WO2007112739A1 - Phytase variants - Google Patents

Phytase variants Download PDF

Info

Publication number
WO2007112739A1
WO2007112739A1 PCT/DK2007/000135 DK2007000135W WO2007112739A1 WO 2007112739 A1 WO2007112739 A1 WO 2007112739A1 DK 2007000135 W DK2007000135 W DK 2007000135W WO 2007112739 A1 WO2007112739 A1 WO 2007112739A1
Authority
WO
WIPO (PCT)
Prior art keywords
phytase
seq
activity
variant
animal
Prior art date
Application number
PCT/DK2007/000135
Other languages
French (fr)
Inventor
Leonardo De Maria
Carsten Andersen
Lars Kobberoee Skov
Mikael Blom Soerensen
Original Assignee
Novozymes A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to JP2009503408A priority Critical patent/JP5221516B2/en
Application filed by Novozymes A/S filed Critical Novozymes A/S
Priority to CN200780020771.5A priority patent/CN101460612B/en
Priority to US12/294,526 priority patent/US8460656B2/en
Priority to EP07711278A priority patent/EP2001999B1/en
Priority to MX2008012632A priority patent/MX2008012632A/en
Priority to CA2647476A priority patent/CA2647476C/en
Priority to BRPI0709732A priority patent/BRPI0709732B1/en
Priority to AU2007234177A priority patent/AU2007234177B2/en
Priority to ES07711278T priority patent/ES2387203T3/en
Publication of WO2007112739A1 publication Critical patent/WO2007112739A1/en
Priority to US13/874,954 priority patent/US8877471B2/en
Priority to US14/504,517 priority patent/US9451783B2/en
Priority to US15/249,856 priority patent/US10041052B2/en

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)

Definitions

  • the present invention relates to a phytase which has at least 74% identity to a phytase derived from Citrobacter braakii ATCC 51113 and comprises at least one alteration as compared to this phytase (i.e., is a variant thereof).
  • the invention also relates to DNA encoding these phytases, methods of their production, as well as the use thereof, e.g. in animal feed and animal feed additives.
  • the mature part of the Citrobacter braakii ATCC 51113 phytase is included in the sequence listing as SEQ ID NO:2.
  • WO-2004/085638 discloses, as SEQ ID NO:7, the amino acid sequence of a phytase from Citrobacter braakii YH-15, deposited as KCCM 10427. The mature part of this amino acid sequence is included herein as SEQ ID NO:3. This sequence is also found in the database
  • WO 2006/037328 discloses the wildtype phytase of Citrobacter braakii ATCC 51113
  • SEQ ID NO:2 herein
  • SEQ ID NO:6 a variant thereof, which is also included in the present sequence listing, viz. as SEQ ID NO:6.
  • WO 2006/038062 and WO 2006/038128 both disclose the amino acid sequence of the phytase gene of Citrobacter freundii P3-42, deposited under accession number NCIMB 41247.
  • Non-limiting examples of such properties are: Thermostability, temperature profile pH profile, specific activity, performance in animal feed, protease-sensibility, and/or glycosylation pattern.
  • the present invention relates to a phytase which has at least 74% identity to SEQ ID
  • the invention also relates to a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 1 H 1 K 1 R, 6OP, 105E, 106A.G,
  • the phytase is not SEQ ID NO:3, not SEQ ID NO:4, not SEQ ID NO:6, and not SEQ ID NO:9 and the variants thereof listed in Fig. 1.
  • the invention also relates to DNA encoding these phytases, methods of their production, as well as the use thereof, e.g. in animal feed and animal feed additives.
  • Fig. 1 corresponds to Table 2 of WO 2006/038062 and discloses a number of variants of the Citrobacter freundii NCIMB 41247 phytase which has the amino acid sequence of SEQ ID NO:9;
  • Fig. 2 is an alignment of the phytases of SEQ ID NO:2 and 9.
  • Fig. 1 refers to the numbering of SEQ ID NO:9.
  • the corresponding SEQ ID NO:2 positions can be found by deduction of 22 (e.g., variant P229S of Fig.1 means variant P207S using the numbering of the present application).
  • the present invention relates to a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:
  • NO:2 in at least one position selected from the following: 1 , 2, 3, 4, 5, 31, 41 , 46, 52, 53, 55,
  • Position numbers refer to the position numbering of SEQ ID NO:2, as described in the section “Position Numbering.” Positions corresponding to these SEQ ID NO:2 position numbers in other phytases are determined as described in the section “Identifying Corresponding Position Numbers.”
  • the phytase of the invention is a variant of the phytase of SEQ ID NO:2, viz. it is not identical to SEQ ID NO:2, as it comprises at least one alteration as compared to SEQ ID NO:2.
  • the phytase of the invention comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1 , 2, 3, 4, 5, 31 , 46, 52, 53, 55, 57, 59, 76, 82, 99, 100, 107, 109, 111 , 114, 115, 116, 117, 118, 1 19, 120, 121 , 122, 123, 124, 137, 141 , 161 , 162, 164, 167, 179, 180, 181 , 182, 183, 184, 185, 186, 196, 199, 200, 202, 218, 223, 241 , 273, 276, 285, 286, 299, 314, 331
  • the phytase of the invention is not SEQ ID NO:9. In a still further particular embodiment, the phytase of the invention is not the variants of SEQ ID NO:9 listed in Fig.1.
  • the phytase of the invention comprises at least one of the following alterations: 1*, 2*, 3*, 4P, 5P, 31C 1 T, 41 P, 46C 1 D 1 E, 52C.E, 53V,Q, 55D 1 I, 57Y, 59C,
  • the phytase of the invention comprises at least one of the following alterations: 1*, 2*, 3*, 4P, 5P, 31 C, 46E, 52C,E, 53V, 55D, 57Y, 59C, 76G, 82E, 99C, 100C, 107D ; E,G, 109A, 1 11 P, 114T 1 115Q, 1 16AT, 1 17D, 118T, 119K 1 R 1 S, 120S, 121 D,P,T,122D, 123P 1 124L, 137P, 141C, 161 P, 162C, 164E, 167Q, 179K, 180E.T, 181D,K, 182H 1 K 1 Q, 183L,V,Q, 184*, 185*, 186*, 196Q, 199C
  • amino acids in position 179, 180, 181 , 182, 183, 184, 185, and 186 have been replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV, or KTDKL.
  • the invention also relates to a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 1 H 1 K 1 R, 60P 1 105E 1 106A 1 G 1
  • the phytase comprises the alteration 1 K.
  • the phytase comprises the following combinations of alterations: 280P/282P/283P, 155F/254Y, and/or 155F/157F/254Y.
  • Preferred phytases of the invention comprise an alteration selected from the following: 52C, 141 C, 162C, 31 C, 52C, 99C, 59C, 100C, 141 C/199C, 4P, 5P, 111 P, 137P, 161 P, 52E, 57Y, 76G, 107D, 107G, 109A, 1*, 1*/2*, 1*/2*/3*, 121T, 273L, 285G, 286Q, 299L, 362K, 331 K/55D, 107E, 46E, 82E, 1 19R, 119K, 164E, 223E, 276R, 276K 1 362R, 379R, 379K, 385D, 410D, 410E, 41 1 R, 411 K, 53V, 121 D, 167Q, 196Q, 200K, 202N, 218Q, 241 Q 1 285N, 314N, 314G, 406A,
  • the phytase of the invention may be a variant of any wildtype or variant phytase. In particular embodiments, it is a variant of the phytase of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, or a variant of any one of the phytase variants related to SEQ ID NO:9 and listed in Fig.1.
  • the phytase of the invention may furthermore comprise an alteration (substitution) or a combination of alterations (substitutions) selected from amongst the alterations (substitutions) and combinations of alterations (substitutions) listed in each row of Fig.1.
  • Particularly preferred variants of the phytase of SEQ ID NO:2 are the following: R339D, N4P, G5P, Q1 1 1 P, E1*, E1*/E2*, E1*/E2*/Q3*, M273L, and N286K; as well as any combination thereof; as well as the corresponding variants of SEQ ID NO:3, 4 and 6.
  • Particularly preferred phytases of the invention comprise at least one of the following alterations: 339D, 4P, 5P, 111 P, 1*, 1*/2*, 1*/2*/3*, 273L, and/or 286K.
  • the invention also relates to a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: (i) 141 C/199C, 91 C/46C, 52C/99C, 31 C/176C, 31 C/177C, 59C/100C, and/or 162C/247C; (ii) 41 P 1 91 P, 136P, 137P, 154P 1 161 P, 355P, 1 11 P 1 240P 1 282P, 283P 1 284P 1 289P 1 4P 1 and/or 5P; (iii) 52E, 551, 57Y, 104A/105F, 107D.G, 109A.G, 76G 1 84Y, 121T 1 362K 1 273L 1 Q, 285G,R,
  • the structure of the C. braakii ATCC 51113 phytase was built by homology modelling, using as a template the structure of the E. coli AppA phytase (Protein Data Bank id.: 1DKO; Lim et al, Nat. Struct. Biol. (2000), vol. 2, pp. 108-113).
  • the structure was subjected to molecular dynamics (MD) simulations and electrostatic calculations. Positions for putative disulfide bridges and prolines were also identified, as well as other positions of potential importance as regards the various desirable enzymatic properties. Finally, putative glycosylation sites (stretches of NXT or NXS) were identified.
  • a phytase is a polypeptide having phytase activity, i.e. an enzyme which catalyzes the hydrolysis of phytate (myo-inositol hexakisphosphate) to (1) myo- inositol and/or (2) mono-, di-, tri-, tetra- and/or penta-phosphates thereof and (3) inorganic phosphate.
  • phytate myo-inositol hexakisphosphate
  • a phytase substrate encompasses, i.a., phytic acid and any phytate (salt of phytic acid), as well as the phosphates listed under (2) above.
  • the ENZYME site at the internet http://www.expasy.ch/enzyme/ is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB) and it describes each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided (Bairoch A.
  • the ENZYME database 2000, Nucleic Acids Res 28:304-305). See also the handbook Enzyme Nomenclature from NC-IUBMB, 1992).
  • phytases According to the ENZYME site, three different types of phytases are known: A so-called 3-phytase (alternative name 1 -phytase; a myo-inositol hexaphosphate 3- phosphohydrolase, EC 3.1.3.8), a so-called 4-phytase (alternative name 6-phytase, name based on 1 L-numbering system and not I D-numbering, EC 3.1.3.26), and a so-called 5- phytase (EC 3.1.3.72). For the purposes of the present invention, all three types are included in the definition of phytase.
  • 3-phytase alternative name 1 -phytase; a myo-inositol hexaphosphate 3- phosphohydrolase, EC 3.1.3.8
  • 4-phytase alternative name 6-phytase, name based on 1 L-numbering system and not I D
  • the phytases of the invention belong to the family of acid histidine phosphatases, which includes the Escherichia coli pH 2.5 acid phosphatase (gene appA) as well as fungal phytases such as Aspergillus awamorii phytases A and B (EC: 3.1.3.8) (gene phyA and phyB).
  • the histidine acid phosphatases share two regions of sequence similarity, each centered around a conserved histidine residue. These two histidines seem to be involved in the enzymes' catalytic mechanism. The first histidine is located in the N-terminal section and forms a phosphor-histidine intermediate while the second is located in the C- terminal section and possibly acts as proton donor.
  • the phytases of the invention have a conserved active site motif, viz. R-H-G-X-R-X-P, wherein X designates any amino acid (see amino acids 16 to 22 of SEQ ID NOs:2, 3, 4, 6 and amino acids 38-44 of SEQ ID NO:9).
  • the conserved active site motif is R-H-G-V-R-A-P 1 i.e. amino acids 16-22 (by reference to SEQ ID NO:2) are RHGVRAP.
  • 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 P 6 Na 12 ) 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.
  • Phytase activity may also be determined using the assays of Example 1 ("Determination of phosphatase activity” or “Determination of phytase activity”).
  • the phytase of the invention is isolated.
  • isolated 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.
  • 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.
  • the relatedness between two amino acid sequences is described by the parameter "identity".
  • the alignment of two amino acid sequences is determined by 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 and the amino acid sequence referred to in the claims (SEQ ID NO:2) is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the "invention sequence,” or the length of the SEQ ID NO:2, whichever is the shortest. The result is expressed in percent identity.
  • An exact match occurs when the "invention sequence 1 ' and SEQ ID NO:2 have identical amino acid residues in the same positions of the overlap (in the alignment example below this is represented by "
  • the length of a sequence is the number of amino acid residues in the sequence (e.g. the length of amino acids 1-411 of SEQ ID NO:2 is 411).
  • Example 13 is an example of an alignment of the phytase of SEQ ID NO:2 and the phytase of SEQ ID NO:9, and the example illustrates how to calculate the percentage of identity between these two backbones.
  • the percentage of identity of an amino acid sequence of a polypeptide with, or to, SEQ ID NO:2 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 number of exact matches is 6, the length of the shortest one of the two amino acid sequences is 12; accordingly the percentage of identity is 50%.
  • the degree of identity to SEQ ID NO:2 is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.
  • the degree of identity is at least 98.0%, 98.2%, 98.4%, 98.6%, 98.8%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9%.
  • the degree of identity is at least 70%, 71%, 72%, or at least 73%.
  • the phytase of the invention has no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, or no more than 10 alterations as compared to SEQ ID NO:2; no more than 11 , 12, 13, 14, 15, 16, 17, 18, 19, or no more than 20 alterations as compared to SEQ ID NO:2; no more than 21, 22, 23, 24, 25, 26, 27, 28, 29, or no more than 30 alterations as compared to SEQ ID NO:2; no more than 31, 32, 33, 34, 35, 36, 37, 38, 39, or not more than 40 alterations as compared to SEQ ID NO:2; no more than 41 , 42, 43, 44, 45, 46, 47, 48, 49, or no more than 50 alterations as compared to SEQ ID NO:2; no more than 51 , 52, 53, 54, 55, 56, 57, 58, 59, or no more than 60 alterations as compared to SEQ ID NO:2; no more than 61 , 62, 63, 64, 65, 66, 67
  • SEQ ID NO:2 Accordingly, in the present context, the basis for numbering positions is SEQ ID NO:2 starting with E1 and ending with E411.
  • the term "mature" part refers to that part of the polypeptide which is secreted by a cell which contains, as part of its genetic equipment, a polynucleotide encoding the polypeptide.
  • the mature polypeptide part refers to that part of the polypeptide which remains after the signal peptide part, as well as a propeptide part, if any, has been cleaved off.
  • the signal peptide part can be predicted by programs known in the art (e.g. SignalP).
  • the expected signal peptide part of SEQ ID NO:2 is included in the present sequence listing as SEQ ID NO:8, which is encoded by SEQ ID NO:7.
  • SEQ ID NO:2 is the expected mature part.
  • the first amino acid of the mature part of an enzyme can be determined by N-terminal sequencing of the purified enzyme. Any difference between the signal peptide part and the mature part must then be due to to the presence of a propeptide.
  • a phytase variant can comprise various types of alterations relative to a template (i.e. a reference or comparative amino acid sequence such as SEQ ID NO:2): An amino acid can be substituted with another amino acid; an amino acid can be deleted; an amino acid can be inserted; as well as any combination of any number of such alterations.
  • a template i.e. a reference or comparative amino acid sequence such as SEQ ID NO:2
  • An amino acid can be substituted with another amino acid; an amino acid can be deleted; an amino acid can be inserted; as well as any combination of any number of such alterations.
  • insertion is intended to cover also N- and/or C-terminal extensions.
  • the position number (“D") is counted from the first amino acid residue of SEQ ID NO:2.
  • "at least one" means one or more, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 alterations; or 12, 14, 15, 16, 18, 20, 22, 24, 25, 28, or 30 alterations; and so on, up to a maximum number of alterations of 125, 130, 140, 150, 160, 170, 180, 190, or of
  • a substitution or extension without any indication of what to substitute or extend with refers to the insertion of any natural, or non-natural, amino acid, except the one that occupies this position in the template.
  • Example 13 provides further illustration of how to apply this nomenclature.
  • NO:2 is used as the standard for position numbering and, thereby, also for the nomenclature.
  • the position corresponding to position D in SEQ ID NO:2 is found by aligning the two sequences as specified above in the section entitled "Phytase polypeptides, percentage of identity". From the alignment, the position in the sequence of the invention corresponding to position D of SEQ ID NO:2 can be clearly and unambiguously identified (the two positions on top of each other in the alignment).
  • Example 13 is an example of an alignment of the phytase of SEQ ID NO:2 and the phytase of SEQ ID NO:9, and the example illustrates how corresponding positions in these two backbones are identified.
  • Table 1 above which in the third column includes a number of alignments of two sequences:
  • Position number 80 refers to amino acid residue G in the template. Amino acid A occupies the corresponding position in the variant. Accordingly, this substitution is designated G80A.
  • Position number 80 again refers to amino acid residue G in the template.
  • the variant has two insertions, viz. TY, after G80 and before V81 in the template.
  • T and Y of course would have their own “real" position number in the variant amino acid sequence, for the present purposes we always refer to the template position numbers, and accordingly the T and the Y are said to be in position number 80a and 80b, respectively.
  • Position number 275 refers to the last amino acid of the template.
  • a C-terminal extension of ST are said to be in position number 275a and 275b, respectively, although, again, of course they have their own “real" position number in the variant amino acid sequence.
  • the phytase of the invention has amended, preferably improved, properties.
  • the terms "amended” and “improved” imply a comparison with another phytase.
  • Examples of such other, reference, or comparative, phytases are: SEQ ID NO:3, and/or SEQ ID NO:4.
  • Still further examples of reference phytases may be SEQ ID NO:2, and/or SEQ ID NO:6.
  • a still further example of a reference phytase may be SEQ ID NO:9, and the variants thereof disclosed in Fig. 1.
  • Non-limiting examples of properties that are amended, preferably improved, are the following: Thermostability, pH profile, specific activity, performance in animal feed, protease- sensibility, and/or glycosylation pattern.
  • the phytase of the invention may also have an amended, preferably improved, temperature profile, and/or it may incorporate a change of a potential protease cleavage site.
  • a phytase of the invention has a residual activity which is higher than the residual activity of a reference phytase, wherein residual activity is determined as follows: A fermentation supernatant is divided in two parts, one part is incubated for 30 minutes at a desired elevated temperature, and the other part for 30 minutes at 5 0 C, following which the activity of both is determined on p-nitrophenyl phosphate at 37 0 C and pH 5.5, and the activity of the sample having been incubated at an elevated temperature is divided by the activity of the same sample having been incubated at 5°C.
  • Preferred elevated temperatures are 50 0 C, 55 0 C, 60 0 C, 65 0 C, 70 0 C, 75°C, 8O 0 C, or 85°C.
  • the enzyme-containing samples may be diluted in 0.1 M NaAc pH 5.5.
  • the residual activity of a phytase of the invention is preferably at least 105%, or at least 110%, 120%, 130%, 140%, 150% of the residual activity of the reference phytase.
  • the residual activity of a phytase of the invention is at least 200%, ' or at least 250%, 300%, 400%, or at least 500% of the residual activity of the reference phytase.
  • the residual activity of a phytase of the invention is at least 2x, 3x, 4x, 5x, 6x, 7x, 10x, 15x, 2Ox, or at least 25x the residual activity of the reference phytase.
  • a phytase of the invention has a residual activity which is higher than the residual activity of a reference phytase, wherein residual activity is determined as follows: A fermentation supernatant is divided in two parts, one part is incubated for 30 minutes at a 5O 0 C, and the other part for 30 minutes at 5°C, following which the activity of both is determined on p-nitrophenyl phosphate at 37 0 C and pH 5.5, and the activity of the sample having been incubated at an elevated temperature is divided by the activity of the same sample having been incubated at 5°C.
  • the enzyme-containing samples may be diluted in 0.1 M NaAc pH 5.5.
  • the residual activity of a phytase of the invention is preferably at least 2x, 3x, 4x, 5x, 6x, 7x, 10x, 15x, 2Ox, or at least 25x the residual activity of the reference phytase of SEQ ID NO:3.
  • the residual activity of a phytase of the invention is preferably at least 105%, or at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or at least 200% of the residual activity of the reference phytase of SEQ ID NO:2.
  • substitutions are particularly preferred as they improve thermostability as compared to the phytase of SEQ ID NO:3 as well as to the phytase of SEQ ID NO:2 (see Table 3): 4P, 5P 1 111 P 1 1 * , 1 * /2 * . 1 * /2*/3*. 273L, and/or 286Q.
  • a phytase of the invention has a residual activity which is higher than the residual activity of a reference phytase, wherein residual activity is determined as follows: A fermentation supernatant is divided in two parts, one part is incubated for 30 minutes at a 60°C, and the other part for 30 minutes at 5 0 C 1 following which the activity of both is determined on p-nitrophenyl phosphate at 37°C and pH 5.5, and the activity of the sample having been incubated at an elevated temperature is divided by the activity of the same sample having been incubated at 5 0 C.
  • the enzyme-containing samples may be diluted in 0.1 M NaAc pH 5.5, optionally including 0.005% Tween-20.
  • the phytase of the invention and the reference phytase may be expressed in a Bacillus subtilis host strain.
  • the host strain may be grown in 100ml PS1 medium (100g/L sucrose, 40g/L Soy flakes, 10g/L Na 2 HPO 4 .12H 2 O, 0.1ml/L Dowfax 63N10 (Dow)) in 500ml shake flasks for four days at 30°C at 300 rpm.
  • the residual activity of a phytase of the invention is preferably at least 32%, or at least 34%, 36%, 38%, or at least 40% of the residual activity of the reference phytase of SEQ ID NO:2. More preferably, the residual activity of a phytase of the invention is at least 50%, or at least 60%, 70%, 80%, 90%, or at least 100% of the residual activity of the reference phytase of SEQ ID NO:2. Even more preferably the residual activity of a phytase of the invention is at least 120%, 140%, 160%, 180%, or at least 200% of the residual activity of the reference phytase of SEQ ID NO:2.
  • the residual activity of a phytase of the invention is at least 2x, or at least 3x, 4x, or at least 5x the residual activity of the reference phytase of SEQ ID NO:2.
  • the following substitutions are particularly preferred (see Table 5): (i) 409E, 136P; (ii) 411 K, 331K/55D, 167Q, 179K/180T/181D/182K/183L/184*/185*/186*, 107E; (iii) 196Q, 276R, 285G, 299L, 200K;
  • thermostability 141 C/199C, 52C/99C.
  • Thermostability may also be determined as described in Example 9, i.e. using DSC measurements to determine the denaturation temperature, Td, of the purified phytase protein.
  • Td is indicative of the thermostability of the protein: The higher the Td, the higher the thermostability.
  • the phytase of the invention has a Td which is higher than the Td of a reference phytase, wherein Td is determined on purified phytase samples (preferably with a purity of at least 95%, determined by SDS-PAGE), after dialysis in 2OmM Na-acetate pH4.0 (preferably in a 2-3 h step followed by an over night step), followed by 0.45um filtration and dilution with dialysis buffer to a protein concentration corresponding to approximately 2 absorbancy units (A 280 ), using Differential Scanning Calorimetry at a 90°C/h scan rate from 20-90 0 C in 20 mM Na-acetate buffer, pH 4.0.
  • Td is determined on purified phytase samples (preferably with a purity of at least 95%, determined by SDS-PAGE), after dialysis in 2OmM Na-acetate pH4.0 (preferably in a 2-3 h step followed by an over night step), followed by 0.45um filtration and
  • the Td of the phytase of the invention is higher than the Td of the phytase of SEQ ID NO:4, more preferably at least 101% thereof, or at least 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or at least 110% thereof. Even more preferably, the Td of the phytase of the invention is at least 120%, 130%, 140%, 150%, 160%, 170%, 180%, or at least 190% of the Td of the phytase of SEQ ID NO:4. The following substitutions are particularly preferred (see Table 6): 362K, 362R, 111P 1 and/or 273L.
  • thermostable phytase of the invention has a melting temperature, Tm (or a denaturation temperature, Td), as determined using Differential Scanning Calorimetry (DSC) as described in Example 2 (i.e. in 20 mM sodium acetate, pH 4.0), of at least 5O 0 C.
  • Tm is at least 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 62.5.
  • DSC measurements may also be performed as described in Example 1 ("DSC measurements"), or Example 2 ("Thermostability by DSC").
  • the phytase of the invention after incubation for 60 minutes at 70 0 C and pH 4.0, has an improved residual activity as compared to the residual activity of a reference phytase treated in the same way, the residual activity being calculated for each phytase relative to the activity found before the incubation (at 0 minutes).
  • the residual activity is preferably measured on sodium phytate at pH 5.5 and 37°C.
  • the incubation is preferably in 0.1 M sodium acetate, pH 4.0.
  • the phytase is preferably purified, more preferably to a purity of at least 95%, determined by SDS-PAGE.
  • a preferred phytase activity assay buffer is 0.25 M Na-acetate pH 5.5.
  • the residual activity of the phytase of the invention is preferably at least 105% of the residual activity of the reference phytase, more preferably at least 110%, 115%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or at least 200%.
  • the residual activity relative to the activity at 0 minutes is preferably at least 31%, or at least 32%.
  • substitutions providing improved thermostability stability are preferred (see Table 9): 273L, 46E, 362R, and/or 53V.
  • the phytase variant of the invention is more thermostable than the reference phytase, wherein thermostability is determined using any of the above- mentioned four tests (based on Example 1 , 5, 8, 9, or 12).
  • thermostability is expected of the following variants of the phytase of SEQ ID NO:2 (in order of preference, within each grouping):
  • HQPEIGKMDPV TQADTSSPDPL, HQQDIKQADPL, TQTDTSSPDPL, NQADLKKTDPL;
  • a phytase of the invention has an amended temperature profile as compared to a reference phytase may be determined as described in Example 10. Accordingly, in a particular embodiment the phytase of the invention has an amended temperature profile as compared to a reference phytase, wherein the temperature profile is determined as phytase activity as a function of temperature on sodium phytate at pH 5.5 in the temperature range of 20-90 0 C (in 10 c C steps).
