de distillation entier
WO2021126966A1 (fr) 2019-12-16 2021-06-24 Novozymes A/S Procédés de production de produits de fermentation
CN114395484A (zh) * 2021-12-21 2022-04-26 上海市水产研究所（上海市水产技术推广站） 一株腐质霉菌KC0924g及其生产的菌剂和应用
CN114395484B (zh) * 2021-12-21 2023-06-30 上海市水产研究所（上海市水产技术推广站） 一株腐质霉菌KC0924g及其生产的菌剂和应用
WO2024137250A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Polypeptides de la famille 3 de gludice estérase (ce3) présentant une activité acétyl xylane estérase et polynucléotides codant pour ceux-ci
WO2024137252A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Procédé de réduction de la viscosité du sirop à la fin d'un processus de production d'un produit de fermentation
WO2024137248A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Compositions contenant des arabinofuranosidases et une xylanase, et leur utilisation pour augmenter la solubilisation de fibres hémicellulosiques
WO2024137704A2 (fr) 2022-12-19 2024-06-27 Novozymes A/S Procédés de production de produits de fermentation faisant appel à des enzymes de dégradation de fibres avec levure modifiée
WO2024137246A1 (fr) 2022-12-19 2024-06-27 Novozymes A/S Polypeptides de la famille 1 d'estérase de glucide (ce1) présentant une activité d'estérase d'acide férulique et/ou d'estérase d'acétyl xylane et polynucléotides codant pour ceux-ci
WO2024258820A2 (fr) 2023-06-13 2024-12-19 Novozymes A/S Procédés de fabrication de produits de fermentation à l'aide d'une levure modifiée exprimant une bêta-xylosidase

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