  • a preferred buffer is in 0.25 M Na-acetate buffer pH 5.5.
  • the activity at each temperature is preferably indicated as relative activity (in %) normalized to the value at optimum temperature. The optimum temperature is that temperature within the tested temperatures (i.e. those with 10 0 C jumps) where the activity is highest.
  • the phytase of the invention has a relative activity at 70 0 C of at least 18%, or at least 19%, 20%, 21%, 22%, 23%, 24%, or at least 25%. As explained above, this is relative to the activity at the optimum temperature. More preferably, the phytase of the invention has a relative activity at 7O 0 C of at least 26%, 27%, 28%, 29%, 30%, 31%, or at least 32%.
  • Preferred substitutions which provide an amended temperature profile are (see Table 7): 57Y, 76G, 107G, 273L, 362K, 46E, 362R, 53V, and/or 241 Q.
  • Their relative activity at 70 0 C is higher as compared to the reference phytase of SEQ ID NO:3 and 4, and in some instances (57Y, 76G, 107G, 273L, 362K, 362R, and/or 53V) also as compared to the reference phytase of SEQ ID NO:2.
  • a phytase of the invention has an amended pH profile as compared to a reference phytase, wherein the pH profile is determined as phytase activity as a function of pH on sodium phytate at 37°C in the pH range of 2.0 to 7.5 (in 0.5 pH-unit steps).
  • a preferred buffer is a cocktail of 5OmM glycine, 5OmM acetic acid and 5OmM Bis-Tris. Another preferred buffer is 0.25M sodium acetate.
  • the activity at each pH is preferably indicated as relative activity (in %) normalized to the value at optimum pH.
  • An example of an amended pH profile is where the pH curve (relative activity as a function of pH) is shifted towards higher, or lower, pH.
  • Preferred substitutions which provide a shift of 0.5 pH units towards a higher pH as compared to the reference phytase of SEQ ID NO:2, 3 or 4 are (see Table 8): 46E, and/or 218Q.
  • Another example of an amended pH profile is where the optimum pH is changed, in the upward or the downward direction.
  • Preferred substitutions which provide a lower optimum pH as compared to SEQ ID NO:2, 3, and 4 are (see Table 8): 46E, 121 D, and/or 200K.
  • Preferred substitutions which provide a higher optimum pH as compared to SEQ ID NO:2,3, and 4 are
  • An amended pH profile may also be determined as described in Example 1 ("Amended pH profile: Determination of pH 3.5/5.5 activity ratio"), viz. by comparing phosphatase activity at pH 3.5 and 5.5. Alternatively, the activity at pH 3.5 may be compared with the activity at pH
  • phytase activities are compared instead of phosphatase activities.
  • the phytase of the invention has an amended pH profile as compared to a reference phytase. More in particular, the pH profile is amended in the pH- range of 3.5-5.5. Still more in particular, the activity at pH 4.0, 4.5, 5.0, and/or 5.5 is at a level of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of the activity at the pH-optimum (pH 3.5).
  • the pH profile, as well as the pH-optimum, of a polypeptide may be determined by incubating it at various pH-values, using a substrate in a pre-determined concentration and a fixed incubation temperature.
  • the pH profile is a graphical representation of phytase activity versus pH, the pH-optimum is determined from the pH profile.
  • the phosphatase or phytase assay of Example 1 is used, e.g. the substrate is 5mM sodium phytate, the reaction temperature 37 0 C 1 and the activity is determined at various pH-values, for example pH 2-12, replacing the pH 5.5 acetate buffer with a suitable buffer.
  • buffers examples include: 0.1 M glyc/ne/HCI (pH 2.0-3.5), 0.1 M NaAc/Ac (pH 4.0-5.0), 0.1 M Bis- Tris/HCI (pH 5.5-6.5), 0.1 M Tris/HCI (pH 7.0).
  • Other examples of buffers are: 10OmM succinic acid, 10OmM HEPES, 10OmM CHES, 10OmM CABS adjusted to pH-values 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 with HCI or NaOH.
  • an amended pH profile is expected of the following variants of the phytase of SEQ ID NO:2 (in order of preference, within each grouping): (i) E218Q, D324N, T200R,K, N121D, E196Q, D202N, E406A, E167Q, E53V,Q, E241Q, D314N,G, E239Q, E285N;
  • the phytase of the invention has an improved specific activity relative to a reference phytase. More in particular, the specific activity of a phytase of the invention is at least 105%, relative to the specific activity of a reference phytase determined by the same procedure. In still further particular embodiments, the relative specific activity is at least 110, 115, 120, 125, 130, 140, 145, 150, 160, 170, 180, 190, 200, 220, 240,
  • high specific activity refers to a specific activity of at least
  • the specific activity is at least 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,
  • Specific activity is measured on highly purified samples (an SDS poly acryl amide gel should show the presence of only one component).
  • the enzyme protein concentration may be determined by amino acid analysis, and the phytase activity in the units of FYT, determined as described in Example 1.
  • Specific activity is a characteristic of the specific phytase variant in question, and it is calculated as the phytase activity measured in FYT units per mg phytase variant enzyme protein. See Example 7 for further details.
  • an amended specific activity is expected of the following variants of the phytase of SEQ ID NO:2, in which, in order of preference, the loop between residues 114 and 124 (YQKDEEKNDPL) which faces the active site is replaced with a loop selected from, e.g., HQEKMGTMDPT, HQQDIKQVDSL, HQPEIGKMDPV, TQADTSSPDPL,
  • the phytase of the invention has an improved performance in animal feed as compared to a reference phytase.
  • the performance in animal feed may be determined by the in vitro model of Example 6.
  • the phytase of the invention has an improved performance in animal feed, wherein the performance is determined in an in vitro model, by preparing feed samples composed of 30% soybean meal and 70% maize meal with added CaCI 2 to a concentration of 5 g calcium per kg feed; pre-incubating them at 40°C and pH 3.0 for 30 minutes followed by addition of pepsin (3000 U/g feed) and phytase; incubating the samples at 40 0 C and pH 3.0 for 60 minutes followed by pH 4.0 for 30 minutes; stopping the reactions; extracting phytic acid and inositol- phosphates by addition of HCI to a final concentration of 0.5M and incubation at 40 0 C for 2 hours, followed by one freeze-thaw cycle and 1 hour incubation
  • the phytase of the invention and the reference phytase are of course dosed in the same amount, preferably based on phytase activity units (FYT).
  • a preferred dosage is 125 FYT/kg feed.
  • Another preferred dosage is 250 FYT/kg feed.
  • the phytases may be dosed in the form of purified phytases, or in the form of fermentation supematants. Purified phytases preferably have a purity of at least 95%, as determined by SDS-PAGE.
  • the degraded IP6-P value of the purified phytase of the invention, relative to the degraded IP6-P value of the reference phytase is at least 101 %, or at least 102%, 103%, 104%, 105%, 110%, 115%, or at least 120%.
  • the degraded IP6-P value of the purified phytase of the invention, relative to the degraded IP6-P value of the reference phytase is at least 125%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or at least 200%.
  • the degraded IP6-P value of the phytase of the invention is at least 105%, 110%, 113%, 115%, 120%, 125%, or at least 130%.
  • substitutions provide an improved or at least as good performance in animal feed in vitro (see Table 4A) as compared to the phytase of SEQ ID NO:3: 4P, 5P, 111 P 1 1*, V 12*, 1*/2*/3*, 273L, 286Q.
  • substitutions also provide an improved or at least as good performance in animal feed in vitro (see Table 4B) as compared to the phytase of SEQ ID NO:3: 57Y, 76G, 107G, 362K, 362R, 121 D, 196Q, 200K, 202N, 314N, 406A, and 114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L
  • the relative performance of a phytase of the invention may also be calculated as the percentage of the phosphorous released by the reference phytase. in a still further particular embodiment, the relative performance of the phytase of the invention may be calculated as the percentage of the phosphorous released by the phytase of the invention, relative to the amount of phosphorous released by the reference phytase.
  • the relative performance of the phytase of the invention is at least 105%, preferably at least 110, 120, 130, 140, 150, 160, 170, 180, 190, or at least 200%.
  • the phytase of the invention has a reduced protease- sensibility. More in particular, it has a reduced sensibility towards the Kex2 protease, meaning a reduced tendency to become cleaved by this protease.
  • Variant 339D is an example of a phytase of the invention with a reduced protase-sensibility.
  • Glycosylation is a phenomenon which is only observed when expressing proteins in eukaryotes such as fungi and transgenic plants, but not in prokaryotes such as bacteria.
  • N- glycosylation i.e. the asparagine-linked glycosylation where sugars are attached to a protein, starting from an N-acetyg!ucosamine molecule attached to asparagines.
  • N-glycosylation has been found to occur only to asparagines that in the sequence are part of the following tripeptides: N-X-T or N-X-S, where X designates any amino acid.
  • thermostability was observed when the phytase of SEQ ID NO:2 was expressed in the fungus (yeast) Pichia pastoris, as compared to when it was expressed in Bacillus subtilis, see Example 2.
  • thermostability may be improved for phytases expressed in fungi by altering potential glycosylation sites.
  • the present invention accordingly also relates to phytase variants having an amended glycosylation pattern, preferably amended N-glycosylation sites.
  • the amended glycosylation is expected to confer an improved thermostability upon the phytase variant, when expressed in a fungus.
  • phytases are bacterial phytases, e.g. Gram-negative phytases, such as E.coli and Citrobacter phytases and variants thereof, including the phytases of the present invention as well as the phytases of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:9 herein.
  • fungal expression hosts are Pichia, Saccharomyces, and Aspergillus species.
  • an amended glycosylation pattern is expected of the following phytases of the invention (e.g.
  • variants of SEQ ID NO:2) in order of preference: N31T, N74A, N171T, N203T, N281H, N316D, N308A.
  • the following are replacing an N-X-T type pattern: N31T, N74A, N281 H.
  • the phytases of the present invention are (also) low- allergenic variants, designed to invoke a reduced immunological response when exposed to animals, including man.
  • the term immunological response is to be understood as any reaction by the immune system of an animal exposed to the phytase variant.
  • One type of immunological response is an allergic response leading to increased levels of IgE in the exposed animal.
  • Low-allergenic variants may be prepared using techniques known in the art.
  • the phytase variant may be conjugated with polymer moieties shielding portions or epitopes of the phytase variant involved in an immunological response. Conjugation with polymers may involve in vitro chemical coupling of polymer to the phytase variant, e.g.
  • Conjugation may in addition or alternatively thereto involve in vivo coupling of polymers to the phytase variant.
  • Conjugation may be achieved by genetic engineering of the nucleotide sequence encoding the phytase variant, inserting consensus sequences encoding additional glycosylation sites in the phytase variant and expressing the phytase variant in a host capable of glycosylating the phytase variant, see e.g. WO 00/26354.
  • Another way of providing low- allergenic variants is genetic engineering of the nucleotide sequence encoding the phytase variant so as to cause the phytase variants to self-oligomerize, effecting that phytase variant monomers may shield the epitopes of other phytase variant monomers and thereby lowering the antigenicity of the oligomers.
  • Such products and their preparation is described e.g. in WO 96/16177.
  • Epitopes involved in an immunological response may be identified by various methods such as the phage display method described in WO 00/26230 and WO 01/83559, or the random approach described in EP 561907.
  • an epitope Once an epitope has been identified, its amino acid sequence may be altered to produce altered immunological properties of the phytase variant by known gene manipulation techniques such as site directed mutagenesis (see e.g. WO 00/26230, WO 00/26354 and/or WO 00/22103) and/or conjugation of a polymer may be done in sufficient proximity to the epitope for the polymer to shield the epitope.
  • site directed mutagenesis see e.g. WO 00/26230, WO 00/26354 and/or WO 00/22103
  • the present invention also relates to nucleic acid sequences comprising a nucleic acid sequence which encodes a phytase variant of the invention.
  • isolated nucleic acid sequence refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90% pure as determined by agarose electrophoresis.
  • an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced.
  • the cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated.
  • the nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
  • the nucleic acid sequences of the invention can be prepared by introducing at least one mutation into a template phytase coding sequence or a subsequence thereof, wherein the mutant nucleic acid sequence encodes a variant phytase.
  • the introduction of a mutation into the nucleic acid sequence to exchange one nucleotide for another nucleotide may be accomplished by any of the methods known in the art, e.g. by site-directed mutagenesis, by random mutagenesis, or by doped, spiked, or localized random mutagenesis.
  • Random mutagenesis is suitably performed either as localized or region-specific random mutagenesis in at least three parts of the gene translating to the amino acid sequence shown in question, or within the whole gene.
  • the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions which are to be changed. The doping or spiking may be performed so that codons for unwanted amino acids are avoided.
  • the doped or spiked oligonucleotide can be incorporated into the DNA encoding the phytase enzyme by any technique, using, e.g., PCR, LCR or any DNA polymerase and ligase as deemed appropriate.
  • the doping is carried out using "constant random doping", in which the percentage of wild-type and mutation in each position is predefined.
  • the doping may be directed toward a preference for the introduction of certain nucleotides, and thereby a preference for the introduction of one or more specific amino acid residues.
  • the doping may be made, e.g., so as to allow for the introduction of 90% wild type and 10% mutations in each position.
  • An additional consideration in the choice of a doping scheme is based on genetic as well as protein-structural constraints.
  • the random mutagenesis may be advantageously localized to a part of the parent phytase in question. This may, e.g., be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme.
  • Alternative methods for providing variants of the invention include gene shuffling e.g. as described in WO 95/22625 or in WO 96/00343, and the consensus derivation process as described in EP 897985.
  • a nucleic acid construct comprises a nucleic acid sequence 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. Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.
  • 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 Vector 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.
  • a nucleic acid sequence encoding a phytase variant of the invention can be expressed using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
  • the recombinant expression vector carrying the DNA sequence encoding a phytase variant of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the phytase variant may also be co-expressed together with at least one other enzyme of animal feed interest, such as a phytase, phosphatase, xylanase, galactanase, aipha- galactosidase, protease, phospholipase, amylase, and/or beta-glucanase.
  • the enzymes may be co-expressed from different vectors, from one vector, or using a mixture of both techniques.
  • the vectors may have different selectable markers, and different origins of replication.
  • the genes can be expressed from one or more promoters.
  • the phytase variant may also be expressed as a fusion protein, i.e. that the gene encoding the phytase variant has been fused in frame to the gene encoding another protein.
  • This protein may be another enzyme or a functional domain from another enzyme.
  • 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.
  • 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, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans and Streptomyces murinus, or gram negative bacteria such as E.
  • the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell.
  • the Bacillus cell is an alkalophilic Bacillus.
  • the introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), using competent cells (see, e.g., Young and Spizizin, 1961 , Journal of Bacteriology 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thome, 1987, Journal of Bacteriology 169: 5771-5278).
  • protoplast transformation see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115
  • competent cells see, e.g., Young and Spizizin, 1961 , Journal of Bacteriology 81 : 823-829,
  • 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). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S. M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
  • the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.
  • the yeast host cell is a Pichia pastoris, Pichia methanolica,
  • Saccharomyces carlsbergensis Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces dougfasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell.
  • the yeast host cell is a Kluyveromyces lactis cell.
  • the yeast host cell is a Yarrowia lipolytica 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 fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides.
  • Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.
  • vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
  • the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusar ⁇ um, 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,
  • the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell.
  • the filamentous fungal host cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, or Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koning
  • 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. and Simon, M.
  • the present invention also relates to methods for producing a phytase of the present invention comprising (a) cultivating a host cell under conditions conducive for production of the phytase; and (b) recovering the phytase.
  • 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 resulting polypeptide may be recovered using methods known in the art.
  • 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 nucleotide sequence encoding a polypeptide having phytase activity of the present invention so as to express and produce the polypeptide in recoverable quantities.
  • 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.
  • 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 having phytase activity of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
  • Transgenic Animals 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.
  • transgenic animal selected cells of a selected animal are transformed with a nucleic acid sequence encoding a polypeptide having phytase activity 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 the polypeptide.
  • the transgene expression vector is 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. et al (1999): Expression of recombinant proteins in the milk of transgenic animals, Gene expression systems: Using nature for the art of expression. J. M. Fernandez and J. P. Hoeffler (eds.), Academic Press.
  • 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.
  • the present invention relates to compositions comprising a polypeptide of the present invention, as well as methods of using these.
  • the 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 granulates or microgranulates.
  • the polypeptide to be included in the composition may be stabilized in accordance with methods known in the art.
  • the phytase of the invention can be used for degradation, in any industrial context, of, for example, phytate, phytic acid, and/or the mono-, di-, tri-, tetra- and/or penta-phosphates of myo-inositol.
  • the phosphate moieties of these compounds 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 manganese, copper and molybdenum.
  • the phytic acid also to a certain extent binds proteins by electrostatic interaction. Accordingly, preferred uses of the polypeptides of the invention are in animal feed preparations (including human food) or in additives for such preparations.
  • the polypeptide of the invention can be used for improving the nutritional value of an animal feed.
  • improving the nutritional value of animal feed including human food
  • the nutritional value of the feed is therefore increased, and the growth rate and/or weight gain and/or feed conversion (i.e. the weight of ingested feed relative to weight gain) of the animal may be improved.
  • polypeptide of the invention can be used for reducing phytate level of manure.
  • animal includes all animals, including human beings. Examples of animals are non-ruminants, and ruminants. Ruminant animals include, for example, animals such as sheep, goat, and cattle, e.g. cow such as beef cattle and dairy cows. In a particular embodiment, the animal is a non-ruminant animal. Non-ruminant animals include mono-gastric animals, e.g.
  • pig or swine including, but not limited to, piglets, growing pigs, and sows
  • poultry such as turkeys, ducks and chickens (including but not limited to broiler chicks, layers)
  • fish including but not limited to salmon, trout, tilapia, catfish and carp
  • crustaceans including but not limited to shrimp and prawn
  • feed or feed composition means any compound, preparation, mixture, or composition suitable for, or intended for intake by an animal.
  • 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 substantially pure.
  • it is well-defined.
  • the term "well-defined” means that the phytase preparation is at least 50% pure as determined by Size-exclusion chromatography (see Example 12 of WO 01/58275).
  • the phytase preparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined by this method.
  • a substantially pure, and/or 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 optimising dosage based upon the desired effect.
  • the phytase polypeptide of the invention need not be that pure; it may e.g. include other polypeptides, in which case it could be termed a phytase preparation.
  • the phytase preparation can be (a) added directly to the feed (or used directly in a treatment process of 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.
  • Such polypeptide preparation may of course be mixed with other polypeptides.
  • the polypeptide can be added to the feed in any form, be it as a relatively pure polypeptide, 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.
  • the present invention relates to compositions for use in animal feed, such as animal feed, and animal feed additives, e.g. premixes.
  • 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.
  • the feed additive 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 polypeptide selected from amongst phytase (EC 3.1.3.8 or 3.1.3.26); phosphatase (EC
  • 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 3.1.4.3
  • phospholipase D EC 3.1.4.4
  • amylase such as, for example, alpha-amylase (EC 3.2.1.1); and/or beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).
  • these other polypeptides are well-defined (as defined above for phytase preparations).
  • the phytase of the invention may also be combined with other phytases, for example ascomycete phytases such as Aspergillus phytases, for example derived from Aspergillus ficuum, Aspergillus niger, or Aspergillus awamori; or basidiomycete phytases, for example derived from Peniophora lycii, Agrocybe pediades, Trametes pubescens, or Paxillus involutus; or derivatives, fragments or variants thereof which have phytase activity.
  • the phytase of the invention is combined with such phytases.
  • 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 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 polypeptides such as an oxidase, an oxygenase or a syntethase.
  • chemicals such as perborate, persulphate, or percarbonate
  • polypeptides such as an oxidase, an oxygenase or a syntethase.
  • Usally fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed.
  • Either of these composition types, when enriched with a polypeptide of the invention, is an animal feed additive of the invention.
  • 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 pantothenate, e.g. Ca-D-panthothenate.
  • trace minerals are manganese, zinc, iron, copper, iodine, selenium, and cobalt.
  • the animal feed additive of the invention comprises at least one of the individual components specified in Table A of WO 01/58275. At least one means either of, one or more of, one, or two, or three, or four and so forth up to all thirteen, or up to all fifteen individual components. More specifically, this at least one individual component is included in the additive of the invention in such an amount as to provide an in-feed-concentration within the range indicated in column four, or column five, or column six of Table A.
  • 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 characterised as indicated in Table B of WO 01/58275, columns 2-3.
  • Fish diets can be characterised 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 (R. 2-5).
  • the nitrogen content is determined by the Kjeldahf 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.
  • the animal feed composition of the invention contains at least one protein.
  • the protein may be an animal protein, such as meat and bone meal, and/or fish meal; or it may be a vegetable protein.
  • 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.
  • Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, triticale, and sorghum.
  • 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.
  • Polypeptides can be added as solid or liquid polypeptide formulations.
  • a solid polypeptide formulation is typically added before or during the mixing step; and a liquid polypeptide preparation is typically added after the pelleting step.
  • the polypeptide may also be incorporated in a feed additive or premix.
  • the final polypeptide concentration in the diet is within the range of 0.01-200 mg polypeptide protein per kg diet, for example in the range of 5-30 mg polypeptide protein per kg animal diet.
  • the phytase of the invention should of course be applied in an effective amount, i.e. in an amount adequate for improving solubilization and/or improving nutritional value of feed. It is at present contemplated that the polypeptide 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; 10-100; 0.05-50; or 0.10-
  • the phytase is purified from the feed composition, and the specific activity of the purified phytase is determined using a relevant assay.
  • the phytase 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 phytase protein per kg feed is calculated.
  • a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1 , 2, 3, 4, 5, 31 , 41 , 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105,
  • a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1, 2, 3, 4, 5, 41 , 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91 , 99, 100, 104, 105, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 136, 137, 141, 154, 161, 162, 164, 167, 171 , 176, 177, 179, 180, 181 , 182, 183, 184, 185, 186, 196, 199, 200, 202, 203,
  • a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1, 2, 3, 4, 5, 31, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111 , 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 136, 137, 141, 154, 161 , 162, 164, 167, 171 , 176, 177, 179, 180, 181 , 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241 , 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 31
  • a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1 , 2, 3, 4, 5, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111 , 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 136, 137, 141 , 154, 161 , 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241 , 247, 273, 276, 281 , 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 324
  • a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 4, 5, 41 , 46, 59, 82, 84, 91 , 99, 105, 107, 109, 111 , 115, 116, 117, 119, 122, 123, 124, 136, 137, 141 , 161, 162, 164, 167, 171, 176, 179, 180, 186, 196, 199, 200, 218, 223, 239, 240, 241 , 247, 273, 276, 281, 282, 283, 284, 289, 294, 299, 308, 314, 324, 339, 351 , 355, 379, 385, 406, 409, 410, and 411; preferably in at least one position selected from the following: 4, 5, 46, 59, 82, 99, 107, 109, 111 , 115,
  • a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 1*, 2 * , 3*, 4P, 5P, 31 C 1 T, 41 P, 46C,D,E, 52C,E, 53V,Q, 55D,I, 57Y, 59C, 74A, 76G, 82E, 84Y, 91 C.P, 99C, 100C, 104A, 105F, 107D,E,G, 109A,G, 111P, 114H,N,T, 115Q, 116A 1 E 1 P 1 T 1 Q 1 117D 1 E 1 K 118I,L,M,T, 119G, K 1 R 1 S, 120K 1 S 1 T 1 Q, 121A,D,M,P,T,V, 122D, 123P.S, 124L,T,V, 136P, 137P, 141C, 154P, 161 P,
  • a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations:: 1*, 2*, 3*, 4P 1 5P 1 31 C 1 T, 41P 1 46C 1 D 1 E, 52C 1 E 1 53V 1 Q 1 55I 1 D 1 57Y, 59C, 74A 1 76G, 82E, 84Y, 91C 1 P, 99C, 100C, 104A, 105F, 107D 1 E 1 G 1 109A 1 G 1 111 P 1 114H 1 N 1 T 1 115Q, 116A 1 E 1 P 1 T 1 Q 117D 1 E 1 K, 118I 1 M 1 L 1 T, 119G 1 K 1 R 1 S, 120K 1 S 1 T 1 Q 1 121A 1 D 1 M 1 P 1 V, 122D 1 123P 1 S 1 124L 1 T 1 V, 136P, 137P 1 141C 1 154P 1 161P 1 16
  • a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 1*, 2*, 3 * , 4P 1 5P 1 31C 1 T, 41P 1 46C 1 D 1 E, 52C 1 E 1 53V 1 Q 1 55D 1 I 1 57Y 1 59C, 74A, 76G, 82E 1 84Y, 91C 1 P 1 99C, 100C, 104A, 105F, 107D 1 E 1 G, 109A 1 G 1 111P 1 114H 1 N 1 T, 115Q 1 116A 1 E 1 P 1 T 1 Q 1 117D 1 E 1 K, 118I 1 L 1 M 1 T 1 119G 1 K 1 R 1 S 1 120K 1 S 1 T 1 Q 1 121A 1 D 1 M 1 PJ 1 V 1 122D, 123P 1 S, 124LJ 1 V 1 136P, 137P 1 141 C 1 154P 1 161 P 1
  • a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations:: 1*, 2*, 3*, 4P 1 5P 1 31 C 1 T 1 41 P, 46C 1 D 1 E, 52C.E, 53V 1 Q, 551, D, 57Y 1 59C, 74A 1 76G 1 82E 1 84Y, 91C 1 P 1 99C, 100C, 104A, 105F, 107D 1 E 1 G, 109A 1 G, 111 P, 114H 1 N 1 T 1 115Q 1 116A 1 E 1 P 1 T 1 Q 117D 1 E 1 K 1 1181,M 1 L 1 T 1 119G 1 K 1 R 1 S 1 120K 1 S 1 T 1 Q 1 121A 1 D 1 M 1 P 1 V 1 122D 1 123P 1 S, 124LJ.V, 136P, 137P, 141C 1 154P, 161P 1 162C 1
  • a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 1 H 1 K 1 R, 6OP, 105E, 106A 1 G, 155F 1 157F, 173P, 175L 1 188P, 205P, 215M 1 231 P, 254Y, 280P, 330D, and/or 371 P; preferably 1 K; with the proviso that the phytase is not SEQ ID NO:3, not SEQ ID NO:4, not SEQ ID
  • XII A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 1H 1 R, 60P 1 105E 1 106A 1 G 1 157F, 173P, 175L, 188P 1 205P 1 215M, 231 P 1 254Y, 280P. XIII.
  • a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 52C, 141C 1 162C, 31 C, 52C, 99C, 59C, 100C, 141C/199C, 4P, 5P, 111 P, 137P, 161 P 1 52E, 57Y, 76G, 107D 1 107G 1 109A 1 1*, 1*/2*, 1*/2*/3*, 121T 1 273L, 285G, 286Q, 299L, 362K, 331 K/55D, 107E, 46E, 82E, 119R, 119K 1 164E 1 223E, 276R, 276K, 362R, 379R 1 379K, 385D, 410D, 410E, 411 R 1 411 K, 53V, 121 D, 167Q, 196Q, 200K, 202N, 218Q, 241 Q, 285N
  • XV The phytase of any one of embodiments 1-13 which is a variant of the phytase of SEQ ID NO:3.
  • XVI The phytase of any one of embodiments 1-13 which is a variant of the phytase of SEQ ID NO:4.
  • HXX The phytase of any one of embodiments 1-13 which is a variant of the phytase of SEQ ID NO:9.
  • XXI The phytase of any one of embodiments 1-20, which has an improved thermostability, an improved pH profile, an improved specific activity, an amended glycosylation pattern, an improved temperature profile, an improved performance in animal feed, and/or which incorporates a change of a potential protease cleavage site.
  • XXII An isolated nucleic acid sequence comprising a nucleic acid sequence which encodes the phytase of any of embodiments I-XXI.
  • XXIII A nucleic acid construct comprising the nucleic acid sequence of embodiment XXII operably linked to one or more control sequences that direct the production of the phytase in a suitable expression host.
  • XXIV A recombinant expression vector comprising the nucleic acid construct of embodiment
  • a recombinant host cell comprising the nucleic acid construct of embodiment XXIII and/or the expression vector of embodiment XXIV.
  • a method for producing the phytase of any one of embodiments I-XXI comprising (a) cultivating the host cell of embodiment XXV to produce a supernatant comprising the phytase; and (b) recovering the phytase.
  • XXVII A transgenic plant, or plant part, capable of expressing a phytase of any one of embodiments I-XXI.
  • I IXXX A transgenic, non-human animal, or products, or elements thereof, being capable of expressing a phytase of any one of embodiments I-XXI.
  • composition comprising at least one phytase of any one of embodiments I-XXI, and (a) at least one fat soluble vitamin; (b) at least one water soluble vitamin; and/or
  • composition of embodiment IXX further comprising at least one enzyme selected from the following group of enzymes: amylase, phytase, phosphatase, xylanase, galactanase, alpha-galactosidase, protease, phospholipase, and/or beta-glucanase.
  • composition of any one of embodiments IXX-XXX which is an animal feed additive.
  • XXXII An animal feed composition having a crude protein content of 50 to 800 g/kg and comprising the phytase of any one of embodiments I-XXI or the composition of any one of embodiments IXXX-XXXI.
  • XXXIII A method for improving the nutritional value of an animal feed, wherein the phytase of any one of embodiments I-XXI or the composition of any one of embodiments IXXX-XXXI is added to the feed.
  • XXXIV A process for reducing phytate levels in animal manure comprising feeding an animal with an effective amount of the feed of embodiment XXXI I .
  • a method for the treatment of vegetable proteins comprising the step of adding the phytase of any one of embodiments I-XXI or the composition of any one of embodiments IXXX-XXXI to at least one vegetable protein or protein source.
  • XXXVI Use of the phytase of any one of embodiments I-XXI or the composition of any one of embodiments IXXX-XXXI in animal feed; in the preparation of animal feed; for improving the nutritional value of animal feed; for reducing phytate levels in animal manure; for the treatment of vegetable proteins; or for liberating phosphorous from a phytase substrate.
  • a phytase which has at least 70% identity to SEQ ID NO:2 and which comprises at (east one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1 , 2, 3, 4, 5, 31, 41 , 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105,
  • a phytase which has at least 70% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1 , 2, 3, 4, 5, 31 , 41 , 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111 , 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 136, 137, 141 , 154,
  • a phytase which has at least 70% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1, 2, 3, 4, 5, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111 , 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 136, 137, 141 , 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241 , 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 324, 339, 351, 355,
  • a4) The phytase of any one of embodiments a2)-a3), which comprises at least one of the following alterations: (i) 31C, 46C 1 52C, 59C, 91C, 99C 1 100C 1 141C 1 162C 1 176C 1 177C 1 199C, and/or 247C; (ii) 4P, 5P, 41 P, 91 P, 111P, 136P, 137P, 154P, 161 P, 240P, 282P 1 283P, 284P, 289P, and/or 355P;
  • the phytase of embodiment c) or c1) which comprises at least one of the one or more alterations of feature (x) of embodiment c) and has an improved specific activity. h).
  • the phytase of embodiment c) or c1) which comprises at least one of the one or more alterations of feature (xi) of embodiment c) and has an amended glycosylation pattern.
  • the phytase of embodiment c) or c1) which comprises the alteration of feature (xii) of embodiment c) which changes a potential protease cleavage site.
  • T284P, G289P, N4P, and/or G5P are T284P, G289P, N4P, and/or G5P;
  • T284P, G289P, N4P, and/or G5P are T284P, G289P, N4P, and/or G5P;
  • the phytase of embodiment m) which is a variant of SEQ ID NO:4. o).
  • T284P, G289P, N4P, and/or G5P are T284P, G289P, N4P, and/or G5P;
  • T284P, G289P, N4P, and/or G5P are T284P, G289P, N4P, and/or G5P;
  • T284P, G289P, N4P, and/or G5P are T284P, G289P, N4P, and/or G5P;
  • the phytase of embodiment s) which is a variant of SEQ ID NO:9.
  • An isolated nucleic acid sequence comprising a nucleic acid sequence which encodes the phytase of any of embodiment a)-t) including a1)-a5) and d).
  • a nucleic acid construct comprising the nucleic acid sequence of embodiment u) operably linked to one or more control sequences that direct the production of the phytase in a suitable expression host.
  • a recombinant expression vector comprising the nucleic acid construct of embodiment v). x).
  • a recombinant host cell comprising the nucleic acid construct of embodiment v) and/or the expression vector of embodiment w). y).
  • a composition comprising at least one phytase of any one of embodiment a)-t) including a1)-a5) and d), and
  • composition of embodiment oe) further comprising at least one enzyme selected from the following group of enzymes: amylase, phytase, phosphatase, xylanase, galactanase, alpha-galactosidase, protease, phospholipase, and/or beta-glucanase.
  • enzymes selected from the following group of enzymes: amylase, phytase, phosphatase, xylanase, galactanase, alpha-galactosidase, protease, phospholipase, and/or beta-glucanase.
  • bb The composition of any one of embodiment oe)-aa) which is an animal feed additive.
  • An animal feed composition having a crude protein content of 50 to 800 g/kg and comprising the phytase of any one of embodiment a)-t) including a1)-a5) and d) or the composition of any one of embodiment oe)-aa). dd).
  • a method for improving the nutritional value of an animal feed wherein the phytase of any one of embodiment a)-t) including a1)-a5) and c1) or the composition of any one of embodiment oe)-aa) is added to the feed.
  • a process for reducing phytate levels in animal manure comprising feeding an animal with an effective amount of the feed of embodiment cc). ff).
  • a method for the treatment of vegetable proteins comprising the step of adding the phytase of any one of embodiment a)-t) including a1)-a5) and d) or the composition of any one of embodiment oe)-aa) to at least one vegetable protein or protein source.
  • gg Use of the phytase of any one of embodiment a)-t) including a1)-a5) and d) or the composition of any one of embodiment oe)-aa) in animal feed; in the preparation of animal feed; for improving the nutritional value of animal feed; for reducing phytate levels in animal manure; for the treatment of vegetable proteins; or for liberating phosphorous from a phytase substrate.
  • Example 1 Preparation of variants, and test of thermostability and pH profile
  • NO:2 is generated by methods known in the art, and the constructs are fused by PCR to the DNA coding for the signal peptide described by Takami et al in Biosci. Biotechnol. Biochem. 56:1455 (1992) and integrated by homologous recombination into the genome of a Bacillus subtilis host cell (see Diderichsen et al (1990), J. Bacterid., 172, 4315-4321) using standard techniques. The genes are expressed under the control of a triple promoter system (as described in WO 99/43835) and the resulting phytase proteins purified using conventional methods.
  • a triple promoter system as described in WO 99/43835
  • the temperature stability of a phytase variant may be determined in the following way:
  • 500 microliter protein solution of the variant and of the reference protein (SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:6) having approximately 10 microgram protein per ml, and being dissolved in 0.1 IVI Na-acetate buffer, pH 5.5, are split in two portions, one portion is incubated at a desired elevated temperature (e.g. 50 0 C, 55 0 C, 60 0 C, 65°C, 70 0 C, 75 0 C, 80°C, or 85 0 C) in plastic containers, the other is stored at 5°C.
  • a desired elevated temperature e.g. 50 0 C, 55 0 C, 60 0 C, 65°C, 70 0 C, 75 0 C, 80°C, or 85 0 C
  • the protein solutions are transferred to an ice-bath and the activity of the cooled as well as the heated sample is measured by the phosphatase assay described below (buffer blind subtracted).
  • the residual activity is defined as the activity after heat-treatment divided by the activity of the cooled sample (in %).
  • a variant is considered to be more temperature stable (thermostable) if the residual activity in the phosphatase, or phytase, assay is higher, as compared to the reference.
  • 75 microliter phytase-containing enzyme solution is dispensed in a microtiter plate well, e. g. NUNC 269620 and 75 microliter substrate is added (for preparing the substrate, two 5 mg p-nitrophenyl phosphate tablets (Sigma, Cat.No. N-9389) are dissolved in 10 ml 0.1 M Na- acetate buffer, pH 5.5). The plate is sealed and incubated 15 min., shaken with 750 rpm at 37 0 C. After the incubation time 75 microliter stop reagent is added (the stop reagent is 0.1 M di-sodiumtetraborate in water) and the absorbance at 405 nm is measured in a microtiter plate spectrophotometer.
  • the stop reagent is 0.1 M di-sodiumtetraborate in water
  • One phosphatase unit is defined as the enzyme activity that releases 1 micromol phosphate/min under the given reaction conditions (buffer blind subtracted).
  • Differential Scanning Calorimetry may be performed at various pH-values using the VP-DSC from Micro CaI. Scans are performed at a constant scan rate of 1.5°C/min from 20-90 0 C.
  • the phytases are desalted using NAP-5 columns (Pharmacia) equilibrated in the appropriate buffers (e.g. 0.1M glycine-HCI, pH 2.5 or 3.0; 2OmM sodium acetate pH 4.0; 0.1 M sodium acetate, pH 5.5; 0.1 M Tris-HCI, pH 7.0).
  • Data- handling is performed using the MicroCal Origin software (version 4.10), and the denaturation temperature, Td (also called the melting temperature, Tm) is defined as the temperature at the apex of the peak in the thermogram.
  • An amendment of the pH profile of a phytase variant may be determined as follows: The activity is measured at pH 3.5 (0.1 M acetate buffer, pH 3.5) and at pH 5.5 (0.1 M acetate buffer, pH 5.5), in both cases the buffer blind is subtracted. The activity determined at pH 3.5 is divided by the activity determined at pH 5.5, i.e. the two absorbancy measurements are divided (see below). To measure the activity, supernatants of the variants and references are appropriately diluted (e.g. 1 :5000) in the respective buffer. 75 microliter of the respective enzyme solution is dispensed in a microtiter plate well, e. g.
  • NUNC 269620 and 75 microliter substrate with the corresponding pH is added (the substrate is prepared by dissolving two 5 mg p-nitrophenyl phosphate tablets (Sigma, Cat. No. N-9389) in 10 ml 0.1 M Na-acetate buffer, pH 5.5 and 10 ml 0.1 M acetate buffer, pH 3.5, respectively).
  • the plate is sealed and incubated 15 min., shaken with 750 rpm at 37°C. After the incubation time 75 microliter stop (0.1 M di-sodiumtetraborate in water) reagent is added and the absorbance at 405 nm is measured in a microtiter plate spectrophotometer.
  • 75 microliter phytase-containing enzyme solution is dispensed in a microtiter plate well, e. g. NUNC 269620, and 75 microliter substrate is added (prepared by dissolving 100 mg sodium phytate from rice (Aldrich Cat.No. 274321) in 10 ml 0.25 M Na-acetate buffer, pH 5.5). The plate is sealed and incubated 15 min. shaken with 750 rpm at 37°C.
  • stop reagent is added (the stop reagent being prepared by mixing 10 ml molybdate solution (10% (w/v) Ammonium hepta-molybdate in 0.25% (w/v) ammonia solution); 10 ml ammonium vanadate (0.24% commercial product from Bie&Berntsen, Cat.No. LAB17650) and 21.7 % (w/v) nitric acid)
  • the absorbance at 405 nm is measured in a microtiter plate spectrophotometer.
  • the phytase activity is expressed in the unit of FYT, one FYT being the amount of enzyme that liberates 1 micromol inorganic ortho-phosphate per min. under the conditions above.
  • An absolute value for the measured phytase activity is obtained by reference to a standard curve prepared from appropriate dilutions of inorganic phosphate or to a standard curve made from dilutions of a phytase enzyme preparation with known activity (such standard enzyme preparation with a known activity is available on request from Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd).
  • the phytase of SEQ ID NO:2 was expressed in Bacillus subtilis as described in Example 1 , and purified using conventional methods: Centrifugation, germ filtration, ammonium sulphate precipitation (80% ammonium sulphate saturation), centrifugation, re- suspension of pellets in buffer A (50 mM sodium acetate, 1.5 M ammonium sulphate pH 4.5), filtration, hydrophobic interaction chromatography (Phenyl Toyopearl, loading with buffer A, eluting with buffer B (50 mM sodium acetate pH 4.5)), and cation exchange chromatography (SP-sepharose, loading with 10 mM sodium citrate pH 4.0, eluting with a linear salt gradient (10 mM sodium citrate pH 4.0 + 1 M NaCI).
  • the phytase of SEQ ID NO:2 was expressed in Pichia pastoris as generally described by Rodriguez et al in Archives of Biochemistry and Biophysics, vol. 382, no. 1, 2000, pp. 105-112.
  • the phytase was purified from the supernatant of the fermentation broth as follows: Precipitation with ammonium sulfate (80% saturation), re-dissolution in 10 ml 25mM sodium acetate buffer pH4.5, dialysis against the same buffer, and filtration through a 0.45 mm filter.
  • the Pichia- and the Bacillus-expressed phytase of SEQ ID NO:2 were subjected to thermostability measurements by Differential Scanning Calorimetry (DSC).
  • Samples (less than 3 ml in volume) were dialyzed in a cold room (approx. 5 degrees centigrade) for a minimum of 1 hour against 500 ml of 20 mM sodium acetate buffer pH 4.0. The sample was transferred to 500 ml of fresh, cold buffer preparation and left to dialyze overnight. The samples were filtered using a 0.45 micrometer syringe filter, volume adjusted to approx. 1.5 ml using the dialysis buffer, and A 28 o (absorbancy at 280nm) recorded. The dialysis buffer was used as reference in the DSC scans. The samples were degassed using vacuum suction and stirring for approx. 10 minutes.
  • the enzyme concentrations of the samples were approx. 1 - 1.5 mg/ml as estimated by
  • the thermal unfolding temperature (Td) was evaluated using MicroCal Origin software (version 4.10) and the denaturation temperature determined as the temperature at the apex in the thermogram.
  • the Pichia-expressed phytase was heavily glycosylated, as visualized by a broad range of molecular masses using mass spectrometry (Maldi-TOF), whereas the Bacillus- expressed phytase was not glycosylated.
  • a protein-engineered variant of the phytase of SEQ ID NO:2 having the substitution R339D was prepared and expressed in Aspergillus oryzae using methods known in the art. Its denaturation temperature, Td, was determined to 62.5°C (20 mM sodium acetate, pH 4.0), using DSC as described in Example 2.
  • the R339D substitution furthermore serves to remove a Kex2 protease cleavage site of potential relevance for expression in Aspergillus.
  • Example 4 Animal feed and animal feed additives comprising a phytase variant
  • a formulation of phytase variant R339D of SEQ ID NO:2 containing 0.15 g phytase enzyme protein is added to the following premix (per kilo of premix):
  • the ingredients are mixed, and the feed is pelleted at the desired temperature, e.g. 60, 65, 75, 80, 85, 90 or even 95 0 C.
  • SEQ ID NO:2 Eight variants of SEQ ID NO:2 (the alterations as compared to SEQ ID NO:2 are shown in Table 3 below) were prepared as described in Example 1.
  • the two reference phytases having SEQ ID NO:2 and SEQ ID NO:3 were prepared in the same manner.
  • the temperature stability was determined as follows:
  • the performance in animal feed of a phytase variant is compared, in an in vitro model, to the performance of a reference protein such as SE ⁇ Q ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:6.
  • the in vitro model simulates gastro-intestinal conditions in a monogastric animal and correlates well with results obtained in animal trials in vivo. The comparison is performed as follows:
  • Phytase activity in the variant sample is determined as described in Example 1 under "Determination of phytase activity”.
  • Feed samples composed of 30% soybean meal and 70% maize meal with added CaCI 2 to a concentration of 5 g calcium per kg feed are then prepared and pre-incubated at 40°C and pH 3.0 for 30 minutes followed by addition of pepsin (3000 U/g feed) and suitable dosages of the phytases (identical dosages are used for all phytases to be tested to allow comparison), for example between 0.25 to 0.75 phytase units FYT/g feed.
  • a blank with no phytase activity is also included as reference.
  • the samples are then incubated at 40 0 C and pH 3.0 for 60 minutes followed by pH 4.0 for 30 minutes.
  • the reactions are stopped and phytic acid and inositol-phosphates extracted by addition of HCI to a final concentration of 0.5 M and incubation at 40 0 C for 2 hours, followed by one freeze-thaw cycle and 1 hour incubation at 40 0 C.
  • Phytic acid and inositol-phosphates are separated by high performance ion chromatography as described by Chen et al in Journal of Chromatography A (2003) vol. 1018, pp. 41- 52 and quantified as described by Skoglund et al in J. Agric. Food Chem. (1997), vol. 45, pp. 431-436.
  • IP-P inositol-phosphate bound phosphorous
  • the blank and the reference (SEQ ID NO:2) values are averages from a number of independent determinations, whereas the other values are based on single determinations.
  • the blank value is average from a number of independent determinations, wheres the other values are based on single determinations.
  • Variants 015, 016, 018, 040, 041 , 042, 044, and 050 appear to have an in vitro performance which is at least as good or better than the phytases of SEQ ID NO:2 and 3.
  • Variants 030, 031 , 037, 056, 072, 085, 087, 089, 090, 095, 098, and 125 appear to perform at least as good in vitro as the phytase of SEQ ID NO:3.
  • the specific activity of a phytase variant is determined on highly purified samples dialysed against 250 mM sodium acetate, pH 5.5. The purity is checked beforehand on an SDS poly acryl amide gel showing the presence of only one component.
  • the protein concentration is determined by amino acid analysis as follows: An aliquot of the sample is hydrolyzed in 6N HCI, 0.1 % phenol for 16 h at 11 O 0 C in an evacuated glass tube. The resulting amino acids are quantified using an Applied Biosystems 420A amino acid analysis system operated according to the manufacturer's instructions. From the amounts of the amino acids the total mass - and thus also the concentration - of protein in the hydrolyzed aliquot can be calculated. The phytase activity is determined in the units of FYT as described in Example 1
  • Determination of phytase activity is calculated as the phytase activity measured in FYT units per mg phytase variant enzyme protein.
  • the specific activity for the phytase of SEQ ID NO:2 and variant 072 was determined as described above.
  • the specific activity of variant 072 was 86% of the specific activity of the phytase of SEQ ID NO:2.
  • the uncertainty (standard deviation) is estimated to approximately 10%, which is mainly due to the phytase activity assay based on a complex substrate.
  • Example 8 Temperature stability A number of variants of SEQ ID NO:2 were prepared as described in Example 1 , and the Bacillus subtilis host strains grown in 100ml PS1 medium (100g/L sucrose, 40g/L Soy flakes, 10g/L Na 2 HPO 4 .12H 2 O, 0.1ml/L Dowfax 63N10 (Dow)) in 500ml shake flasks for four days at 3O 0 C at 300 rpm.
  • PS1 medium 100g/L sucrose, 40g/L Soy flakes, 10g/L Na 2 HPO 4 .12H 2 O, 0.1ml/L Dowfax 63N10 (Dow)
  • Two reference phytases were prepared in the same manner, viz. the phytase having SEQ ID NO:3 (corresponding to variant N31 D/Q139K/L197F/N316K of SEQ ID NO:2), and the phytase having SEQ ID NO:4 (corresponding to variant N31 D/N121T/K132T/Q139K of SEQ ID NO:2).
  • the phytase having SEQ ID NO:9 was included for comparison (corresponding to variant Q3P/N31 D/N121T/K132T/Q139K of SEQ ID NO:2).
  • the temperature stability of the variants and the reference phytases was determined as follows:
  • the supematants were diluted ten times by adding 2OuI (microliter) supernatant to 18OuI 0.1 M Na-acetate buffer, pH5.5 + 0.005% Tween-20.
  • the diluted enzymes were split in two portions, one portion was incubated at 6O 0 C in plastic containers, and the other portion was stored at 5°C. After 30 minutes incubation at 60 0 C the protein solutions were transferred to an ice-bath. After dilution 1:10 in 0.1 M Na-acetate buffer, pH5.5, and 0.005% Tween-20, the activity of the cooled and heated sample was measured by the phosphatase assay of Example 1 ("Determination of phosphatase activity"), buffer blind subtracted.
  • Table 5 is a list of variants with improved temperature stability as compared to the reference phytases. For each variant, the table also specifies the alterations as compared to SEQ ID NO:2.
  • the enzyme activity in absorption units (AU)) after incubation for 30 minutes at 5°C and 6O 0 C, respectively, was determined, and the residual activity (RA) calculated as the activity of the heat-treated sample (60°C incubation) divided by the activity of the cooled sample (5 0 C incubation).
  • the residual activity results were normalized to the residual activity of the phytase of SEQ ID NO:2, having been expressed and treated in the same manner.
  • the resulting Improvement Factor (IF) Is shown in Table 5.
  • the IF is 1.0, whereas the two reference phytases of SEQ ID NO:3 and 4 were less thermostable than the phytase of SEQ ID NO:2, which is apparent from the fact that the IF for these two phytases was only 0.1 and 0.3, respectively.
  • a number of purified variants of SEQ ID NO:2 were prepared as generally described in Example 1.
  • Two reference phytases were prepared in the same manner, viz. the phytase having SEQ ID NO:3 (corresponding to variant N31D/Q139K/L197F/N316K of SEQ ID NO:2), and the phytase having SEQ ID NO:4 (corresponding to variant N31D/N121T/K132T/Q139K of SEQ ID NO:2).
  • the phytase having SEQ ID NO:9 was included for comparison (corresponding to variant Q3P/N31D/N121T/K132T/Q139K of SEQ ID NO:2).
  • Td denaturation temperatures
  • Example 10 Purification and temperature profile
  • the phytase variants and reference and comparative phytases used herein were purified as follows: The fermentation supernatant with the phytase was first centrifuged at 7200rpm and 5 0 C for one hour and filtered through a sandwich of four Whatman glass microfibre filters (2.7, 1.6, 1.2 and 0.7 micrometer). Following this the solution was sterile filtered (either through a Fast PES Bottle top filter with a 0.22 ⁇ m cut-off or through a Seitz- EKS depth filter using pressure). The solution was added solid ammonium sulfate giving a final concentration of 1.5M and the pH was adjusted to 6.0 using 6M HCI.
  • the phytase-containing solution was applied to a butyl-sepharose column, approximately 50ml in a XK26 column, using as buffer A 25mM bis-tris (Bis-(2- hydroxyethyl)imino-tris(hydroxymethyl)methan)) + 1.5M ammonium sulfate pH ⁇ .O, and as buffer B 25mM bis-tris pH ⁇ .O.
  • the fractions from the column were analyzed for activity using the phosphatase assay (see Example 1 , "Determination of phosphatase activity") and fractions with activity were pooled. The pooled fractions were dialyzed extensively against 1OmM sodium acetate pH4.5.
  • the phytase-containing solution was purified by chromatography on S Sepharose, approximately 75ml in a XK26 column, using as buffer A 5OmM sodium acetate pH4.5, and as buffer B 5OmM sodium acetate + 1M NaCI pH4.5. Again the fractions from the column were analyzed for activity and fractions with activity were pooled. Finally, the solution containing the purified phytase was concentrated using an Amicon ultra-15 filtering device with a 1OkDa cut-off membrane.
  • the temperature profile (phytase activity as a function of temperature) of the variants was determined in the temperature range of 20-90°C essentially as described in Example 1 ("Determination of phytase activity"), however, the enzymatic reactions (100 microliter phytase-containing enzyme solution + 100 microliter substrate) were performed in PCR tubes instead of microtiter plates. After a 15 minute reaction period at desired temperature the tubes were cooled to 20 0 C for 20 seconds and 150 microliter of the reaction mixture was transferred to a microtiter plate. 75 microliter stop reagent was added and the absorbance at 405 nm was measured in a microtiter plate spectrophotometer. The results are summarized in Table 7 below. The numbers given for each temperature (20-90 0 C in 10 0 C steps) are relative activity (in %) normalized to the value at optimum.
  • Variants 030, 031 , 037, 044, 056, 062, 072, 083, and 093 have a higher relative activity at 7O 0 C as compared to the reference phytases 026 and 102.
  • Example 11 pH profile
  • the pH profiles (phytase activity as a function of pH) of a number of variants and the same reference and comparative phytases as used in the previous examples were determined at 37 0 C in the pH range of 2.0 to 7.5 (in 0.5 pH-unit steps) as described in Example 1 ("Determination of phytase activity"), except that a buffer cocktail (5OmM glycine, 5OmM acetic acid and 5OmM Bis-Tris was used instead of the 0.25M sodium acetate pH5.5 buffer.
  • a buffer cocktail 5OmM glycine, 5OmM acetic acid and 5OmM Bis-Tris was used instead of the 0.25M sodium acetate pH5.5 buffer.
  • Table 8 The results are summarized in Table 8 below.
  • the numbers given for each pH (2.0-7.5) are relative activity (in %) normalized to the value at optimum.
  • the temperature stability of a number of purified variants and the same reference and comparative phytases as in the previous examples was determined by measuring residual phytase activity after incubation at 70 0 C and pH 4.0 (0.1 M sodium acetate). The phytases were incubated and samples were withdrawn after 0, 10, 30 and 60 minutes and cooled on ice. The residual activity at pH 5.5 was determined using the method described in Example 1 ("Determination of phytase activity"). The results, normalized to the activity found at 0 minutes, are shown in Table 9 below.
  • Example 13 Calculating percentage of identity and identifying corresponding positions
  • SEQ ID NO:9 was aligned with SEQ ID NO:2 using the Needle program from the
  • EMBOSS package version 2.8.0 The substitution matrix used was BLOSUM62, the gap opening penalty was 10.0, and the gap extension penalty was 0.5.
  • Fig. 2 The alignment of Fig. 2 is also used for deriving corresponding positions as follows: Amino acids on top of each other in this alignment are in corresponding positions. E.g. amino acid Q in position 3 of SEQ ID NO:2 corresponds to amino acid P in position number 25 of
  • SEQ ID NO:9 may be considered a variant of SEQ ID NO:2 which comprises the substitution Q3P.
  • Other differences in the form of substitutions within the overlap of the alignment are found in positions 31 , 121 , 132, and 139, viz. N31D, N121T, K132T, and Q139K. Additional differences are found in the N-terminus, where SEQ ID NO:9 has an extension of 22 amino acids as compared to SEQ ID NO:2.
  • SEQ ID NO:9 may therefore be considered the following variant of SEQ ID NO:2:

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Food Science & Technology (AREA)
  • Animal Husbandry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Fodder In General (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Detergent Compositions (AREA)

Abstract

The present invention relates to a phytase which has at least 74% identity to a phytase derived from Citrobacter braakii and comprises at least one alteration as compared to this phytase. These phytase variants have amended, preferably improved, properties, such as thermostability, temperature profile, pH profile, specific activity, performance in animal feed, reduced protease sensitiliby, and/or an amended glycosylation pattern. The invention also relates to DNA encoding these phytases, methods of their production, as well as the use thereof, e.g. in animal feed and animal feed additives.

Description

PHYTASE VARIANTS
Reference to sequence listing This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.
Field of the Invention
The present invention relates to a phytase which has at least 74% identity to a phytase derived from Citrobacter braakii ATCC 51113 and comprises at least one alteration as compared to this phytase (i.e., is a variant thereof). The invention also relates to DNA encoding these phytases, methods of their production, as well as the use thereof, e.g. in animal feed and animal feed additives. The mature part of the Citrobacter braakii ATCC 51113 phytase is included in the sequence listing as SEQ ID NO:2.
Background of the Invention
Background art
The sequence of the phyA gene from Citrobacter freundii has been submitted by Zinin et al to the EMBL/GenBank/DDBJ databases with accession no. AY390262. The corresponding phytase amino acid sequence is found in the UniProt/TrEMBL databases with accession no. Q676V7. The expected mature part of Q676V7 is included in the present sequence listing as SEQ ID NO:4.
WO-2004/085638 discloses, as SEQ ID NO:7, the amino acid sequence of a phytase from Citrobacter braakii YH-15, deposited as KCCM 10427. The mature part of this amino acid sequence is included herein as SEQ ID NO:3. This sequence is also found in the database
Geneseqp with accession no. ADU50737.
WO 2006/037328 discloses the wildtype phytase of Citrobacter braakii ATCC 51113
(i.e., SEQ ID NO:2 herein), as well as a variant thereof, which is also included in the present sequence listing, viz. as SEQ ID NO:6. WO 2006/038062 and WO 2006/038128 both disclose the amino acid sequence of the phytase gene of Citrobacter freundii P3-42, deposited under accession number NCIMB 41247.
This amino acid sequence is included herein as SEQ ID NO:9.
It is an object of the invention to provide phytases of amended, preferably, improved properties. Non-limiting examples of such properties are: Thermostability, temperature profile pH profile, specific activity, performance in animal feed, protease-sensibility, and/or glycosylation pattern. Summary of the Invention
The present invention relates to a phytase which has at least 74% identity to SEQ ID
NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1, 2, 3, 4, 5, 31 , 41 , 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111 , 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124,
136, 137, 141 , 154, 161 , 162, 164, 167, 171 , 176, 177, 179, 180, 181 , 182, 183, 184, 185,
186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241 , 247, 273, 276, 281 , 282, 283, 284,
285, 286, 289, 294, 299, 308, 314, 316, 324, 331 , 339, 351 , 355, 362, 379, 385, 406, 409,
410, and 411; with the proviso that the phytase is not SEQ ID NO:3, not SEQ ID NO:4, and not SEQ ID NO:6.
The invention also relates to a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 1 H1K1R, 6OP, 105E, 106A.G,
155F, 157F, 173P, 175L, 188P, 205P, 215M, 231 P, 254Y, 280P, 330D, and/or 371 P; with the proviso that the phytase is not SEQ ID NO:3, not SEQ ID NO:4, not SEQ ID NO:6, and not SEQ ID NO:9 and the variants thereof listed in Fig. 1.
The invention also relates to DNA encoding these phytases, methods of their production, as well as the use thereof, e.g. in animal feed and animal feed additives.
Brief Description of the Drawings Fig. 1 corresponds to Table 2 of WO 2006/038062 and discloses a number of variants of the Citrobacter freundii NCIMB 41247 phytase which has the amino acid sequence of SEQ ID NO:9; and
Fig. 2 is an alignment of the phytases of SEQ ID NO:2 and 9.
The position numbers in Fig. 1 refer to the numbering of SEQ ID NO:9. The corresponding SEQ ID NO:2 positions can be found by deduction of 22 (e.g., variant P229S of Fig.1 means variant P207S using the numbering of the present application).
Detailed Description of the Invention
In a first aspect, the present invention relates to a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID
NO:2 in at least one position selected from the following: 1 , 2, 3, 4, 5, 31, 41 , 46, 52, 53, 55,
57, 59, 74, 76, 82, 84, 91 , 99, 100, 104, 105, 107, 109, 111 , 114, 115, 116, 117, 118, 119,
120, 121 , 122, 123, 124, 136, 137, 141 , 154, 161 , 162, 164, 167, 171 , 176, 177, 179, 180,
181 , 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241 , 247, 273, 276, 281 , 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316, 324, 331 , 339, 351 , 355,
362, 379, 385, 406, 409, 410, and 411 ; with the proviso that the phytase is not SEQ ID NO:3, not SEQ ID NO:4, and not SEQ ID NO:6. The percentage of identity is determined as described in the section "Phytase Polypeptides, Percentage of Identity".
The position numbers refer to the position numbering of SEQ ID NO:2, as described in the section "Position Numbering." Positions corresponding to these SEQ ID NO:2 position numbers in other phytases are determined as described in the section "Identifying Corresponding Position Numbers."
The phytase of the invention is a variant of the phytase of SEQ ID NO:2, viz. it is not identical to SEQ ID NO:2, as it comprises at least one alteration as compared to SEQ ID NO:2. In a particular embodiment, the phytase of the invention comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1 , 2, 3, 4, 5, 31 , 46, 52, 53, 55, 57, 59, 76, 82, 99, 100, 107, 109, 111 , 114, 115, 116, 117, 118, 1 19, 120, 121 , 122, 123, 124, 137, 141 , 161 , 162, 164, 167, 179, 180, 181 , 182, 183, 184, 185, 186, 196, 199, 200, 202, 218, 223, 241 , 273, 276, 285, 286, 299, 314, 331 , 339, 362, 379, 385, 406, 410, and 41 1.
In another particular embodiment the phytase of the invention is not SEQ ID NO:9. In a still further particular embodiment, the phytase of the invention is not the variants of SEQ ID NO:9 listed in Fig.1.
In a preferred embodiment, the phytase of the invention comprises at least one of the following alterations: 1*, 2*, 3*, 4P, 5P, 31C1T, 41 P, 46C1D1E, 52C.E, 53V,Q, 55D1I, 57Y, 59C,
74A, 76G, 82E, 84Y, 91 C1P, 99C, 100C, 104A, 105F, 107D,E,G, 109A1G1 111 P1 1 14H1N1T,
115Q1 116A,E,P,T,Q, 117D,E,K 1181,L1M1T1 119G1K1R1S, 120K,S,T,Q, 121A,D,M,P,T,V, 122D,
123P,S, 124L1T1V, 136P, 137P, 141 C, 154P, 161 P, 162C, 164D,E, 167Q, 171T, 176C, 177C,
179G.I, K1N1Q, 180A,E,G,T, 181 D,G,I,K, 182H1K1S1Q, 183A1L1P1S1V1Q, 184*, 185*, 186*, 196Q, 199C, 200K, R, 202N, 203T, 218Q, 223E, 239Q, 240P, 241 Q, 247C, 273L,Q, 276K,R,
281 H, 282P, 283P, 284P, 285G,N,R, 286K,Q, 289P, 294T, 299L, 308A, 314G.N, 316D, 324N,
331K, 339D, 351 Y, 355P, 362K.R, 379K,R, 385D, 406A, 409D,E, 410D1E, and/or 411R,K.
The nomenclature used herein for alterations is described in detail in the section "Alterations, such as Substitutions, Deletions, Insertions." Preferably the phytase of the invention comprises at least one of the following alterations: 1*, 2*, 3*, 4P, 5P, 31 C, 46E, 52C,E, 53V, 55D, 57Y, 59C, 76G, 82E, 99C, 100C, 107D;E,G, 109A, 1 11 P, 114T1 115Q, 1 16AT, 1 17D, 118T, 119K1R1S, 120S, 121 D,P,T,122D, 123P1 124L, 137P, 141C, 161 P, 162C, 164E, 167Q, 179K, 180E.T, 181D,K, 182H1K1Q, 183L,V,Q, 184*, 185*, 186*, 196Q, 199C, 200K, 202N, 218Q, 223E, 241 Q, 273L, 276K,R, 285G1R1 286Q1 299L, 314G1N1 331 K1 339D, 362K1R1 379K1R, 385D, 406A, 41OD1E, and/or 411 R,K; and/or wherein the amino acids in position 179, 180, 181 , 182, 183, 184, 185, and 186 have been replaced by KEKHQ, KEKQQ, KEKKV, or KTDKL. In another preferred embodiment, the amino acids in position 179, 180, 181 , 182, 183, 184, 185, and 186 have been replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV, or KTDKL.
The invention also relates to a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 1 H1K1R, 60P1 105E1 106A1G1
155F1 157F1 173P1 175L1 188P1 205P1 215M1 231 P, 254Y, 280P, 330D1 and/or 371 P; with the proviso that the phytase is not SEQ ID NO:3, not SEQ ID NO:4, not SEQ ID NO:6, and not
SEQ ID NO:9 and the variants thereof listed in Fig. 1. In a preferred embodiment the phytase comprises the alteration 1 K. In additional preferred embodiments, the phytase comprises the following combinations of alterations: 280P/282P/283P, 155F/254Y, and/or 155F/157F/254Y.
Preferred phytases of the invention comprise an alteration selected from the following: 52C, 141 C, 162C, 31 C, 52C, 99C, 59C, 100C, 141 C/199C, 4P, 5P, 111 P, 137P, 161 P, 52E, 57Y, 76G, 107D, 107G, 109A, 1*, 1*/2*, 1*/2*/3*, 121T, 273L, 285G, 286Q, 299L, 362K, 331 K/55D, 107E, 46E, 82E, 1 19R, 119K, 164E, 223E, 276R, 276K1 362R, 379R, 379K, 385D, 410D, 410E, 41 1 R, 411 K, 53V, 121 D, 167Q, 196Q, 200K, 202N, 218Q, 241 Q1 285N, 314N, 314G, 406A, 179K/180E/181 K/182H/183Q/184*/185*/186*.
179K/180E/181 K/182Q/183Q/184*/185*/186*, 179K/180E/181 K/182K/183V/184*/185*/186*, 179K/180T/181 D/182K/183L/184*/185*/186*, 111 P/241 Q, 1 K1 114T/1 15Q/116A/117D/1 18T/119S/120S/121 P/122D/123P/124L1 and 114T/1 15Q/116T/117D/1 18T/119S/120S/121 P/122D/123P/124L.
The phytase of the invention may be a variant of any wildtype or variant phytase. In particular embodiments, it is a variant of the phytase of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, or a variant of any one of the phytase variants related to SEQ ID NO:9 and listed in Fig.1. The phytase of the invention may furthermore comprise an alteration (substitution) or a combination of alterations (substitutions) selected from amongst the alterations (substitutions) and combinations of alterations (substitutions) listed in each row of Fig.1.
Particularly preferred variants of the phytase of SEQ ID NO:2 are the following: R339D, N4P, G5P, Q1 1 1 P, E1*, E1*/E2*, E1*/E2*/Q3*, M273L, and N286K; as well as any combination thereof; as well as the corresponding variants of SEQ ID NO:3, 4 and 6.
Particularly preferred phytases of the invention comprise at least one of the following alterations: 339D, 4P, 5P, 111 P, 1*, 1*/2*, 1*/2*/3*, 273L, and/or 286K.
The invention also relates to a phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: (i) 141 C/199C, 91 C/46C, 52C/99C, 31 C/176C, 31 C/177C, 59C/100C, and/or 162C/247C; (ii) 41 P1 91 P, 136P, 137P, 154P1 161 P, 355P, 1 11 P1 240P1 282P, 283P1 284P1 289P1 4P1 and/or 5P; (iii) 52E, 551, 57Y, 104A/105F, 107D.G, 109A.G, 76G1 84Y, 121T1 362K1 273L1Q, 285G,R,
286K,Q, 294T1 299L, 331K/55D, and/or 351 Y;
(iv) 1*, 1*/2*, or 1*/2*/3*;
(V) wherein K179, T180, T181, E182, K183, S184, T185, and K186 have been replaced by QADKP1 GEDKP, NGISA1 IAGKS1 KEKHQ1 KEKQQ, KEKKV1 or KTDKL;
(vi) 119R.K, and/or 411 R1K;
(vii) 107E1 and/or 164E1D;
(viii) 362R1K1 276R1K, 379R1K, 409D1E1 223E, 385D, 46D1E1 410D1E, and/or 82E;
(ix) 218Q, 324N1 200R.K, 121D1 196Q, 202N, 406A, 167Q, 53V,Q, 241Q1 314N1G1 239Q1 and/or 285N;
(X) 114H/115Q/116E/117K/118M/119G/120T/121 M/122D/123P/124T,
114H/115Q/116Q/117D/1181/119K/120Q/121 V/122D/123S/124L1
114H/115Q/116P/117E/1181/119G/120K/121 M/122D/123P/124V,
114T/115Q/116A/117D/118T/119S/120S/121 P/122D/123P/124L1 114H/115Q/116Q/117D/118I/119K/120Q/121A/122D/123P/124L,
114T/115Q/116T/117D/118T/119S/120S/121 P/122D/123P/124L, or
114N/115Q/116A/117D/118Lyi 19K/120K/121 T/122D/123P/124L;
(xi) 31T1 74A1 171T1 203T1 281H1 316D, and/or 308A; and/or
(xii) 339D.
Strategy for Preparing Variants
The structure of the C. braakii ATCC 51113 phytase was built by homology modelling, using as a template the structure of the E. coli AppA phytase (Protein Data Bank id.: 1DKO; Lim et al, Nat. Struct. Biol. (2000), vol. 2, pp. 108-113). The structure was subjected to molecular dynamics (MD) simulations and electrostatic calculations. Positions for putative disulfide bridges and prolines were also identified, as well as other positions of potential importance as regards the various desirable enzymatic properties. Finally, putative glycosylation sites (stretches of NXT or NXS) were identified.
All these suggestions were evaluated within the framework of the modelled structure and the simulation results, for the thermostability property with particular emphasis at the high temperature end.
The corresponding phytase variants were prepared by methods known in the art and tested as described in the experimental part.
Phytase Polypeptides, Percentage of Identity
In the present context a phytase is a polypeptide having phytase activity, i.e. an enzyme which catalyzes the hydrolysis of phytate (myo-inositol hexakisphosphate) to (1) myo- inositol and/or (2) mono-, di-, tri-, tetra- and/or penta-phosphates thereof and (3) inorganic phosphate.
In the present context the term a phytase substrate encompasses, i.a., phytic acid and any phytate (salt of phytic acid), as well as the phosphates listed under (2) above. The ENZYME site at the internet (http://www.expasy.ch/enzyme/) is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB) and it describes each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided (Bairoch A. The ENZYME database, 2000, Nucleic Acids Res 28:304-305). See also the handbook Enzyme Nomenclature from NC-IUBMB, 1992).
According to the ENZYME site, three different types of phytases are known: A so- called 3-phytase (alternative name 1 -phytase; a myo-inositol hexaphosphate 3- phosphohydrolase, EC 3.1.3.8), a so-called 4-phytase (alternative name 6-phytase, name based on 1 L-numbering system and not I D-numbering, EC 3.1.3.26), and a so-called 5- phytase (EC 3.1.3.72). For the purposes of the present invention, all three types are included in the definition of phytase.
In a particular embodiment, the phytases of the invention belong to the family of acid histidine phosphatases, which includes the Escherichia coli pH 2.5 acid phosphatase (gene appA) as well as fungal phytases such as Aspergillus awamorii phytases A and B (EC: 3.1.3.8) (gene phyA and phyB). The histidine acid phosphatases share two regions of sequence similarity, each centered around a conserved histidine residue. These two histidines seem to be involved in the enzymes' catalytic mechanism. The first histidine is located in the N-terminal section and forms a phosphor-histidine intermediate while the second is located in the C- terminal section and possibly acts as proton donor.
In a further particular embodiment, the phytases of the invention have a conserved active site motif, viz. R-H-G-X-R-X-P, wherein X designates any amino acid (see amino acids 16 to 22 of SEQ ID NOs:2, 3, 4, 6 and amino acids 38-44 of SEQ ID NO:9). In a preferred embodiment, the conserved active site motif is R-H-G-V-R-A-P1 i.e. amino acids 16-22 (by reference to SEQ ID NO:2) are RHGVRAP.
For the purposes of the present invention 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 370C; substrate: sodium phytate (C6 H6O24P6Na12) 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. Phytase activity may also be determined using the assays of Example 1 ("Determination of phosphatase activity" or "Determination of phytase activity"). In a particular embodiment the phytase of the invention is isolated. The term "isolated" as used herein 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. In particular, it is preferred that 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.
The relatedness between two amino acid sequences is described by the parameter "identity". For purposes of the present invention, the alignment of two amino acid sequences is determined by 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. The degree of identity between an amino acid sequence of the present invention
("invention sequence") and the amino acid sequence referred to in the claims (SEQ ID NO:2) is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the "invention sequence," or the length of the SEQ ID NO:2, whichever is the shortest. The result is expressed in percent identity. An exact match occurs when the "invention sequence1' and SEQ ID NO:2 have identical amino acid residues in the same positions of the overlap (in the alignment example below this is represented by "|"). The length of a sequence is the number of amino acid residues in the sequence (e.g. the length of amino acids 1-411 of SEQ ID NO:2 is 411).
Example 13 is an example of an alignment of the phytase of SEQ ID NO:2 and the phytase of SEQ ID NO:9, and the example illustrates how to calculate the percentage of identity between these two backbones.
In another, purely hypothetical, alignment example below, the overlap is the amino acid sequence "HTWGER-NL" of Sequence 1 ; or the amino acid sequence "HGWGEDANL" of Sequence 2. In the example a gap is indicated by a "-". Hypothetical alignment example:
Sequence 1: ACMSHTWGER-NL
Sequence 2: HGWGEDANliAMNPS
In a particular embodiment, the percentage of identity of an amino acid sequence of a polypeptide with, or to, SEQ ID NO:2 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.
In the above hypothetical example, the number of exact matches is 6, the length of the shortest one of the two amino acid sequences is 12; accordingly the percentage of identity is 50%.
In particular embodiments of the phytase of the invention, the degree of identity to SEQ ID NO:2 is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In still further particular embodiments, the degree of identity is at least 98.0%, 98.2%, 98.4%, 98.6%, 98.8%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9%. In alternative embodiments, the degree of identity is at least 70%, 71%, 72%, or at least 73%.
In still further particular embodiments, the phytase of the invention has no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, or no more than 10 alterations as compared to SEQ ID NO:2; no more than 11 , 12, 13, 14, 15, 16, 17, 18, 19, or no more than 20 alterations as compared to SEQ ID NO:2; no more than 21, 22, 23, 24, 25, 26, 27, 28, 29, or no more than 30 alterations as compared to SEQ ID NO:2; no more than 31, 32, 33, 34, 35, 36, 37, 38, 39, or not more than 40 alterations as compared to SEQ ID NO:2; no more than 41 , 42, 43, 44, 45, 46, 47, 48, 49, or no more than 50 alterations as compared to SEQ ID NO:2; no more than 51 , 52, 53, 54, 55, 56, 57, 58, 59, or no more than 60 alterations as compared to SEQ ID NO:2; no more than 61 , 62, 63, 64, 65, 66, 67, 68, 69, or no more than 70 alterations as compared to SEQ ID NO:2; no more than 71 , 72, 73, 74, 75, 76, 77, 78, 79, or no more than 80 alterations as compared to SEQ ID NO:2; no more than 81 , 82, 83, 84, 85, 86, 87, 88, 89, or no more than 90 alterations as compared to SEQ ID NO:2; no more than 91, 92, 93, 94, 95, 96, 97, 98, 99, or no more than 100 alterations as compared to SEQ ID NO:2; no more than 101 , 102, 103, 104, 105, 106, 107, 108, 109, or no more than 110 alterations as compared to SEQ ID NO:2; no more than 111, 112, 113, 114, 115, 116, 117, 118, 119, or no more than 120 alterations as compared to SEQ ID NO:2; or no more than 121 , 122, 123, or 124 alterations as compared to SEQ ID NO:2.
Position Numbering
The nomenclature used herein for defining amino acid positions is based on the amino acid sequence of the phytase derived from Citrobacter braakii ATCC 51113, the mature sequence of which is given in the sequence lisiting as SEQ ID NO:2 (amino acids 1-411 of
SEQ ID NO:2). Accordingly, in the present context, the basis for numbering positions is SEQ ID NO:2 starting with E1 and ending with E411.
When used herein the term "mature" part (or sequence) refers to that part of the polypeptide which is secreted by a cell which contains, as part of its genetic equipment, a polynucleotide encoding the polypeptide. In other words, the mature polypeptide part refers to that part of the polypeptide which remains after the signal peptide part, as well as a propeptide part, if any, has been cleaved off. The signal peptide part can be predicted by programs known in the art (e.g. SignalP). The expected signal peptide part of SEQ ID NO:2 is included in the present sequence listing as SEQ ID NO:8, which is encoded by SEQ ID NO:7. SEQ ID NO:2 is the expected mature part. Generally, the first amino acid of the mature part of an enzyme can be determined by N-terminal sequencing of the purified enzyme. Any difference between the signal peptide part and the mature part must then be due to to the presence of a propeptide.
Alterations, such as Substitutions, Deletions, Insertions
A phytase variant can comprise various types of alterations relative to a template (i.e. a reference or comparative amino acid sequence such as SEQ ID NO:2): An amino acid can be substituted with another amino acid; an amino acid can be deleted; an amino acid can be inserted; as well as any combination of any number of such alterations. In the present context the term "insertion" is intended to cover also N- and/or C-terminal extensions.
The general nomenclature used herein for a single alteration is the following: XDcY, where "X" and "Y" independently designate a one-letter amino acid code, or a "*" (deletion of an amino acid), "D" designates a number, and "c" designates an alphabetical counter (a, b, c, and so forth), which is only present in insertions. Reference is made to Table 1 below which describes purely hypothetical examples of applying this nomenclature to various types of alterations.
Table 1
Figure imgf000010_0001
Figure imgf000011_0001
As explained above, the position number ("D") is counted from the first amino acid residue of SEQ ID NO:2.
Several alterations in the same sequence are separated by "/" (slash), e.g. the designation "1*/2*/3*" means that the amino acids in position number 1 , 2, and 3 are all deleted, and the designation "104A/105F" means that the amino acid in position number 104 is substituted by A, and the amino acid in position number 105 is substituted by F.
Alternative alterations are separated by "," (comma), e.g., the designation "1 19R,K" means that the amino acid in position 1 19 is substituted with R or K. The commas used herein in various other enumerations of possibilities mean what they usually do grammatically, viz. often and/or. E.g., the first comma in the listing "53V,Q, 121 D, and/or 167Q" denotes an alternative (V or Q), whereas the two next commas should be interpreted as and/or options: 53 V or Q, and/or 121 D, and/or 167Q.
In the present context, "at least one" (e.g. alteration) means one or more, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 alterations; or 12, 14, 15, 16, 18, 20, 22, 24, 25, 28, or 30 alterations; and so on, up to a maximum number of alterations of 125, 130, 140, 150, 160, 170, 180, 190, or of
200. The phytase variants of the invention, however, still have to be at least 74% identical to
SEQ ID NO:2, this percentage being determined as described above.
A substitution or extension without any indication of what to substitute or extend with refers to the insertion of any natural, or non-natural, amino acid, except the one that occupies this position in the template.
Example 13 provides further illustration of how to apply this nomenclature.
Identifying Corresponding Position Numbers As explained above, the mature phytase of Citrobacter braakii ATCC 51113 (SEQ ID
NO:2) is used as the standard for position numbering and, thereby, also for the nomenclature.
For another phytase, in particular a phytase variant of the invention, the position corresponding to position D in SEQ ID NO:2 is found by aligning the two sequences as specified above in the section entitled "Phytase polypeptides, percentage of identity". From the alignment, the position in the sequence of the invention corresponding to position D of SEQ ID NO:2 can be clearly and unambiguously identified (the two positions on top of each other in the alignment).
Example 13 is an example of an alignment of the phytase of SEQ ID NO:2 and the phytase of SEQ ID NO:9, and the example illustrates how corresponding positions in these two backbones are identified. Below some additional, purely hypothetical, examples are included which are derived from Table 1 above which in the third column includes a number of alignments of two sequences:
Consider the third cell in the first row of Table 1 : The upper sequence is the template, the lower the variant. Position number 80 refers to amino acid residue G in the template. Amino acid A occupies the corresponding position in the variant. Accordingly, this substitution is designated G80A.
Consider now the third cell in the second row of Table 1 : The upper sequence is again the template and the lower the variant. Position number 80 again refers to amino acid residue G in the template. The variant has two insertions, viz. TY, after G80 and before V81 in the template. Whereas the T and Y of course would have their own "real" position number in the variant amino acid sequence, for the present purposes we always refer to the template position numbers, and accordingly the T and the Y are said to be in position number 80a and 80b, respectively.
Finally, consider the third cell in the last row of Table 1 : Position number 275 refers to the last amino acid of the template. A C-terminal extension of ST are said to be in position number 275a and 275b, respectively, although, again, of course they have their own "real" position number in the variant amino acid sequence.
Amended Properties, Reference Phytase In a particular embodiment, the phytase of the invention has amended, preferably improved, properties. The terms "amended" and "improved" imply a comparison with another phytase. Examples of such other, reference, or comparative, phytases are: SEQ ID NO:3, and/or SEQ ID NO:4. Still further examples of reference phytases may be SEQ ID NO:2, and/or SEQ ID NO:6. A still further example of a reference phytase may be SEQ ID NO:9, and the variants thereof disclosed in Fig. 1.
Non-limiting examples of properties that are amended, preferably improved, are the following: Thermostability, pH profile, specific activity, performance in animal feed, protease- sensibility, and/or glycosylation pattern. The phytase of the invention may also have an amended, preferably improved, temperature profile, and/or it may incorporate a change of a potential protease cleavage site.
Thermostability Thermostability, or temperature stability, may be determined as described in Example 1 under the heading of "Determination of temperature stability." Accordingly, in a preferred embodiment, a phytase of the invention has a residual activity which is higher than the residual activity of a reference phytase, wherein residual activity is determined as follows: A fermentation supernatant is divided in two parts, one part is incubated for 30 minutes at a desired elevated temperature, and the other part for 30 minutes at 50C, following which the activity of both is determined on p-nitrophenyl phosphate at 370C and pH 5.5, and the activity of the sample having been incubated at an elevated temperature is divided by the activity of the same sample having been incubated at 5°C. Preferred elevated temperatures are 500C, 550C, 600C, 650C, 700C, 75°C, 8O0C, or 85°C. If desired, the enzyme-containing samples may be diluted in 0.1 M NaAc pH 5.5. The residual activity of a phytase of the invention is preferably at least 105%, or at least 110%, 120%, 130%, 140%, 150% of the residual activity of the reference phytase. In still further embodiments, the residual activity of a phytase of the invention is at least 200%,' or at least 250%, 300%, 400%, or at least 500% of the residual activity of the reference phytase. In still further embodiments, the residual activity of a phytase of the invention is at least 2x, 3x, 4x, 5x, 6x, 7x, 10x, 15x, 2Ox, or at least 25x the residual activity of the reference phytase.
Thermostability may also be determined as described in Example 5. Accordingly, in a preferred embodiment, a phytase of the invention has a residual activity which is higher than the residual activity of a reference phytase, wherein residual activity is determined as follows: A fermentation supernatant is divided in two parts, one part is incubated for 30 minutes at a 5O0C, and the other part for 30 minutes at 5°C, following which the activity of both is determined on p-nitrophenyl phosphate at 370C and pH 5.5, and the activity of the sample having been incubated at an elevated temperature is divided by the activity of the same sample having been incubated at 5°C. If desired, the enzyme-containing samples may be diluted in 0.1 M NaAc pH 5.5. The residual activity of a phytase of the invention is preferably at least 2x, 3x, 4x, 5x, 6x, 7x, 10x, 15x, 2Ox, or at least 25x the residual activity of the reference phytase of SEQ ID NO:3. The residual activity of a phytase of the invention is preferably at least 105%, or at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or at least 200% of the residual activity of the reference phytase of SEQ ID NO:2. The following substitutions are particularly preferred as they improve thermostability as compared to the phytase of SEQ ID NO:3 as well as to the phytase of SEQ ID NO:2 (see Table 3): 4P, 5P1 111 P1 1*, 1*/2*. 1*/2*/3*. 273L, and/or 286Q.
Thermostability may also be determined as described in Example 8. Accordingly, in a preferred embodiment, a phytase of the invention has a residual activity which is higher than the residual activity of a reference phytase, wherein residual activity is determined as follows: A fermentation supernatant is divided in two parts, one part is incubated for 30 minutes at a 60°C, and the other part for 30 minutes at 50C1 following which the activity of both is determined on p-nitrophenyl phosphate at 37°C and pH 5.5, and the activity of the sample having been incubated at an elevated temperature is divided by the activity of the same sample having been incubated at 50C. If desired, the enzyme-containing samples may be diluted in 0.1 M NaAc pH 5.5, optionally including 0.005% Tween-20. The phytase of the invention and the reference phytase may be expressed in a Bacillus subtilis host strain. The host strain may be grown in 100ml PS1 medium (100g/L sucrose, 40g/L Soy flakes, 10g/L Na2HPO4.12H2O, 0.1ml/L Dowfax 63N10 (Dow)) in 500ml shake flasks for four days at 30°C at 300 rpm. The residual activity of a phytase of the invention is preferably at least 32%, or at least 34%, 36%, 38%, or at least 40% of the residual activity of the reference phytase of SEQ ID NO:2. More preferably, the residual activity of a phytase of the invention is at least 50%, or at least 60%, 70%, 80%, 90%, or at least 100% of the residual activity of the reference phytase of SEQ ID NO:2. Even more preferably the residual activity of a phytase of the invention is at least 120%, 140%, 160%, 180%, or at least 200% of the residual activity of the reference phytase of SEQ ID NO:2. Most preferably, the residual activity of a phytase of the invention is at least 2x, or at least 3x, 4x, or at least 5x the residual activity of the reference phytase of SEQ ID NO:2. The following substitutions are particularly preferred (see Table 5): (i) 409E, 136P; (ii) 411 K, 331K/55D, 167Q, 179K/180T/181D/182K/183L/184*/185*/186*, 107E; (iii) 196Q, 276R, 285G, 299L, 200K;
(iv) 119R1 121D, 107D, 179K/180E/181K/182H/183Q/184*/185*/186*;
(V) 314N, 161P, 410D, 141 C1 179K/180E/181 K/182Q/183Q/184V1857186*. 285N;
(vi) 164E, 411R1 52C, 137P, 314G;
(vii) 1K1 1*/2*/3*. 121 T, 406A, 82E, 109A; (Six) 5P1 57Y, 379R, 172*; CDC) 410E, 1*, 119K. 52E; (X) 4P, 362K, 202N, 276K, 385D;
(Xi) 111 P/241 Q, 162C, 179K/180E/181 K/182K/183V/18471857186*, 241 Q; (xii) 223E1 286Q, 107G, 114T/115Q/116A/117D/ 118T/119S/120S/121 P/122D/123P/124L, 379K, 273L;
(xiii) 31 C, 53V, 59C/100C;
(Xiv) 46E1 111 P, 114T/115Q/116T/117D/118T/119S/120S/121 P/122D/123P/124L,
76G, 362R;
(XV) 141 C/199C, 52C/99C. Thermostability may also be determined as described in Example 9, i.e. using DSC measurements to determine the denaturation temperature, Td, of the purified phytase protein. The Td is indicative of the thermostability of the protein: The higher the Td, the higher the thermostability. Accordingly, in a preferred embodiment, the phytase of the invention has a Td which is higher than the Td of a reference phytase, wherein Td is determined on purified phytase samples (preferably with a purity of at least 95%, determined by SDS-PAGE), after dialysis in 2OmM Na-acetate pH4.0 (preferably in a 2-3 h step followed by an over night step), followed by 0.45um filtration and dilution with dialysis buffer to a protein concentration corresponding to approximately 2 absorbancy units (A280), using Differential Scanning Calorimetry at a 90°C/h scan rate from 20-900C in 20 mM Na-acetate buffer, pH 4.0. In a preferred embodiment, the Td of the phytase of the invention is higher than the Td of the phytase of SEQ ID NO:4, more preferably at least 101% thereof, or at least 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or at least 110% thereof. Even more preferably, the Td of the phytase of the invention is at least 120%, 130%, 140%, 150%, 160%, 170%, 180%, or at least 190% of the Td of the phytase of SEQ ID NO:4. The following substitutions are particularly preferred (see Table 6): 362K, 362R, 111P1 and/or 273L. In still further particular embodiments, the thermostable phytase of the invention has a melting temperature, Tm (or a denaturation temperature, Td), as determined using Differential Scanning Calorimetry (DSC) as described in Example 2 (i.e. in 20 mM sodium acetate, pH 4.0), of at least 5O0C. In still further particular embodiments, the Tm is at least 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 62.5. 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or at least 1000C. DSC measurements may also be performed as described in Example 1 ("DSC measurements"), or Example 2 ("Thermostability by DSC").
Thermostability may also be determined as described in Example 12. Accordingly, in a preferred embodiment the phytase of the invention, after incubation for 60 minutes at 700C and pH 4.0, has an improved residual activity as compared to the residual activity of a reference phytase treated in the same way, the residual activity being calculated for each phytase relative to the activity found before the incubation (at 0 minutes). The residual activity is preferably measured on sodium phytate at pH 5.5 and 37°C. The incubation is preferably in 0.1 M sodium acetate, pH 4.0. The phytase is preferably purified, more preferably to a purity of at least 95%, determined by SDS-PAGE. A preferred phytase activity assay buffer is 0.25 M Na-acetate pH 5.5. Using this method, the residual activity of the phytase of the invention is preferably at least 105% of the residual activity of the reference phytase, more preferably at least 110%, 115%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or at least 200%. In the alternative, the residual activity relative to the activity at 0 minutes is preferably at least 31%, or at least 32%. The following substitutions providing improved thermostability stability are preferred (see Table 9): 273L, 46E, 362R, and/or 53V.
In a particular embodiment, the phytase variant of the invention is more thermostable than the reference phytase, wherein thermostability is determined using any of the above- mentioned four tests (based on Example 1 , 5, 8, 9, or 12).
In particular embodiments, an improved thermostability is expected of the following variants of the phytase of SEQ ID NO:2 (in order of preference, within each grouping):
(i) K141C/V199C, Q91C/W46C, G52C/A99C, N31C/E176C, N31C/T177C, G59C/F100C, S162C/S247C;
(ii) D41 P, Q91 P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P, T283P,
T284P, G289P, N4P, G5P;
(iii) G52E, V55I, E57Y, L104A/A105F, K107D,G, Q109A,G, T76G, A84Y, N121T, I362K,
M273L.Q, E285G,R, N286Q, V294T, I299L, E331KΛ/55D, F351Y; (iv) E1*, E1*/E2*, E1*/E2*/Q3*;
(v) replacing the loop comprised between C178 and C187 with shorter loops selected from, e.g., QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV, KTDKL;
(vi) E119R1K, E411R.K;
(vil) K107E, R164E,D; (iix) I362R.K, T276R.K, I379R.K, V409D.E, Q223E, N385D, W46D.E, T410D.E, Q82E.
(ix) replacing the loop between residues 114 and 124 (YQKDEEKNDPL) which faces the active site with a loop selected from, e.g., HQEKMGTMDPT, HQQDIKQVDSL,
HQPEIGKMDPV, TQADTSSPDPL, HQQDIKQADPL, TQTDTSSPDPL, NQADLKKTDPL;
(X) R339D.
Temperature profile
Whether or not a phytase of the invention has an amended temperature profile as compared to a reference phytase may be determined as described in Example 10. Accordingly, in a particular embodiment the phytase of the invention has an amended temperature profile as compared to a reference phytase, wherein the temperature profile is determined as phytase activity as a function of temperature on sodium phytate at pH 5.5 in the temperature range of 20-900C (in 10cC steps). A preferred buffer is in 0.25 M Na-acetate buffer pH 5.5. The activity at each temperature is preferably indicated as relative activity (in %) normalized to the value at optimum temperature. The optimum temperature is that temperature within the tested temperatures (i.e. those with 100C jumps) where the activity is highest.
In a preferred embodiment, the phytase of the invention has a relative activity at 700C of at least 18%, or at least 19%, 20%, 21%, 22%, 23%, 24%, or at least 25%. As explained above, this is relative to the activity at the optimum temperature. More preferably, the phytase of the invention has a relative activity at 7O0C of at least 26%, 27%, 28%, 29%, 30%, 31%, or at least 32%. Preferred substitutions which provide an amended temperature profile (in the form of a higher relative activity at 7O0C) are (see Table 7): 57Y, 76G, 107G, 273L, 362K, 46E, 362R, 53V, and/or 241 Q. Their relative activity at 700C is higher as compared to the reference phytase of SEQ ID NO:3 and 4, and in some instances (57Y, 76G, 107G, 273L, 362K, 362R, and/or 53V) also as compared to the reference phytase of SEQ ID NO:2.
pH profile
Whether or not a phytase of the invention has an amended pH profile as compared to a reference phytase may be determined as described in Example 11. Accordingly, in a particular embodiment the phytase of the invention has an amended pH profile as compared to a reference phytase, wherein the pH profile is determined as phytase activity as a function of pH on sodium phytate at 37°C in the pH range of 2.0 to 7.5 (in 0.5 pH-unit steps). A preferred buffer is a cocktail of 5OmM glycine, 5OmM acetic acid and 5OmM Bis-Tris. Another preferred buffer is 0.25M sodium acetate. The activity at each pH is preferably indicated as relative activity (in %) normalized to the value at optimum pH.
An example of an amended pH profile is where the pH curve (relative activity as a function of pH) is shifted towards higher, or lower, pH. Preferred substitutions which provide a shift of 0.5 pH units towards a higher pH as compared to the reference phytase of SEQ ID NO:2, 3 or 4 are (see Table 8): 46E, and/or 218Q.
Another example of an amended pH profile is where the optimum pH is changed, in the upward or the downward direction. Preferred substitutions which provide a lower optimum pH as compared to SEQ ID NO:2, 3, and 4 are (see Table 8): 46E, 121 D, and/or 200K. Preferred substitutions which provide a higher optimum pH as compared to SEQ ID NO:2,3, and 4 are
(see Table 8): 218Q, and/or 241 Q.
An amended pH profile may also be determined as described in Example 1 ("Amended pH profile: Determination of pH 3.5/5.5 activity ratio"), viz. by comparing phosphatase activity at pH 3.5 and 5.5. Alternatively, the activity at pH 3.5 may be compared with the activity at pH
4.0, 4.5, or 5.0. In a still further alternative embodiment, phytase activities are compared instead of phosphatase activities.
In a particular embodiment, the phytase of the invention has an amended pH profile as compared to a reference phytase. More in particular, the pH profile is amended in the pH- range of 3.5-5.5. Still more in particular, the activity at pH 4.0, 4.5, 5.0, and/or 5.5 is at a level of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of the activity at the pH-optimum (pH 3.5).
The pH profile, as well as the pH-optimum, of a polypeptide may be determined by incubating it at various pH-values, using a substrate in a pre-determined concentration and a fixed incubation temperature. The pH profile is a graphical representation of phytase activity versus pH, the pH-optimum is determined from the pH profile. In a particular embodiment, the phosphatase or phytase assay of Example 1 is used, e.g. the substrate is 5mM sodium phytate, the reaction temperature 370C1 and the activity is determined at various pH-values, for example pH 2-12, replacing the pH 5.5 acetate buffer with a suitable buffer. Examples of suitable buffers are: 0.1 M glyc/ne/HCI (pH 2.0-3.5), 0.1 M NaAc/Ac (pH 4.0-5.0), 0.1 M Bis- Tris/HCI (pH 5.5-6.5), 0.1 M Tris/HCI (pH 7.0). Other examples of buffers are: 10OmM succinic acid, 10OmM HEPES, 10OmM CHES, 10OmM CABS adjusted to pH-values 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 with HCI or NaOH.
In particular embodiments, an amended pH profile is expected of the following variants of the phytase of SEQ ID NO:2 (in order of preference, within each grouping): (i) E218Q, D324N, T200R,K, N121D, E196Q, D202N, E406A, E167Q, E53V,Q, E241Q, D314N,G, E239Q, E285N;
(ii) replacing the loop between residues 114 and 124 (YQKDEEKNDPL) which faces the active site with a loop selected from, e.g., HQEKMGTMDPT, HQQDIKQVDSL, HQPEIGKMDPV, TQADTSSPDPL, HQQDIKQADPL, TQTDTSSPDPL, NQADLKKTDPL.
Specific Activity
In a particular embodiment, the phytase of the invention has an improved specific activity relative to a reference phytase. More in particular, the specific activity of a phytase of the invention is at least 105%, relative to the specific activity of a reference phytase determined by the same procedure. In still further particular embodiments, the relative specific activity is at least 110, 115, 120, 125, 130, 140, 145, 150, 160, 170, 180, 190, 200, 220, 240,
260, 280, 300, 350 or even 400%, still relative to the specific activity of the reference phytase as determined by the same procedure.
In the alternative, the term high specific activity refers to a specific activity of at least
200 FYT/mg Enzyme Protein (EP). In particular embodiments, the specific activity is at least 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,
1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000 FYT/mg EP.
Specific activity is measured on highly purified samples (an SDS poly acryl amide gel should show the presence of only one component). The enzyme protein concentration may be determined by amino acid analysis, and the phytase activity in the units of FYT, determined as described in Example 1. Specific activity is a characteristic of the specific phytase variant in question, and it is calculated as the phytase activity measured in FYT units per mg phytase variant enzyme protein. See Example 7 for further details.
In particular embodiments, an amended specific activity is expected of the following variants of the phytase of SEQ ID NO:2, in which, in order of preference, the loop between residues 114 and 124 (YQKDEEKNDPL) which faces the active site is replaced with a loop selected from, e.g., HQEKMGTMDPT, HQQDIKQVDSL, HQPEIGKMDPV, TQADTSSPDPL,
HQQDIKQADPL, TQTDTSSPDPL, NQADLKKTDPL. Performance in animal feed
In a particular embodiment the phytase of the invention has an improved performance in animal feed as compared to a reference phytase. The performance in animal feed may be determined by the in vitro model of Example 6. Accordingly, in a preferred embodiment the phytase of the invention has an improved performance in animal feed, wherein the performance is determined in an in vitro model, by preparing feed samples composed of 30% soybean meal and 70% maize meal with added CaCI2 to a concentration of 5 g calcium per kg feed; pre-incubating them at 40°C and pH 3.0 for 30 minutes followed by addition of pepsin (3000 U/g feed) and phytase; incubating the samples at 400C and pH 3.0 for 60 minutes followed by pH 4.0 for 30 minutes; stopping the reactions; extracting phytic acid and inositol- phosphates by addition of HCI to a final concentration of 0.5M and incubation at 400C for 2 hours, followed by one freeze-thaw cycle and 1 hour incubation at 40°C; separating phytic acid and inositol-phosphates by high performance ion chromatography; determining the amount of residual phytate phosphorus (IP6-P); calculating the difference in residual IP6-P between the phytase-treated and a non-phytase-treated blank sample (this difference is degraded IP6-P); and expressing the degraded IP6-P of the phytase of the invention relative to degraded IP6-P of the reference phytase (e.g. the phytases having SEQ ID NO:3 and 4).
The phytase of the invention and the reference phytase are of course dosed in the same amount, preferably based on phytase activity units (FYT). A preferred dosage is 125 FYT/kg feed. Another preferred dosage is 250 FYT/kg feed. The phytases may be dosed in the form of purified phytases, or in the form of fermentation supematants. Purified phytases preferably have a purity of at least 95%, as determined by SDS-PAGE.
In preferred embodiments, the degraded IP6-P value of the purified phytase of the invention, relative to the degraded IP6-P value of the reference phytase, is at least 101 %, or at least 102%, 103%, 104%, 105%, 110%, 115%, or at least 120%. In still further preferred embodiments, the degraded IP6-P value of the purified phytase of the invention, relative to the degraded IP6-P value of the reference phytase, is at least 125%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or at least 200%. Preferably, the degraded IP6-P value of the phytase of the invention, relative to the degraded IP6-P value of the SEQ ID NO:2 phytase, is at least 105%, 110%, 113%, 115%, 120%, 125%, or at least 130%.
The following substitutions provide an improved or at least as good performance in animal feed in vitro (see Table 4A) as compared to the phytase of SEQ ID NO:3: 4P, 5P, 111 P1 1*, V 12*, 1*/2*/3*, 273L, 286Q. The following substitutions also provide an improved or at least as good performance in animal feed in vitro (see Table 4B) as compared to the phytase of SEQ ID NO:3: 57Y, 76G, 107G, 362K, 362R, 121 D, 196Q, 200K, 202N, 314N, 406A, and 114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L
Even more preferred substitutions when it comes to animal feed performance are: 57Y, 76G1 362K, 362R, 121 D, 196Q, 200K, 202N, and 406A.
The relative performance of a phytase of the invention may also be calculated as the percentage of the phosphorous released by the reference phytase. in a still further particular embodiment, the relative performance of the phytase of the invention may be calculated as the percentage of the phosphorous released by the phytase of the invention, relative to the amount of phosphorous released by the reference phytase.
In still further particular embodiments, the relative performance of the phytase of the invention is at least 105%, preferably at least 110, 120, 130, 140, 150, 160, 170, 180, 190, or at least 200%.
Reduced protease-sensibility
In a particular embodiment, the phytase of the invention has a reduced protease- sensibility. More in particular, it has a reduced sensibility towards the Kex2 protease, meaning a reduced tendency to become cleaved by this protease.
Variant 339D, preferably R339D, is an example of a phytase of the invention with a reduced protase-sensibility.
Glycosylation pattern
Glycosylation is a phenomenon which is only observed when expressing proteins in eukaryotes such as fungi and transgenic plants, but not in prokaryotes such as bacteria. There are various types of glycosylation, but in the present context the most relevant is the N- glycosylation, i.e. the asparagine-linked glycosylation where sugars are attached to a protein, starting from an N-acetyg!ucosamine molecule attached to asparagines. N-glycosylation has been found to occur only to asparagines that in the sequence are part of the following tripeptides: N-X-T or N-X-S, where X designates any amino acid.
Surprisingly, a lower thermostability was observed when the phytase of SEQ ID NO:2 was expressed in the fungus (yeast) Pichia pastoris, as compared to when it was expressed in Bacillus subtilis, see Example 2.
This observation has led to the proposal of the present invention that thermostability may be improved for phytases expressed in fungi by altering potential glycosylation sites.
The present invention accordingly also relates to phytase variants having an amended glycosylation pattern, preferably amended N-glycosylation sites. The amended glycosylation is expected to confer an improved thermostability upon the phytase variant, when expressed in a fungus.
Examples of phytases are bacterial phytases, e.g. Gram-negative phytases, such as E.coli and Citrobacter phytases and variants thereof, including the phytases of the present invention as well as the phytases of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:9 herein. Examples of fungal expression hosts are Pichia, Saccharomyces, and Aspergillus species. In particular embodiments, an amended glycosylation pattern is expected of the following phytases of the invention (e.g. variants of SEQ ID NO:2), in order of preference: N31T, N74A, N171T, N203T, N281H, N316D, N308A. The following are replacing an N-X-T type pattern: N31T, N74A, N281 H. The following are replacing an N-X-S type pattern: N171T, N203T, N308A, N316D.
Low-allergenic variants
In a specific embodiment, the phytases of the present invention are (also) low- allergenic variants, designed to invoke a reduced immunological response when exposed to animals, including man. The term immunological response is to be understood as any reaction by the immune system of an animal exposed to the phytase variant. One type of immunological response is an allergic response leading to increased levels of IgE in the exposed animal. Low-allergenic variants may be prepared using techniques known in the art. For example the phytase variant may be conjugated with polymer moieties shielding portions or epitopes of the phytase variant involved in an immunological response. Conjugation with polymers may involve in vitro chemical coupling of polymer to the phytase variant, e.g. as described in WO 96/17929, WO 98/30682, WO 98/35026, and/or WO 99/00489. Conjugation may in addition or alternatively thereto involve in vivo coupling of polymers to the phytase variant. Such conjugation may be achieved by genetic engineering of the nucleotide sequence encoding the phytase variant, inserting consensus sequences encoding additional glycosylation sites in the phytase variant and expressing the phytase variant in a host capable of glycosylating the phytase variant, see e.g. WO 00/26354. Another way of providing low- allergenic variants is genetic engineering of the nucleotide sequence encoding the phytase variant so as to cause the phytase variants to self-oligomerize, effecting that phytase variant monomers may shield the epitopes of other phytase variant monomers and thereby lowering the antigenicity of the oligomers. Such products and their preparation is described e.g. in WO 96/16177. Epitopes involved in an immunological response may be identified by various methods such as the phage display method described in WO 00/26230 and WO 01/83559, or the random approach described in EP 561907. Once an epitope has been identified, its amino acid sequence may be altered to produce altered immunological properties of the phytase variant by known gene manipulation techniques such as site directed mutagenesis (see e.g. WO 00/26230, WO 00/26354 and/or WO 00/22103) and/or conjugation of a polymer may be done in sufficient proximity to the epitope for the polymer to shield the epitope. Nucleic Acid Sequences and Constructs
The present invention also relates to nucleic acid sequences comprising a nucleic acid sequence which encodes a phytase variant of the invention. The term "isolated nucleic acid sequence" refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90% pure as determined by agarose electrophoresis. For example, an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
The nucleic acid sequences of the invention can be prepared by introducing at least one mutation into a template phytase coding sequence or a subsequence thereof, wherein the mutant nucleic acid sequence encodes a variant phytase. The introduction of a mutation into the nucleic acid sequence to exchange one nucleotide for another nucleotide may be accomplished by any of the methods known in the art, e.g. by site-directed mutagenesis, by random mutagenesis, or by doped, spiked, or localized random mutagenesis.
Random mutagenesis is suitably performed either as localized or region-specific random mutagenesis in at least three parts of the gene translating to the amino acid sequence shown in question, or within the whole gene. When the mutagenesis is performed by the use of an oligonucleotide, the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions which are to be changed. The doping or spiking may be performed so that codons for unwanted amino acids are avoided. The doped or spiked oligonucleotide can be incorporated into the DNA encoding the phytase enzyme by any technique, using, e.g., PCR, LCR or any DNA polymerase and ligase as deemed appropriate.
Preferably, the doping is carried out using "constant random doping", in which the percentage of wild-type and mutation in each position is predefined. Furthermore, the doping may be directed toward a preference for the introduction of certain nucleotides, and thereby a preference for the introduction of one or more specific amino acid residues. The doping may be made, e.g., so as to allow for the introduction of 90% wild type and 10% mutations in each position. An additional consideration in the choice of a doping scheme is based on genetic as well as protein-structural constraints.
The random mutagenesis may be advantageously localized to a part of the parent phytase in question. This may, e.g., be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme. Alternative methods for providing variants of the invention include gene shuffling e.g. as described in WO 95/22625 or in WO 96/00343, and the consensus derivation process as described in EP 897985.
Nucleic Acid Constructs A nucleic acid construct comprises a nucleic acid sequence 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. Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.
The term "nucleic acid construct" as used herein 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. The term 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.
The term "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. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, 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.
The term "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. When used herein the term "coding sequence" (CDS) 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 Vector The term "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.
The term "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.
A nucleic acid sequence encoding a phytase variant of the invention can be expressed using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes. The recombinant expression vector carrying the DNA sequence encoding a phytase variant of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. The vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
The phytase variant may also be co-expressed together with at least one other enzyme of animal feed interest, such as a phytase, phosphatase, xylanase, galactanase, aipha- galactosidase, protease, phospholipase, amylase, and/or beta-glucanase. The enzymes may be co-expressed from different vectors, from one vector, or using a mixture of both techniques. When using different vectors, the vectors may have different selectable markers, and different origins of replication. When using only one vector, the genes can be expressed from one or more promoters. If cloned under the regulation of one promoter (di- or multi- cistronic), the order in which the genes are cloned may affect the expression levels of the proteins. The phytase variant may also be expressed as a fusion protein, i.e. that the gene encoding the phytase variant has been fused in frame to the gene encoding another protein. This protein may be another enzyme or a functional domain from another enzyme.
Host Cells
The term "host cell", as used herein, 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.
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, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans and Streptomyces murinus, or gram negative bacteria such as E. coli and Pseudomonas sp. In a preferred aspect, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell. In another preferred aspect, the Bacillus cell is an alkalophilic Bacillus.
The introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), using competent cells (see, e.g., Young and Spizizin, 1961 , Journal of Bacteriology 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thome, 1987, Journal of Bacteriology 169: 5771-5278).
The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
In a preferred aspect, 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).
In a more preferred aspect, 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). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S. M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
. In an even more preferred aspect, the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell. In a most preferred aspect, the yeast host cell is a Pichia pastoris, Pichia methanolica,
Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces dougfasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. In another most preferred aspect, the yeast host cell is a Kluyveromyces lactis cell. In another most preferred aspect, the yeast host cell is a Yarrowia lipolytica cell.
In another more preferred aspect, 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 fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
In an even more preferred aspect, the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarϊum, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. In a most preferred aspect, the filamentous fungal host cell is an Aspergillus awamori,
Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred aspect, the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell. In another most preferred aspect, the filamentous fungal host cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, or Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma Iongibrachiatum, Trichoderma reesei, or Trichoderma viride strain 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. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; lto et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.
Methods of Production
The present invention also relates to methods for producing a phytase of the present invention comprising (a) cultivating a host cell under conditions conducive for production of the phytase; and (b) recovering the phytase. In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods well known in the art. For example, 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 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.
The 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).
Transgenic Plants
The present invention also relates to a transgenic plant, plant part, or plant cell which has been transformed with a nucleotide sequence encoding a polypeptide having phytase activity of the present invention so as to express and produce the polypeptide in recoverable quantities. The polypeptide may be recovered from the plant or plant part. Alternatively, 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.
In a particular embodiment, 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. Examples of 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). Examples of 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.
Examples of 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. Also included within the scope of the present invention are the progeny of such plants, plant parts and plant cells. 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.
Conveniently, 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. Furthermore, 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).
The choice of 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. For instance, 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. For constitutive expression, 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. 24: 863-878), 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. Furthermore, 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). Likewise, 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.
A promoter enhancer element may also be used to achieve higher expression of the polypeptide in the plant. For instance, 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. For instance, Xu et al., 1993, supra disclose the use of the first intron of the rice actin 1 gene to enhance expression.
Still further, the 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).
Presently, 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. Presently, 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.
Following transformation, the transformants having incorporated therein the expression construct are selected and regenerated into whole plants according to methods well-known in the art. Often 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 having phytase activity of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
Transgenic Animals 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. 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. Generally speaking, to prepare a transgenic animal, selected cells of a selected animal are transformed with a nucleic acid sequence encoding a polypeptide having phytase activity 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.
To produce a transgenic animal with a view to recovering the polypeptide from the milk of the animal, 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 the polypeptide. The transgene expression vector is 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. et al (1999): Expression of recombinant proteins in the milk of transgenic animals, Gene expression systems: Using nature for the art of expression. J. M. Fernandez and J. P. Hoeffler (eds.), Academic Press.
In the alternative, 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 still further aspects, the present invention relates to compositions comprising a polypeptide of the present invention, as well as methods of using these. The 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. For instance, the polypeptide composition may be in the form of granulates or microgranulates. The polypeptide to be included in the composition may be stabilized in accordance with methods known in the art.
The phytase of the invention can be used for degradation, in any industrial context, of, for example, phytate, phytic acid, and/or the mono-, di-, tri-, tetra- and/or penta-phosphates of myo-inositol. It is well known that the phosphate moieties of these compounds 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 manganese, copper and molybdenum. Besides, the phytic acid also to a certain extent binds proteins by electrostatic interaction. Accordingly, preferred uses of the polypeptides of the invention are in animal feed preparations (including human food) or in additives for such preparations.
In a particular embodiment, the polypeptide of the invention can be used for improving the nutritional value of an animal feed. Non-limiting examples of improving the nutritional value of animal feed (including human food), are: Improving feed digestibility; promoting growth of the animal; improving feed utilization; improving bio-availability of proteins; increasing the level of digestible phosphate; improving the release and/or degradation of phytate; improving bioavailability of trace minerals; improving bio-availability of macro minerals; eliminating the need for adding supplemental phosphate, trace minerals, and/or macro minerals; and/or improving egg shell quality. The nutritional value of the feed is therefore increased, and the growth rate and/or weight gain and/or feed conversion (i.e. the weight of ingested feed relative to weight gain) of the animal may be improved.
Furthermore, the polypeptide of the invention can be used for reducing phytate level of manure.
Animals, Animal Feed, and Animal Feed Additives
The term animal includes all animals, including human beings. Examples of animals are non-ruminants, and ruminants. Ruminant animals include, for example, animals such as sheep, goat, and cattle, e.g. cow such as beef cattle and dairy cows. In a particular embodiment, the animal is a non-ruminant animal. Non-ruminant animals include mono-gastric animals, e.g. pig or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chickens (including but not limited to broiler chicks, layers); fish (including but not limited to salmon, trout, tilapia, catfish and carp); and crustaceans (including but not limited to shrimp and prawn).
The term feed or feed composition means any compound, preparation, mixture, or composition suitable for, or intended for intake by an animal. In the use according to the invention the polypeptide can be fed to the animal before, after, or simultaneously with the diet. The latter is preferred.
In a particular embodiment, the polypeptide, in the form in which it is added to the feed, or when being included in a feed additive, is substantially pure. In a particular embodiment it is well-defined. The term "well-defined" means that the phytase preparation is at least 50% pure as determined by Size-exclusion chromatography (see Example 12 of WO 01/58275). In other particular embodiments the phytase preparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined by this method.
A substantially pure, and/or 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. The term dose correctly refers in particular to the objective of obtaining consistent and constant results, and the capability of optimising dosage based upon the desired effect.
For the use in animal feed, however, the phytase polypeptide of the invention need not be that pure; it may e.g. include other polypeptides, in which case it could be termed a phytase preparation.
The phytase preparation can be (a) added directly to the feed (or used directly in a treatment process of 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. Such polypeptide preparation may of course be mixed with other polypeptides.
The polypeptide can be added to the feed in any form, be it as a relatively pure polypeptide, 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.
In a further aspect the present invention relates to compositions for use in animal feed, such as animal feed, and animal feed additives, e.g. premixes. Apart from the polypeptide of the invention, 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. The feed additive may also contain at least one macro mineral.
Further, optional, 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 polypeptide selected from amongst phytase (EC 3.1.3.8 or 3.1.3.26); phosphatase (EC
3.1.3.1 ; EC 3.1.3.2; EC 3.1.3.39); 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 (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase such as, for example, alpha-amylase (EC 3.2.1.1); and/or beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).
In a particular embodiment these other polypeptides are well-defined (as defined above for phytase preparations). The phytase of the invention may also be combined with other phytases, for example ascomycete phytases such as Aspergillus phytases, for example derived from Aspergillus ficuum, Aspergillus niger, or Aspergillus awamori; or basidiomycete phytases, for example derived from Peniophora lycii, Agrocybe pediades, Trametes pubescens, or Paxillus involutus; or derivatives, fragments or variants thereof which have phytase activity. Thus, in preferred embodiments of the use in animal feed of the invention, and in preferred embodiments of the animal feed additive and the animal feed of the invention, the phytase of the invention is combined with such phytases.
Examples of antimicrobial peptides (AMP's) are 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.
Examples of antifungal polypeptides (AFP's) are the 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.
Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma- linoleic acid.
Examples of reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and polypeptides such as an oxidase, an oxygenase or a syntethase. Usally fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed. Either of these composition types, when enriched with a polypeptide of the invention, is an animal feed additive of the invention. In a particular embodiment, 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.
The following are non-exclusive lists of examples of these components: Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin K, e.g. vitamin K3.
Examples of water-soluble vitamins are vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and pantothenate, e.g. Ca-D-panthothenate.
Examples of 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 (exemplified with poultry and piglets/pigs) are listed in Table A of WO 0.1/58275. Nutritional requirement means that these components should be provided in the diet in the concentrations indicated. In the alternative, the animal feed additive of the invention comprises at least one of the individual components specified in Table A of WO 01/58275. At least one means either of, one or more of, one, or two, or three, or four and so forth up to all thirteen, or up to all fifteen individual components. More specifically, this at least one individual component is included in the additive of the invention in such an amount as to provide an in-feed-concentration within the range indicated in column four, or column five, or column six of Table A.
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 characterised as indicated in Table B of WO 01/58275, columns 2-3. Fish diets can be characterised 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
50-800 g/kg, and furthermore comprises at least one polypeptide as claimed herein. Furthermore, or in the alternative (to the crude protein content indicated above), 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.
In particular embodiments, 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 (R. 2-5).
Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25, i.e. Crude protein (g/kg)= N (g/kg) x 6.25. The nitrogen content is determined by the Kjeldahf 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. In a particular embodiment, the animal feed composition of the invention contains at least one protein. The protein may be an animal protein, such as meat and bone meal, and/or fish meal; or it may be a vegetable protein. The term vegetable proteins as used herein 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. In particular embodiments, 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.
In a particular embodiment, the vegetable protein source is material from one or more plants of the family Fabaceae, e.g. soybean, lupine, pea, or bean.
In another particular embodiment, 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. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, triticale, and sorghum.
In still further particular embodiments, 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. Typically, 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. Polypeptides can be added as solid or liquid polypeptide formulations. For example, a solid polypeptide formulation is typically added before or during the mixing step; and a liquid polypeptide preparation is typically added after the pelleting step. The polypeptide may also be incorporated in a feed additive or premix.
The final polypeptide concentration in the diet is within the range of 0.01-200 mg polypeptide protein per kg diet, for example in the range of 5-30 mg polypeptide protein per kg animal diet.
The phytase of the invention should of course be applied in an effective amount, i.e. in an amount adequate for improving solubilization and/or improving nutritional value of feed. It is at present contemplated that the polypeptide 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; 10-100; 0.05-50; or 0.10-
10 - all these ranges being in mg phytase polypeptide protein per kg feed (ppm).
For determining mg phytase polypeptide protein per kg feed, the phytase is purified from the feed composition, and the specific activity of the purified phytase is determined using a relevant assay. The phytase 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 phytase protein per kg feed is calculated.
The same principles apply for determining mg phytase polypeptide protein in feed additives. Of course, if a sample is available of the phytase used for preparing the feed additive or the feed, the specific activity is determined from this sample (no need to purify the phytase from the feed composition or the additive).
Particular embodiments
The invention also relates to the following particular embodiments:
I. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1 , 2, 3, 4, 5, 31 , 41 , 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105,
107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 136, 137, 141 , 154, 161 , 162, 164, 167, 171 , 176, 177, 179, 180, 181 , 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241 , 247, 273, 276, 281 , 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316, 324, 331, 339, 351, 355, 362, 379, 385, 406, 409, 410, and 411 ; preferably in at least one position selected from the following: 1, 2, 3, 4, 5, 31 , 46, 52, 53, 55, 57, 59, 76, 82, 99, 100, 107, 109, 111 , 114, 115, 116, 117, 118, 119, 120, 121 , 122,
123, 124, 137, 141 , 161 , 162, 164, 167, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199,
200, 202, 218, 223, 241, 273, 276, 285, 286, 299, 314, 331 , 339, 362, 379, 385, 406, 410, and
411 ; with the proviso that the phytase is not SEQ ID NO:3, not SEQ ID NO:4, and not SEQ ID NO:6.
II. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1, 2, 3, 4, 5, 41 , 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91 , 99, 100, 104, 105, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 136, 137, 141, 154, 161, 162, 164, 167, 171 , 176, 177, 179, 180, 181 , 182, 183, 184, 185, 186, 196, 199, 200, 202, 203,
218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308,
314, 324, 339, 351, 355, 362, 379, 385, 406, 409, 410, and 411; preferably in at least one position selected from the following: 1, 2, 3, 4, 5, 46, 52, 53,
55, 57, 59, 76, 82, 99, 100, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 137, 141 , 161 , 162, 164, 167, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202,
218, 223, 241 , 273, 276, 285, 286, 299, 314, 339, 362, 379, 385, 406, 410, and 411.
III. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1, 2, 3, 4, 5, 31, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111 , 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 136, 137, 141, 154, 161 , 162, 164, 167, 171 , 176, 177, 179, 180, 181 , 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241 , 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316, 324, 331 , 339, 351 , 355, 362, 379, 385, 406, 409, 410, and 411 ; preferably in at least one position selected from the following preferably in at least one position selected from the following: 1, 2, 3, 4, 5, 31 , 46, 52, 53, 55, 57, 59, 76, 82, 99, 100, 107, 109, 111 , 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 137, 141 , 161, 162, 164, 167, 179, 180, 181 , 182, 183, 184, 185, 186, 196, 199, 200, 202, 218, 223, 241 , 273, 276, 285, 286, 299, 314, 331, 339, 362, 379, 385, 406, 410, and 411; with the proviso that the phytase is not SEQ ID NO:3, not SEQ ID NO:4, not SEQ (D NO:6, and not SEQ ID NO:9 and the variants thereof listed in Fig. 1.
IV. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1 , 2, 3, 4, 5, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111 , 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 136, 137, 141 , 154, 161 , 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241 , 247, 273, 276, 281 , 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 324, 339, 351, 355, 362, 379, 385, 406, 409, 410, and 411 preferably in at least one position selected from the following: 1 , 2, 3, 4, 5, 46, 52, 53, 55, 57, 59, 76, 82, 99, 100, 107, 109, 111 , 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 137, 141 , 161, 162, 164, 167, 179, 180, 181 , 182, 183, 184, 185, 186, 196, 199, 200, 202, 218, 223, 241 , 273, 276, 285, 286, 299, 314, 339, 362, 379, 385, 406, 410, and 411 ; with the proviso that the phytase is not SEQ ID NO:9 and the variants thereof listed in
Fig. 1.
V. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 4, 5, 41 , 46, 59, 82, 84, 91 , 99, 105, 107, 109, 111 , 115, 116, 117, 119, 122, 123, 124, 136, 137, 141 , 161, 162, 164, 167, 171, 176, 179, 180, 186, 196, 199, 200, 218, 223, 239, 240, 241 , 247, 273, 276, 281, 282, 283, 284, 289, 294, 299, 308, 314, 324, 339, 351 , 355, 379, 385, 406, 409, 410, and 411; preferably in at least one position selected from the following: 4, 5, 46, 59, 82, 99, 107, 109, 111 , 115, 116, 117, 119, 122, 123, 124, 137, 141 , 161 , 162, 164, 167, 179, 180, 186, 196, 199, 200, 218, 223, 241 , 273, 276, 299, 314, 339, 379, 385, 406, 410, and 411.
VI. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 1*, 2*, 3*, 4P, 5P, 31 C1T, 41 P, 46C,D,E, 52C,E, 53V,Q, 55D,I, 57Y, 59C, 74A, 76G, 82E, 84Y, 91 C.P, 99C, 100C, 104A, 105F, 107D,E,G, 109A,G, 111P, 114H,N,T, 115Q, 116A1E1P1T1Q1 117D1E1K 118I,L,M,T, 119G, K1R1S, 120K1S1T1Q, 121A,D,M,P,T,V, 122D, 123P.S, 124L,T,V, 136P, 137P, 141C, 154P, 161 P, 162C, 164D.E, 167Q, 171T, 176C, 177C, 179G, I1K1N1Q, 180A1E1G1T, 181D1G1I1K, 182H1K1S1Q1 183A1L1P1S1V1Q1 184*, 185*, 186*, 196Q, 199C, 20OK1R, 202N1 203T1 218Q, 223E, 239Q, 240P, 241Q, 247C, 273L1Q1 276K1R1 281 H, 282P, 283P, 284P, 285G,N,R, 286K.Q, 289P, 294T, 299L, 308A, 314G.N, 316D, 324N, 331 K, 339D, 351Y, 355P, 362K.R, 379K.R, 385D, 406A, 409D1E1 410D, E, and/or 411R,K; and/or wherein the amino acids in position 179, 180, 181, 182, 183, 184, 185, and 186 have been replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV1 or KTDKL; preferably at least one of the following alterations: 1*, 2*, 3*, 4P1 5P, 31 C, 46E, 52C1E1 53V, 55D, 57Y, 59C, 76G, 82E, 99C, 100C, 107D1E1G1 109A, 111 P1 114T, 115Q, 116AT1 117D, 118T1 119K1R1S, 120S, 121D,P,T,122D, 123P, 124L, 137P, 141C, 161 P, 162C, 164E, 167Q, 179K, 180E1T1 181D1K, 182H1K1Q, 183L,V,Q, 184*, 185*, 186*, 196Q, 199C, 200K, 202N, 218Q, 223E, 241 Q, 273L, 276K.R, 285G.R, 286Q1 299L1 314G1N1 331 K, 339D1 362K1R, 379K.R, 385D1 406A, 410D1E1 and/or 411 R1K; and/or wherein the amino acids in position 179, 180, 181, 182, 183, 184, 185, and 186 have been replaced by KEKHQ1 KEKQQ, KEKKV1 or KTDKL; with the proviso that the phytase is not SEQ ID NO:3, not SEQ ID NO:4, and not SEQ ID NO:6.
VIl. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations:: 1*, 2*, 3*, 4P1 5P1 31 C1T, 41P1 46C1D1E, 52C1E1 53V1Q1 55I1D1 57Y, 59C, 74A1 76G, 82E, 84Y, 91C1P, 99C, 100C, 104A, 105F, 107D1E1G1 109A1G1 111 P1 114H1N1T1 115Q, 116A1E1P1T1Q 117D1E1K, 118I1M1L1T, 119G1K1R1S, 120K1S1T1Q1 121A1D1M1P1V, 122D1 123P1S1 124L1T1V, 136P, 137P1 141C1 154P1 161P1 162C, 164D,E, 167Q1 171T1 176C, 177C1 179G1I1K1N1Q, 180A1E1G1T1 181D1G1I1K1 182H1K1S1Q, 183A1L1P1S1V1Q1 184*, 185*, 186*, 196Q, 199C, 200K1R, 202N1 203T1 218Q, 223E, 239Q, 240P, 241 Q, 247C, 273L.Q, 276K,R, 281H1 282P1 283P, 284P1 285G1N1R1 286K,Q, 289P1 294T, 299L, 308A1 314G1N1 316D1 324N1 339D1 351Y, 355P, 362K1R1 379K1R, 385D, 406A, 409D1E, 41OD1E, and/or 411K1R; and/or wherein the amino acids in position 179, 180, 181 , 182, 183, 184, 185, and 186 have been replaced by QADKP1 GEDKP1 NGISA1 IAGKS1 KEKHQ1 KEKQQ1 KEKKV, or KTDKL; preferably at least one of the following alterations: 1*, 2*, 3*, 4P, 5P1 31 C1 46E1 52C1E1 53V, 55D, 57Y, 59C1 76G1 82E, 99C, 100C1 107D1E1G, 109A1 111 P1 114T1 115Q, 116AT1 117D, 118T1 119K1R1S, 120S, 121D1P1 122D, 123P1 124L1 137P1 141C1 161 P1 162C, 164E, 167Q, 179K, 180EJ1 181D1K1 182H1K1Q1 183L1V1Q, 184*, 185*, 186*, 196Q1 199C1 200K 202N, 218Q1 223E1 241Q1 273L1 276K1R, 285G1R1 286Q1 299L1 314G1N1 339D, 362K1R1 379K1R, 385D, 406A, 410D,E, and/or 411 R,K; and/or wherein the amino acids in position 179,
180, 181 , 182, 183, 184, 185, and 186 have been replaced by KEKHQ, KEKQQ, KEKKV, or KTDKL.
HX. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 1*, 2*, 3*, 4P1 5P1 31C1T, 41P1 46C1D1E, 52C1E1 53V1Q1 55D1I1 57Y1 59C, 74A, 76G, 82E1 84Y, 91C1P1 99C, 100C, 104A, 105F, 107D1E1G, 109A1G1 111P1 114H1N1T, 115Q1 116A1E1P1T1Q1 117D1E1K, 118I1L1M1T1 119G1K1R1S1 120K1S1T1Q1 121A1D1M1PJ1V1 122D, 123P1S, 124LJ1V1 136P, 137P1 141 C1 154P1 161 P1 162C1 164D1E1 167Q1 171T1 176C1 177C1 179G1I1K1N1Q1 18OA1E1GJ, 181 D1G1I1K1 182H, K1S1Q1 183A1L1P1S1V1Q, 184*, 185*, 186*, 196Q, 199C, 200K1R1 202N1 203T1 218Q1 223E, 239Q1 240P, 241 Q, 247C, 273L1Q1 276K1R, 281 H, 282P, 283P, 284P1 285G1N1R1 286K1Q1 289P1 294T, 299L, 308A1 314G1N1 316D1 324N, 331 K, 339D, 351 Y, 355P1 362K1R1 379K1R1 385D1 406A, 409D,E, 410D,E, and/or 411 R1K; and/or wherein the amino acids in position 179, 180,
181 , 182, 183, 184, 185, and 186 have been replaced by QADKP1 GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ1 KEKKV, or KTDKL; preferably at least one of the following alterations: 1*, 2*, 3*, 4P1 5P1 31C, 46E, 52C1E1 53V, 55D1 57Y, 59C, 76G1 82E, 99C, 10OC, 107D1E1G, 109A1 111P1 114T1 115Q1 116AT1 117D1 118T1 119K1R1S1 120S1 121D1P1 122D1 123P1 124L, 137P, 141C1 161P1 162C1 164E1 167Q1 179K1 180E1T, 181D1K1 182H1K1Q1 183L1V1Q, 184*, 185*, 186*, 196Q, 199C, 200K 202N1 218Q1 223E, 241 Q, 273L, 276K1R1 285G1R1 286Q, 299L, 314G.N, 339D, 362K1R1 379K1R1 385D, 406A, 410D1E1 and/or 411R1K; and/or wherein the amino acids in position 179, 180, 181 , 182, 183, 184, 185, and 186 have been replaced by KEKHQ, KEKQQ, KEKKV, or KTDKL. with the proviso that the phytase is not SEQ ID NO:3, not SEQ ID NO:4, not SEQ ID NO:6, and not SEQ ID NO:9 and the variants thereof listed in Fig. 1.
IX. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations:: 1*, 2*, 3*, 4P1 5P1 31 C1T1 41 P, 46C1D1E, 52C.E, 53V1Q, 551, D, 57Y1 59C, 74A1 76G1 82E1 84Y, 91C1P1 99C, 100C, 104A, 105F, 107D1E1G, 109A1G, 111 P, 114H1N1T1 115Q1 116A1E1P1T1Q 117D1E1K1 1181,M1L1T1 119G1K1R1S1 120K1S1T1Q1 121A1D1M1P1V1 122D1 123P1S, 124LJ.V, 136P, 137P, 141C1 154P, 161P1 162C1 164D1E, 167Q, 171T1 176C, 177C, 179G1I1K1N1Q1 180A1E1G1T1 181D1G1I1K1 182H1K1S1Q1 183A1L1P1S1V1Q1 184*, 185*, 186*, 196Q1 199C1 200K1R1 202N1 203T1 218Q1 223E1 239Q1 240P1 241Q1 247C1 273L1Q1 276K.R, 281H1 282P1 283P, 284P1 285G1N1R, 286K1Q1 289P1 294T1 299L1 308A1 314G1N1 316D1 324N1 339D1 351Y1 355P1 362K1R1 379K1R1 385D1 406A1 409D1E1 410D1E1 and/or 411 K1R; and/or wherein the amino acids in position 179, 180, 181 , 182, 183, 184, 185, and 186 have been replaced by QADKP1 GEDKP1 NGISA, IAGKS1 KEKHQ, KEKQQ, KEKKV, or KTDKL; preferably at least one of the following alterations: 1*, 2*, 3*, 4P1 5P1 31C1 46E1 52C1E, 53V, 55D1 57Y1 59C1 76G, 82E, 99C, 100C, 107D1E1G1 109A1 111P1 114T1 115Q1 116AT, 117D1 118T1 119K1R1S1 120S1 121 D1P1 122D, 123P1 124L1 137P1 141C1 161 P1 162C1 164E1 167Q1 179K1 180E1T1 181 D1K1 182H1K1Q1 183L1V1Q1 184*, 185*, 186*, 196Q, 199C1 200K 202N, 218Q, 223E, 241Q, 273L, 276K1R1 285G1R, 286Q, 299L1 314G1N1 339D1 362K1R1 379K1R, 385D, 406A, 410D1E1 and/or 411 R1K; and/or wherein the amino acids in position 179, 180, 181, 182, 183, 184, 185, and 186 have been replaced by KEKHQ, KEKQQ1 KEKKV1 or KTDKL. with the proviso that the phytase is not SEQ ID NO:9 and the variants thereof listed in Fig. 1.
X. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations:: 1*, 2*, 3*, 4P1 5P1 31C1T1 41 P, 46C1D1E, 52C1E, 53Q1 55D, 57Y, 59C1 74A, 82E, 84Y, 91C1P, 99C, 100C, 104A, 105F1 107D1E1G, 109A1G1 111P1 114H1T1 115Q1 116A1E1P1T1Q 117D1E1K1 118I1M1L1T1 119G1K1R1S1 120K1S1T1Q, 121A1D1M1V1 122D1 123P1S, 124L1T1V1 136P, 137P, 141C, 154P, 161 P1 162C, 164D1E, 167Q, 171T1 176C, 177C1 179G1I1K1N1Q1 180A1E1G1T, 181 D1G1K, 182K,S,Q, 183A1L1S1V1Q, 184*, 185*, 186*, 196Q1 199C, 200K1R1 202N, 203T, 218Q, 223E, 239Q, 240P1 241Q, 247C, 273L,Q, 276K,R, 281H1 282P, 283P, 284P, 285G1N1R, 286K1Q1 289P1 294T, 299L1 308A, 314G.N, 316D, 324N, 339D, 351Y, 355P, 362K.R, 379K1R1 385D, 406A1 409D,E, 410D.E, and/or 411 K1R; and/or wherein the amino acids in position 179, 180, 181 , 182, 183, 184, 185, and 186 have been replaced by QADKP1 GEDKP, NGISA, IAGKS, KEKHQ1 KEKQQ1 KEKKV, or KTDKL; preferably at least one of the following alterations: 1*, 2*, 3*, 4P, 5P, 31 C, 46E, 52C.E, 55D, 57Y, 59C, 82E, 99C, 100C, 107D1E1G, 109A, 11 1 P, 114T, 115Q, 116AT1 117D, 1 1ST, 119K1R1S, 120S, 121 D, 122D, 123P1 124L, 137P1 141 C1 161 P1 162C, 164E, 167Q1 179K1 180E1T, 181 D1K, 182K.Q, 183L1V1Q, 184*, 185*, 186*, 196Q, 199C, 200K 202N, 218Q, 223E, 241 Q1 273L1 276K1R1 285G.R, 286Q, 299L1 314G1N, 339D, 362K1R, 379K1R1 385D, 406A, 410D1E, and/or 411 R.K; and/or wherein the amino acids in position 179, 180, 181 , 182, 183, 184, 185, and 186 have been replaced by KEKHQ1 KEKQQ, KEKKV, or KTDKL.
XI. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 1 H1K1R, 6OP, 105E, 106A1G, 155F1 157F, 173P, 175L1 188P, 205P, 215M1 231 P, 254Y, 280P, 330D, and/or 371 P; preferably 1 K; with the proviso that the phytase is not SEQ ID NO:3, not SEQ ID NO:4, not SEQ ID
NO:6, and not SEQ ID NO:9 and the variants thereof listed in Fig. 1.
XII. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 1H1R, 60P1 105E1 106A1G1 157F, 173P, 175L, 188P1 205P1 215M, 231 P1 254Y, 280P. XIII. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 52C, 141C1 162C, 31 C, 52C, 99C, 59C, 100C, 141C/199C, 4P, 5P, 111 P, 137P, 161 P1 52E, 57Y, 76G, 107D1 107G1 109A1 1*, 1*/2*, 1*/2*/3*, 121T1 273L, 285G, 286Q, 299L, 362K, 331 K/55D, 107E, 46E, 82E, 119R, 119K1 164E1 223E, 276R, 276K, 362R, 379R1 379K, 385D, 410D, 410E, 411 R1 411 K, 53V, 121 D, 167Q, 196Q, 200K, 202N, 218Q, 241 Q, 285N, 314N1 314G1 406A1 179K/180E/181 K/182H/183Q/184*/185*/186*, 179K/180E/181 K/182Q/183Q/18471857186*, 179K/180E/181 K/182K/183V/18471857186*, 179K/180T/181 D/182K/183L/18471857186*, 111P/241Q, 1K,
114T/115Q/116A/117D/118T/119S/120S/121 P/122D/123P/124L, 114T/1 15Q/116T/1 17D/118T/119S/120S/121 P/122D/123P/124L. XIV. The phytase of any one of embodiments 1-13 which is a variant of the phytase of SEQ ID NO:2.
XV. The phytase of any one of embodiments 1-13 which is a variant of the phytase of SEQ ID NO:3. XVI. The phytase of any one of embodiments 1-13 which is a variant of the phytase of SEQ ID NO:4.
XVII. The phytase of any one of embodiments 1-13 which is a variant of the phytase of SEQ ID NO:6.
HXX. The phytase of any one of embodiments 1-13 which is a variant of the phytase of SEQ ID NO:9.
IXX. The phytase of any one of embodiments 1-13 which is a variant of any one of the phytase variants related to SEQ ID NO:9 and listed in Fig. 1.
XX. The phytase of any one of embodiments 1-19 which furthermore comprises a substitution or a combination of substitutions selected from amongst the substitutions and combinations of substitutions listed in each row of Fig. 1.
XXI. The phytase of any one of embodiments 1-20, which has an improved thermostability, an improved pH profile, an improved specific activity, an amended glycosylation pattern, an improved temperature profile, an improved performance in animal feed, and/or which incorporates a change of a potential protease cleavage site. XXII. An isolated nucleic acid sequence comprising a nucleic acid sequence which encodes the phytase of any of embodiments I-XXI.
XXIII. A nucleic acid construct comprising the nucleic acid sequence of embodiment XXII operably linked to one or more control sequences that direct the production of the phytase in a suitable expression host. XXIV. A recombinant expression vector comprising the nucleic acid construct of embodiment
XXlII.
XXV. A recombinant host cell comprising the nucleic acid construct of embodiment XXIII and/or the expression vector of embodiment XXIV.
XXVI. A method for producing the phytase of any one of embodiments I-XXI, comprising (a) cultivating the host cell of embodiment XXV to produce a supernatant comprising the phytase; and (b) recovering the phytase.
XXVII. A transgenic plant, or plant part, capable of expressing a phytase of any one of embodiments I-XXI.
I IXXX. A transgenic, non-human animal, or products, or elements thereof, being capable of expressing a phytase of any one of embodiments I-XXI.
IXXX. A composition comprising at least one phytase of any one of embodiments I-XXI, and (a) at least one fat soluble vitamin; (b) at least one water soluble vitamin; and/or
(c) at least one trace mineral.
XXX. The composition of embodiment IXX further comprising at least one enzyme selected from the following group of enzymes: amylase, phytase, phosphatase, xylanase, galactanase, alpha-galactosidase, protease, phospholipase, and/or beta-glucanase.
XXXI. The composition of any one of embodiments IXX-XXX which is an animal feed additive.
XXXII. An animal feed composition having a crude protein content of 50 to 800 g/kg and comprising the phytase of any one of embodiments I-XXI or the composition of any one of embodiments IXXX-XXXI.
XXXIII. A method for improving the nutritional value of an animal feed, wherein the phytase of any one of embodiments I-XXI or the composition of any one of embodiments IXXX-XXXI is added to the feed.
XXXIV. A process for reducing phytate levels in animal manure comprising feeding an animal with an effective amount of the feed of embodiment XXXI I .
XXXV. A method for the treatment of vegetable proteins, comprising the step of adding the phytase of any one of embodiments I-XXI or the composition of any one of embodiments IXXX-XXXI to at least one vegetable protein or protein source.
XXXVI. Use of the phytase of any one of embodiments I-XXI or the composition of any one of embodiments IXXX-XXXI in animal feed; in the preparation of animal feed; for improving the nutritional value of animal feed; for reducing phytate levels in animal manure; for the treatment of vegetable proteins; or for liberating phosphorous from a phytase substrate.
a). A phytase which has at least 70% identity to SEQ ID NO:2 and which comprises at (east one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1 , 2, 3, 4, 5, 31, 41 , 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105,
107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 136, 137, 141 , 154,
161 , 162, 164, 167, 171 , 176, 177, 179, 180, 181 , 182, 183, 184, 185, 186, 196, 199, 200,
202, 203, 218, 223, 239, 240, 241 , 247, 273, 276, 281 , 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316, 324, 331, 339, 351, 355, 362, 379, 385, 406, 409, 410, and 411; with the proviso that the phytase is not SEQ ID NO:3, not SEQ ID NO:4, and not SEQ ID NO:6. a1). A phytase which has at least 70% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1 , 2, 3, 4, 5, 31 , 41 , 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111 , 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 136, 137, 141 , 154,
161, 162, 164, 167, 171 , 176, 177, 179, 180, 181 , 182, 183, 184, 185, 186, 196, 199, 200,
202, 203, 218, 223, 239, 240, 241 , 247, 273, 276, 281 , 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316, 324, 331 , 339, 351 , 355, 362, 379, 385, 406, 409, 410, and 411, with the proviso that the variant does not comprise (i) 31D/121T/316N/331E, and not (ii) 31D/121 N/316K/331E, and not (iii) 31N/121N/316N/331K. a2). A phytase which has at least 70% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1, 2, 3, 4, 5, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111 , 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 136, 137, 141 , 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241 , 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 324, 339, 351, 355, 362, 379, 385, 406, 409, 410, and 411. a3). The phytase of embodiment a2), which comprises at least one of the following alterations: V1 2*, 3*, 4P, 5P, 31 C1T, 41 P, 46C,D,E, 52C.E, 53V.Q, 55I,D, 57Y, 59C, 74A, 76G, 82E, 84Y, 91C1P, 99C1 100C, 104A, 105F, 107D1E1G, 109A,G, 111P1 114H1N1T1 115Q, 116A,E,P,T,Q 117D1E1K, 118I,M,L,T, 119G1K1R1S, 120K1S1T1Q, 121 A1D1M1P1V, 122D, 123P.S, 124L,T,V, 136P, 137P, 141C, 154P, 161 P, 162C, 164D.E, 167Q, 171T1 176C, 177C1 179G, I1K1N1Q, 180A1E1G1T, 181D,G,I,K, 182H1K1S1Q1 183A1L1P1S1V1Q, 184*, 185*, 186*, 196Q, 199C1 20OK1R1 202N1 203T1 218Q1 223E1 239Q, 240P, 241Q1 247C, 273L1Q1 276K1R, 281 H, 282P1 283P1 284P, 285G1N1R, 286K,Q, 289P1 294T1 299L1 308A1 314G1N, 316D, 324N, 339D, 351Y, 355P, 362K.R, 379K1R, 385D, 406A, 409D,E, 41OD1E, and/or 411K,R; and/or wherein the amino acids in position 179, 180, 181, 182, 183, 184, 185, and 186 have been replaced by QADKP1 GEDKP, NGISA1 IAGKS, KEKHQ, KEKQQ, KEKKV1 or KTDKL. a4). The phytase of any one of embodiments a2)-a3), which comprises at least one of the following alterations: (i) 31C, 46C1 52C, 59C, 91C, 99C1 100C1 141C1 162C1 176C1 177C1 199C, and/or 247C; (ii) 4P, 5P, 41 P, 91 P, 111P, 136P, 137P, 154P, 161 P, 240P, 282P1 283P, 284P, 289P, and/or 355P;
(iii) 52E, 55D,I, 57Y1 76G1 84Y1 104A1 105F1 107D1G, 109A.G, 273L1Q, 285G1R, 286Q, 294T, 299L, 351Y, and/or 362K; (iv) 1*, 1*/2*, or 1*/2*/3*; (v) wherein K179, T180, T181 , E182, K183, S184, T185, and K186 have been replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ1 KEKKV, or KTDKL; (vi) 119K1R, and/or 411K1R; (vii) 107E, and/or 164D1E; (viii) 46D,E, 82E, 223E, 276K.R, 362K1R1 379K1R, 385D, 409D,E, and/or 410D1E; (ix) 53V.Q, 121D, 167Q, 196Q, 200K,R, 202N, 218Q, 239Q, 241Q, 285N, 314G1N, 324N, and/or 406A; (X) 114H1N1T 115Q, 116A1E1P1T1Q1 117D1E1K1 118I1L1M1T 119G1K1S1 120K1S1T1Q1 121A1M1P1V, 122D1 123P1S, and/or 124L1T1V
(xi) 31 T, 74A1 171T1 203T1 281H1 308A1 and/or 316D; and/or
(xii) 339D. a5). The phytase of any one of embodiments a2)-a4), which comprises at least one of the following alterations:
(i) 141C/199C, 91C/46C, 52C/99C, 31C/176C, 31C/177C, 59C/100C, and/or 162C/247C;
(ii) 41P1 91 P1 136P, 137P1 154P1 161P1 355P1 111P1 240P1 282P1 283P1 284P1 289P1 4P1 and/or 5P;
(iii) 52E, 551, 57Y, 104A/105F, 107D1G1 109A1G1 76G1 84Y, 362K, 273L1Q1 285G1R1 286Q, 294T, 299L1 331 K/55D, and/or 351 Y;
(iv) 1*, 1*/2*, or 1*/2*/3*;
(V) wherein K179, T180, T181 , E182, K183, S184, T185, and K186 have been replaced by
QADKP1 GEDKP1 NGISA1 IAGKS1 KEKHQ1 KEKQQ1 KEKKV1 or KTDKL;
(vi) 119R1K1 and/or 411R1K; (vii) 107E, and/or 164E,D;
(viii) 362R,K, 276R1K1 379R1K1 409D1E1 223E, 385D, 46D.E, 410D1E1 and/or 82E;
(ix) 218Q, 324N, 200R,K, 121 D, 196Q, 202N, 406A, 167Q, 53V,Q, 241 Q, 314N1G, 239Q, and/or 285N;
(X) 114H/115Q/116E/117K/118M/119G/120T/121 M/122D/123P/124T, 114H/115Q/116Q/117D/1181/119K/120Q/121 V/122D/123S/124L,
114H/115Q/116P/117E/1181/119G/120K/121 M/122D/123P/124V,
114T/115Q/116A/117D/118T/119S/120S/121 P/122D/123P/124L,
114H/115Q/116Q/117D/1181/119K/120Q/121 A/122D/123P/124L,
114T/115Q/116T/117D/118T/119S/120S/121 P/122D/123P/124L1 or 114N/115Q/116A/117D/118L/119K/120K/121T/122D/123P/124L;
(xi) 31T1 74A, 171T1 203T1 281H1 308A1 and/or 316D; and/or
(xii) 339D. b). The phytase of embodiment a) or a1), which comprises at least one of the following alterations: 1*, 2*, 3*. 4P1 5P1 31 C1T, 41 P, 46C1D1E, 52C,E, 53V1Q1 55D.I, 57Y1 59C1 74A1 76G, 82E, 84Y, 91C1P, 99C, 100C1 104A1 105F1 107D1E1G1 109A1G1 111P1 114H1N1T, 115Q,
116A1E1P1T1Q, 117D1E1K 1181,L1M1T 119G1K1R1S, 120K1S1T1Q, 121A1D1M1P1T1V1 122D,
123P1S1 124L1T1V1 136P, 137P, 141C1 154P1 161 P, 162C1 164D1E1 167Q1 171T1 176C, 177C,
179G1I1K1N1Q1 180A1E1G1T, 181D1G1I1K, 182H1K1S1Q, 183A1L1P1S1V1Q1 184*, 185*, 186*,
196Q1 199C1 200K1R1 202N1 203T1 218Q1 223E1 239Q1 240P1 241Q1 247C1 273L1Q1 276K1R1 281H1 282P1 283P1 284P1 285G1N1R, 286K1Q1 289P1 294T1 299L1 308A1 314G.N, 316D, 324N1
331K1 339D1 351Y1 355P1 362K1R1 379K1R1 385D1 406A1 409D1E1 410D1E1 and/or 411R1K; and/or wherein the amino acids in position 179, 180, 181, 182, 183, 184, 185, and 186 have been replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV, or KTDKL. c). The phytase of any one of the above embodiments, which comprises at least one of the following alterations:
(i) 31 C, 46C, 52C1 59C1 91 C, 99C, 10OC, 141 C, 162C, 176C, 177C1 199C, and/or 247C; (ii) 4P1 5P1 41 P, 91 P, 111P1 136P, 137P, 154P, 161 P, 240P, 282P, 283P, 284P, 289P, and/or 355P;
(iii) 52E, 55D1I, 57Y, 76G, 84Y, 104A, 105F, 107D1G, 109A1G, 121T1 273L,Q, 285G.R,
286Q1 294T, 299L, 331 K, 351 Y, and/or 362K;
(iv) 1*, 1*72*. or 1*/2*/3*; (v) wherein K179, T180, T181 , E182, K183, S184, T185, and K186 have been replaced by
QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV, or KTDKL;
(vi) 119K.R, and/or 411 K1R;
(vii) 107E, and/or 164D1E;
(viii) 46D.E, 82E, 223E, 276K,R, 362K1R1 379K1R1 385D, 409D.E, and/or 41 OD1E; (ix) 53V.Q, 121D1 167Q, 196Q, 200K1R, 202N, 218Q, 239Q, 241Q, 285N, 314G1N, 324N, and/or 406A;
(x) 114H1N1T 115Q, 116A1E1P1T1Q, 117D1E1K, 118I1L1M1T 119G1K1S1 120K1S1T1Q1
121A1M1P1T1V, 122D, 123P1S1 and/or 124L,T,V;
(xi) 31 T, 74A, 171 T, 203T, 281 H, 308A, and/or 316D; and/or (xii) 339D. c1). The phytase of any one of the above embodiments, which comprises at least one of the following alterations:
(i) 31 C, 46C, 52C, 59C, 91C1 99C, 100C, 141 C, 162C, 176C, 177C1 199C1 and/or 247C1 preferably 52C1 99C1 141 C1 and/or 199C; (ii) 4P1 5P1 41P1 91P1 111P, 136P1 137P1 154P1 161P1 240P, 282P1 283P1 284P1 289P1 and/or 355P1 preferably 4P1 5P1 111P;
(iii) 52E, 55D1I1 57Y1 76G, 84Y1 104A1 105F, 107D1G1 109A1G1 121T1 273L.Q, 285G,R,
286Q1 294T1 299L, 331 K, 351 Y, and/or 362K, preferably 57Y, 76G, 107G, 273L, 286Q and/or
362K; (iv) 1*, 1*/2*, or 1*/2*/3*;
(V) wherein K179, T180, T181 , E182, K183, S184, T185, and K186 have been replaced by
QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV, or KTDKL, preferably KEKKV;
(vi) 119K1R1 and/or 411 K1R, preferably 119K;
(vii) 107E1 and/or 164D1E; (viii) 46D1E1 82E1 223E1 276K1R1 362K1R1 379K1R1 385D1 409D1E1 and/or 410D.E preferably
46E, , 223E 362K.R, and/or 379K1R;
(ix) 53V1Q1 121 D, 167Q, 196Q1 200K1R1 202N1 218Q1 239Q1 241Q1 285N1 314G1N1 324N1 and/or 406A, preferably 53V, 121D1 196Q, 200K, 202N, 218Q, 241Q1 314N, and/or 406A;
(x) 114H1N1T 115Q1 116A1E1P1T1Q, 117D1E1K, 1181, L1M1T 119G1K1S1 120K1S1T1Q,
121A1M1P1T1V1 122D1 123P1S, and/or 124L1T1V preferably 114T 115Q, 116AJ, 117D, 118T
119K1S1 120S1 121 P1 122D1 123P1 and/or 124L; (xi) 31T1 74A1 171T1 203T1 281H1 308A1 and/or 316D; and/or
(xii) 339D. d). The phytase of any one of the above embodiments, which has improved properties. e). The phytase of embodiment c) or c1), which comprises at least one of the one or more alterations of features (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), (x), (xi) and/or (xii) of embodiment 3 and has an improved thermostability. f). The phytase of embodiment c) or d), which comprises at least one of the one or more alterations of features (ix) and/or (x) of embodiment c) and has an improved pH profile. g). The phytase of embodiment c) or c1), which comprises at least one of the one or more alterations of feature (x) of embodiment c) and has an improved specific activity. h). The phytase of embodiment c) or c1), which comprises at least one of the one or more alterations of feature (xi) of embodiment c) and has an amended glycosylation pattern. i). The phytase of embodiment c) or c1), which comprises the alteration of feature (xii) of embodiment c) which changes a potential protease cleavage site. j). The phytase of any one of embodiment a)-d) including a1)-a5) and c1), which comprises at least one of the following alterations:
(i) 141C/199C, 91C/46C, 52C/99C, 31C/176C, 31C/177C, 59C/100C, and/or 162C/247C;
(ii) 41P1 91P1 136P1 137P1 154P1 161P1 355P1 111P1 240P1 282P1 283P, 284P1 289P1 4P1 and/or 5P;
(iii) 52E1 551, 57Y, 104A/105F, 107D1G, 109A1G1 76G1 84Y1 121T, 362K, 273L,Q, 285G1R, 286Q1 294T1 299L1 331 K/55D, and/or 351 Y;
(iv) 1*. Y 12*, or 1*/2*/3*;
(v) wherein K179, T180, T181 , E182, K183, S184, T185, and K186 have been replaced by
QADKP1 GEDKP1 NGlSA1 IAGKS1 KEKHQ1 KEKQQ, KEKKV1 or KTDKL;
(vi) 119R1K1 and/or 411R1K; (vii) 107E, and/or 164E.D;
(viii) 362R1K1 276R1K, 379R1K1 409D1E1 223E, 385D1 46D1E1 41 OD1E1 and/or 82E;
(ix) 218Q1 324N1 200R1K, 121 D1 196Q1 202N1 406A1 167Q, 53V1Q, 241Q, 314N1G, 239Q, and/or 285N;
(X) 114H/115Q/116E/117K/118M/119G/120T/121 M/122D/123P/124T, 114H/115Q/116Q/117D/1181/119K/120Q/121 V/122D/123S/124L,
114H/115Q/116P/117E/1181/119G/120K/121 M/122D/123P/124V,
114T/115Q/116A/117D/118T/119S/120S/121 P/122D/123P/124L, 114H/115Q/116Q/117D/1181/119K/120Q/121 A/122D/123P/124L,
114T/115Q/116T/117D/118T/119S/120S/121 P/122D/123P/124L, or
114N/115Q/116A/117D/118L/119K/120K/121 T/122D/123P/124L;
(xi) 31T, 74A, 171T, 203T, 281 H, 316D, and/or 308A; and/or (xii) 339D. k). The phytase of any one of embodiment a)-d) including a1)-a5) and c1), which comprises at least one of the following alterations:
(i) K141C/V199C, Q91C/W46C, G52C/A99C, N31C/E176C, N31C/T177C, G59C/F100C, and/or S162C/S247C; (ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P, T283P,
T284P, G289P, N4P, and/or G5P;
(iii) G52E, V55I, E57Y, L104A/A105F, K107D,G, Q109A.G, T76G, A84Y, N121T, I362K,
M273L,Q, E285G,R, N286Q, V294T, I299L, E331K/V55D, and/or F351Y;
(iv) E1*, E1*/E2*, or E1*/E2*/Q3*; (V) wherein K179, T180, T181 , E182, K183, S184, T185, and K186 have been replaced by
QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV, or KTDKL;
(vi) E119R,K, and/or E411R.K;
(vii) K107E, and/or R164E.D;
(viii) I362R.K, T276R.K, I379R.K, V409D.E, Q223E, N385D, W46D.E, T410D.E, and/or Q82E;
(ix) E218Q, D324N, T200R.K, N121D, E196Q, D202N, E406A, E167Q, E53V.Q, E241Q,
D314N.G, E239Q, and/or E285N;
(X) Y114H/K116E/D117K/E118M/E119G/K120T/N121M/L124T,
Y114H/K116Q/E118I/E119K/K120Q/N121V/P123S, Y114H/K116P/D117E/E118I/E119G/N121M/L124V,
Y114T/K116A/E118T/E119S/K120S/N121P, Y114H/K116Q/E118I/E119K/K120Q/N121A,
Y114T/K116T/E118T/E119S/K120S/N121P, or Y114N/K116A/E118L/E119K/N121T;
(xi) N31T, N74A, N171T, N203T, N281H, N316D, and/or N308A; and/or
(xii) R339D. I). The phytase of embodiment k) which is a variant of SEQ ID NO:2. m). The phytase of any one of embodiment a)-d) including a1)-a5) and c1), which comprises at least one of the following alterations:
(i) T141C/V199C, Q91C/W46C, G52C/A99C, D31C/E176C, D31C/T177C, G59C/F100C, and/or S162C/S247C; (ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P, T283P,
T284P, G289P, N4P, and/or G5P;
(iii) G52E, V55I, E57Y, L104A/A105F, K107D.G, Q109A,G, T76G, A84Y, I362K, M273L,Q, E285G.R, N286Q, V294T, I299L, E331K/V55D, and/or F351Y;
(iv) E1*, E1*/E2*, or E1*/E2*/Q3*;
(v) wherein K179, T180, T181 , E182, K183, S184, T185, and K186 have been replaced by
QADKP, GEDKP, NGISA1 IAGKS, KEKHQ, KEKQQ, KEKKV, or KTDKL; (vi) E119R1K, and/or E411 R1K;
(vii) K107E, R164E.D;
(viii) I362R,K, T276R,K, 1379R1K, V409D.E, Q223E, N385D, W46D,E, T41 OD1E1 Q82E;
(ix) E218Q, D324N, T200R.K, T121D, E196Q, D202N, E406A, E167Q, E53V,Q, E241Q,
D314N.G, E239Q, and/or E285N; (x) Y114H/K116E/D117K/E118M/E119G/K120T/T121 M/L124T,
Y114H/K116Q/E118I/E119K/K120Q/T121V/P123S,
Y114H/K116P/D117E/E118I/E119G/T121M/L124V,
Y114T/K116A/E118T/E119S/K120S/T121P/, Y114H/K116Q/E118I/E119K/K120Q/T121A/,
Y114T/K116T/E118T/E119S/K120S/T121 P, or Y114N/K116A/E118L/E119K; (xi) N74A, N171T, N203T, N281H, N316D, and/or N308A; and/or
(xii) R339D. n). The phytase of embodiment m) which is a variant of SEQ ID NO:4. o). The phytase of any one of embodiment a)-d) including a1)-a5) and d), which comprises at least one of the following alterations: (i) K141 C/V199C, Q91C/W46C, G52C/A99C, D31C/E176C, D31C/T177C, G59C/F100C, and/or S 162C/S247C;
(ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P, T283P,
T284P, G289P, N4P, and/or G5P;
(iii) G52E, V55I, E57Y, L104A/A105F, K107D.G, Q109A,G, T76G, A84Y, N121T, I362K, M273L.Q, E285G,R, N286Q, V294T, I299L, E331KΛ/55D, and/or F351Y;
(iv) E1*f E1*/E2*, or E1*/E2*/Q3*;
(v) wherein K179, T180, T181, E182, K183, S184, T185, and K186 have been replaced by
QADKP, GEDKP1 NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV, or KTDKL;
(vi) E119R.K, and/or E411R.K; (vii) K107E, and/or R164E.D;
(viii) I362R,K, T276R.K, I379R,K, V409D.E, Q223E, N385D, W46D.E, T410D.E, andor
Q82E;
(ix) E218Q, D324N, T200R.K, N121D, E196Q, D202N, E406A, E167Q, E53V.Q, E241Q,
D314N,G, E239Q, and/or E285N; (X) Y114H/K116E/D117K/E118M/E119G/K120T/N121IV1/L124T,
Y114H/K116Q/E118I/E119K/K120Q/N121V/P123S,
Y114H/K116P/D117E/E118I/E119G/N121M/L124V, Y114T/K116A/E118T/E119S/K120S/N121 P, Y114H/K116Q/E118I/E119K/K120Q/N121A,
Y114T/K116T/E118T/E119S/K120S/N121P, or Y114N/K116A/E118L/E119K/N121T;
(xi) N74A, N171T, N203T, N281 H, and/or N308A; and/or
(xii) R339D. p). The phytase of embodiment o) which is a variant of SEQ ID NO:3. q). The phytase of any one of embodiment a)-d) including a1)-a5) and c1), which comprises at least one of the following alterations:
(i) K141 C/V199C, Q91 C/W46C, G52C/A99C, N31C/E176C, N31C/T177C, G59C/F100C, and/or S162C/S247C; (ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P, T283P,
T284P, G289P, N4P, and/or G5P;
(iii) G52E, V551, E57Y, L104A/A105F, K107D.G, Q109A.G, T76G, A84Y, N121T, I362K,
M273LA E285G.R, N286Q, V294T, I299L, V55D, and/or F351Y;
(iv) E1*, E1*/E2*, or E1*/E2*/Q3*; (v) wherein K179, T180, T181 , E182, K183, S184, T185, and K186 have been replaced by
QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV, or KTDKL;
(vi) E119R.K, and/or E411 R1K;
(vii) K107E, and/or R164E1D;
(viii) I362R.K, T276R,K, 1379R1K1 V409D,E, Q223E, N385D, W46D,E, T410D.E, and/or Q82E;
(ix) E218Q, D324N, T200R.K, N121 D, E196Q, D202N, E406A, E167Q, E53V,Q, E241Q,
D314N.G, E239Q, and/or E285N;
(X) Y114H/K116E/D117K/E118M/E119G/K120T/N121M/L124T,
Y114H/K116Q/E118I/E119K/K120Q/N121V/P123S, Y114H/K116P/D117E/E118I/E119G/N121M/L124V,
Y114T/K116A/E118T/E119S/K120S/N121P, Y114H/K116Q/E118I/E119K/K120Q/N121A,
Y114T/K116T/E118T/E119S/K120S/N121P, or Y114N/K116A/E118L/E119K/N121T;
(xi) N31T, N74A, N171T, N203T, N281 H, N316D, and/or N308A; and/or
(xii) R339D. r). The phytase of embodiment q) which is a variant of SEQ ID NO:6. s). The phytase of any one of embodiment a)-d) including a1)-a5) and c1), which comprises at least one of the following alterations:
(i) K141C/V199C, Q91C/W46C, G52C/A99C, D31C/E176C, D31C/T177C, G59C/F100C, and/or S 162C/S247C; (ii) D41 P, Q91P, N136P, T137P, L154P, S161 P, T355P, Q111P, K240P, G282P, T283P,
T284P, G289P, N4P, and/or G5P;
(iii) G52E, V55I, E57Y, L104A/A105F, K107D.G, Q109A,G, T76G, A84Y, I362K, M273L.Q, E285G.R, N286Q, V294T, I299L, E331 K/V55D, and/or F351Y;
(iv) E1*, E1*/E2*, or E1*/E2*/P3*;
(v) wherein K179, T180, T181 , E182, K183, S184, T185, and K186 have been replaced by
QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV, or KTDKL; (vi) E119R.K, and/or E411 R1K;
(vii) K107E, and/or R164E.D;
(viii) I362R,K, T276R.K, I379R,K, V409D,E, Q223E, N385D, W46D.E, T410D.E, and/or
Q82E;
(ix) E218Q, D324N, T200R.K, T121 D, E196Q, D202N, E406A, E167Q, E53V,Q, E241Q, D314N,G, E239Q, and/or E285N;
(x) Y114H/K116E/D117K/E118M/E119G/K120T/T121M/L124T,
Y114H/K116Q/E118I/E119K/K120Q/T121V/P123S,
Y114H/K116P/D117E/E118I/E119G/T121M/L124V,
Y114T/K116A/E118T/E119S/K120S/T121 P, Y114H/K116Q/E118I/E119K/K120Q/T121A,
Y114T/K116T/E118T/E119S/K120S/T121 P, or
Y114N/K116A/E118L/E119K;
(xi) D31T, N74A, N171T, N203T, N281 H, N316D, and/or N308A; and/or
(xii) R339D. t). The phytase of embodiment s) which is a variant of SEQ ID NO:9. u). An isolated nucleic acid sequence comprising a nucleic acid sequence which encodes the phytase of any of embodiment a)-t) including a1)-a5) and d). v). A nucleic acid construct comprising the nucleic acid sequence of embodiment u) operably linked to one or more control sequences that direct the production of the phytase in a suitable expression host. w). A recombinant expression vector comprising the nucleic acid construct of embodiment v). x). A recombinant host cell comprising the nucleic acid construct of embodiment v) and/or the expression vector of embodiment w). y). A method for producing the phytase of any one of embodiment a)-t) including a1)-a5) and c1), comprising
(a) cultivating the host cell of embodiment x) to produce a supernatant comprising the phytase; and (b) recovering the phytase. z). A transgenic plant, or plant part, capable of expressing a phytase of any one of embodiment a)-t) including a1)-a5) and c1). ae). A transgenic, non-human animal, or products, or elements thereof, being capable of expressing a phytase of any one of embodiment a)-t) including a1)-a5) and c1). oe). A composition comprising at least one phytase of any one of embodiment a)-t) including a1)-a5) and d), and
(a) at least one fat soluble vitamin;
(b) at least one water soluble vitamin; and/or (c) at least one trace mineral. aa). The composition of embodiment oe) further comprising at least one enzyme selected from the following group of enzymes: amylase, phytase, phosphatase, xylanase, galactanase, alpha-galactosidase, protease, phospholipase, and/or beta-glucanase. bb). The composition of any one of embodiment oe)-aa) which is an animal feed additive. cc). An animal feed composition having a crude protein content of 50 to 800 g/kg and comprising the phytase of any one of embodiment a)-t) including a1)-a5) and d) or the composition of any one of embodiment oe)-aa). dd). A method for improving the nutritional value of an animal feed, wherein the phytase of any one of embodiment a)-t) including a1)-a5) and c1) or the composition of any one of embodiment oe)-aa) is added to the feed. ee). A process for reducing phytate levels in animal manure comprising feeding an animal with an effective amount of the feed of embodiment cc). ff). A method for the treatment of vegetable proteins, comprising the step of adding the phytase of any one of embodiment a)-t) including a1)-a5) and d) or the composition of any one of embodiment oe)-aa) to at least one vegetable protein or protein source. gg). Use of the phytase of any one of embodiment a)-t) including a1)-a5) and d) or the composition of any one of embodiment oe)-aa) in animal feed; in the preparation of animal feed; for improving the nutritional value of animal feed; for reducing phytate levels in animal manure; for the treatment of vegetable proteins; or for liberating phosphorous from a phytase substrate.
The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.
Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.
Examples Chemicals used were commercial products of at least reagent grade.
Example 1 : Preparation of variants, and test of thermostability and pH profile
Preparation of phytase variants DNA encoding a variant of the phytase having the amino acid sequence of SEQ ID
NO:2 is generated by methods known in the art, and the constructs are fused by PCR to the DNA coding for the signal peptide described by Takami et al in Biosci. Biotechnol. Biochem. 56:1455 (1992) and integrated by homologous recombination into the genome of a Bacillus subtilis host cell (see Diderichsen et al (1990), J. Bacterid., 172, 4315-4321) using standard techniques. The genes are expressed under the control of a triple promoter system (as described in WO 99/43835) and the resulting phytase proteins purified using conventional methods.
Determination of temperature stability The temperature stability of a phytase variant may be determined in the following way:
500 microliter protein solution of the variant and of the reference protein (SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:6) having approximately 10 microgram protein per ml, and being dissolved in 0.1 IVI Na-acetate buffer, pH 5.5, are split in two portions, one portion is incubated at a desired elevated temperature (e.g. 500C, 550C, 600C, 65°C, 700C, 750C, 80°C, or 850C) in plastic containers, the other is stored at 5°C. After 30 minutes incubation at the elevated temperature the protein solutions are transferred to an ice-bath and the activity of the cooled as well as the heated sample is measured by the phosphatase assay described below (buffer blind subtracted). The residual activity is defined as the activity after heat-treatment divided by the activity of the cooled sample (in %). A variant is considered to be more temperature stable (thermostable) if the residual activity in the phosphatase, or phytase, assay is higher, as compared to the reference.
Determination of phosphatase activity
75 microliter phytase-containing enzyme solution is dispensed in a microtiter plate well, e. g. NUNC 269620 and 75 microliter substrate is added (for preparing the substrate, two 5 mg p-nitrophenyl phosphate tablets (Sigma, Cat.No. N-9389) are dissolved in 10 ml 0.1 M Na- acetate buffer, pH 5.5). The plate is sealed and incubated 15 min., shaken with 750 rpm at 370C. After the incubation time 75 microliter stop reagent is added (the stop reagent is 0.1 M di-sodiumtetraborate in water) and the absorbance at 405 nm is measured in a microtiter plate spectrophotometer. One phosphatase unit is defined as the enzyme activity that releases 1 micromol phosphate/min under the given reaction conditions (buffer blind subtracted). The absorbance of 1 micromol p-nitrophenol is determined to be 56 AU (AU= absorbancy units) under assay conditions.
DSC measurements
Differential Scanning Calorimetry (DSC) may be performed at various pH-values using the VP-DSC from Micro CaI. Scans are performed at a constant scan rate of 1.5°C/min from 20-900C. Before running the DSC, the phytases are desalted using NAP-5 columns (Pharmacia) equilibrated in the appropriate buffers (e.g. 0.1M glycine-HCI, pH 2.5 or 3.0; 2OmM sodium acetate pH 4.0; 0.1 M sodium acetate, pH 5.5; 0.1 M Tris-HCI, pH 7.0). Data- handling is performed using the MicroCal Origin software (version 4.10), and the denaturation temperature, Td (also called the melting temperature, Tm) is defined as the temperature at the apex of the peak in the thermogram.
Amended pH profile: Determination of pH 3.5/5.5 activity ratio
An amendment of the pH profile of a phytase variant may be determined as follows: The activity is measured at pH 3.5 (0.1 M acetate buffer, pH 3.5) and at pH 5.5 (0.1 M acetate buffer, pH 5.5), in both cases the buffer blind is subtracted. The activity determined at pH 3.5 is divided by the activity determined at pH 5.5, i.e. the two absorbancy measurements are divided (see below). To measure the activity, supernatants of the variants and references are appropriately diluted (e.g. 1 :5000) in the respective buffer. 75 microliter of the respective enzyme solution is dispensed in a microtiter plate well, e. g. NUNC 269620 and 75 microliter substrate with the corresponding pH is added (the substrate is prepared by dissolving two 5 mg p-nitrophenyl phosphate tablets (Sigma, Cat. No. N-9389) in 10 ml 0.1 M Na-acetate buffer, pH 5.5 and 10 ml 0.1 M acetate buffer, pH 3.5, respectively). The plate is sealed and incubated 15 min., shaken with 750 rpm at 37°C. After the incubation time 75 microliter stop (0.1 M di-sodiumtetraborate in water) reagent is added and the absorbance at 405 nm is measured in a microtiter plate spectrophotometer.
Determination of phytase activity
75 microliter phytase-containing enzyme solution, appropriately diluted (e.g. in 0.25M sodium acetate, 0.005% (w/v) Tween-20. pH5.5), is dispensed in a microtiter plate well, e. g. NUNC 269620, and 75 microliter substrate is added (prepared by dissolving 100 mg sodium phytate from rice (Aldrich Cat.No. 274321) in 10 ml 0.25 M Na-acetate buffer, pH 5.5). The plate is sealed and incubated 15 min. shaken with 750 rpm at 37°C. After the incubation time 75 microliter stop reagent is added (the stop reagent being prepared by mixing 10 ml molybdate solution (10% (w/v) Ammonium hepta-molybdate in 0.25% (w/v) ammonia solution); 10 ml ammonium vanadate (0.24% commercial product from Bie&Berntsen, Cat.No. LAB17650) and 21.7 % (w/v) nitric acid) the absorbance at 405 nm is measured in a microtiter plate spectrophotometer. The phytase activity is expressed in the unit of FYT, one FYT being the amount of enzyme that liberates 1 micromol inorganic ortho-phosphate per min. under the conditions above. An absolute value for the measured phytase activity is obtained by reference to a standard curve prepared from appropriate dilutions of inorganic phosphate or to a standard curve made from dilutions of a phytase enzyme preparation with known activity (such standard enzyme preparation with a known activity is available on request from Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd).
Example 2: Influence of expression host / glycosylation on thermostability Expression in Bacillus
The phytase of SEQ ID NO:2 was expressed in Bacillus subtilis as described in Example 1 , and purified using conventional methods: Centrifugation, germ filtration, ammonium sulphate precipitation (80% ammonium sulphate saturation), centrifugation, re- suspension of pellets in buffer A (50 mM sodium acetate, 1.5 M ammonium sulphate pH 4.5), filtration, hydrophobic interaction chromatography (Phenyl Toyopearl, loading with buffer A, eluting with buffer B (50 mM sodium acetate pH 4.5)), and cation exchange chromatography (SP-sepharose, loading with 10 mM sodium citrate pH 4.0, eluting with a linear salt gradient (10 mM sodium citrate pH 4.0 + 1 M NaCI).
Expression in Pichia
Still further, the phytase of SEQ ID NO:2 was expressed in Pichia pastoris as generally described by Rodriguez et al in Archives of Biochemistry and Biophysics, vol. 382, no. 1, 2000, pp. 105-112. The phytase was purified from the supernatant of the fermentation broth as follows: Precipitation with ammonium sulfate (80% saturation), re-dissolution in 10 ml 25mM sodium acetate buffer pH4.5, dialysis against the same buffer, and filtration through a 0.45 mm filter. 150ml of this solution was applied to a 40 ml SP Sepharose FF column (Pharmacia) equilibrated with the same buffer pH 4.5, and the protein was eluted with a linear NaCI gradient (0-0.5M). Fractions from the column were analyzed for phytase activity. Fractions with phytase activity were checked by SDS-PAGE and the pure fractions were pooled. The protein concentration was measured by using BCA kit (Pierce).
Thermostability by DSC
The Pichia- and the Bacillus-expressed phytase of SEQ ID NO:2 were subjected to thermostability measurements by Differential Scanning Calorimetry (DSC).
Sample Preparation:
Samples (less than 3 ml in volume) were dialyzed in a cold room (approx. 5 degrees centigrade) for a minimum of 1 hour against 500 ml of 20 mM sodium acetate buffer pH 4.0. The sample was transferred to 500 ml of fresh, cold buffer preparation and left to dialyze overnight. The samples were filtered using a 0.45 micrometer syringe filter, volume adjusted to approx. 1.5 ml using the dialysis buffer, and A28o (absorbancy at 280nm) recorded. The dialysis buffer was used as reference in the DSC scans. The samples were degassed using vacuum suction and stirring for approx. 10 minutes.
During sample preparation of the Pichia-expressed phytase (dialysis against 20 mM sodium acetate (NaAc) pH 4.0) a precipitate was formed. The supernatant was used for a first experiment. Afterwards the remaining part of the purified stock solution was dialysed against 20 mM NaAc pH 4.0 and this allowed precipitation of some low Mw impurities present in the batch. This batch was used for a second experiment which revealed a Tm very similar to the first experiment (54 vs. 550C).
DSC Experiment: Experimental settings using a MicroCal™ VP-DSC instrument: Scan rate: 90 K/h. Scan interval: 20 - 90 degrees centigrade. Feedback mode: None. Filtering period: 16 s.
The enzyme concentrations of the samples were approx. 1 - 1.5 mg/ml as estimated by
A28O and a theoretically calculated extinction coefficicient at 280 nm (Vector NTI version 9.0.0).
The thermal unfolding temperature (Td) was evaluated using MicroCal Origin software (version 4.10) and the denaturation temperature determined as the temperature at the apex in the thermogram.
The results are summarized in Table 2 below.
Table 2
Figure imgf000057_0001
From Table 2 it clearly appears that the Pichia-expressed phytase is much less thermostable than the Bacillus-expressed phytase.
The Pichia-expressed phytase was heavily glycosylated, as visualized by a broad range of molecular masses using mass spectrometry (Maldi-TOF), whereas the Bacillus- expressed phytase was not glycosylated.
Example 3: Phytase variant R339D
A protein-engineered variant of the phytase of SEQ ID NO:2 having the substitution R339D was prepared and expressed in Aspergillus oryzae using methods known in the art. Its denaturation temperature, Td, was determined to 62.5°C (20 mM sodium acetate, pH 4.0), using DSC as described in Example 2.
The R339D substitution furthermore serves to remove a Kex2 protease cleavage site of potential relevance for expression in Aspergillus.
Example 4: Animal feed and animal feed additives comprising a phytase variant
Animal Feed Additive
A formulation of phytase variant R339D of SEQ ID NO:2 containing 0.15 g phytase enzyme protein is added to the following premix (per kilo of premix):
5000000 IE Vitamin A
1000000 IE Vitamin D3
13333 mg Vitamin E
1000 mg Vitamin K3
750 mg Vitamin B1
2500 mg Vitamin B2
1500 mg Vitamin B6
7666 meg Vitamin B12
12333 mg Niacin
33333 meg Biotin
300 mg Folic Acid
3000 mg Ca-D-Panthothenate
1666 mg Cu
16666 mg Fe
16666 mg Zn
23333 mg Mn
133 mg Co
66 mg I
66 mg Se
5.8 % Calcium
25 % Sodium
Animal Feed
This is an example of an animal feed (broiler feed) comprising 1.5 mg/kg (1.5 ppm) of phytase variant R339D of SEQ ID NO:2 (calculated as phytase enzyme protein): 62.55 % Maize 33.8% Soybean meal (50% crude protein, CP) 1.0% Soybean oil
0.2% DL-Methionine
0.22% DCP (dicalcium phosphate)
0.76% CaCO3 (calcium carbonate)
0.32% Sand
0.15% NaCI (sodium chloride)
1 % of the above Premix
The ingredients are mixed, and the feed is pelleted at the desired temperature, e.g. 60, 65, 75, 80, 85, 90 or even 950C.
Example 5: Determination of temperature stability
Eight variants of SEQ ID NO:2 (the alterations as compared to SEQ ID NO:2 are shown in Table 3 below) were prepared as described in Example 1. The two reference phytases having SEQ ID NO:2 and SEQ ID NO:3 were prepared in the same manner.
The temperature stability was determined as follows:
200 microliter supematants of each of the variants and the reference proteins were split in two portions, one portion was incubated at 500C in plastic containers, the other was stored at 5°C. After 30 minutes incubation at 5O0C the protein solutions were transferred to an ice-bath. After dilution 1:100 in 0.1 M Na-acetate buffer, pH 5.5 the activity of the cooled and heated sample was measured by the phosphatase assay of Example 1 ("Determination of phosphatase activity"), buffer blind subtracted. The results are shown in Table 3 below as enzyme activity (in absorption units (AU)) after incubation for 30 minutes at 5°C and 5O0C, respectively, and the residual activity (RA) is calculated as activity of the heat-treated sample (5O0C incubation) divided by the activity of the cooled sample (5°C incubation), in %.
Table 3: Phvtase variants with improved thermostability
Figure imgf000059_0001
N286Q of SEQ ID NO:2 0.062 0.024 39
Example 6: Performance in animal feed in an in vitro model
The performance in animal feed of a phytase variant is compared, in an in vitro model, to the performance of a reference protein such as SEΞQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:6. The in vitro model simulates gastro-intestinal conditions in a monogastric animal and correlates well with results obtained in animal trials in vivo. The comparison is performed as follows:
Phytase activity in the variant sample is determined as described in Example 1 under "Determination of phytase activity". Feed samples composed of 30% soybean meal and 70% maize meal with added CaCI2 to a concentration of 5 g calcium per kg feed are then prepared and pre-incubated at 40°C and pH 3.0 for 30 minutes followed by addition of pepsin (3000 U/g feed) and suitable dosages of the phytases (identical dosages are used for all phytases to be tested to allow comparison), for example between 0.25 to 0.75 phytase units FYT/g feed. A blank with no phytase activity is also included as reference. The samples are then incubated at 400C and pH 3.0 for 60 minutes followed by pH 4.0 for 30 minutes.
The reactions are stopped and phytic acid and inositol-phosphates extracted by addition of HCI to a final concentration of 0.5 M and incubation at 400C for 2 hours, followed by one freeze-thaw cycle and 1 hour incubation at 400C. Phytic acid and inositol-phosphates are separated by high performance ion chromatography as described by Chen et al in Journal of Chromatography A (2003) vol. 1018, pp. 41- 52 and quantified as described by Skoglund et al in J. Agric. Food Chem. (1997), vol. 45, pp. 431-436.
Released phosphorous is then calculated as the difference in inositol-phosphate bound phosphorous (IP-P) between phytase-treated and non-treated samples. The relative performance of the variant is calculated as the percentage of the phosphorous released by the reference phytase.
The in vitro performance of a number of phytase variants of SEQ ID NO:2 was determined as described above, in a dosage of 125 FYT/kg feed. The results are shown in Tables 4A and 4B below, for supernatants and purified phytases, respectively. Residual IP6-P designates the amount of IP6-P (phytate phosphorous) left after the in vitro incubation and it is indicated in mg/g DM (Dry Matter). Degraded IP6-P is determined as the difference between residual IP6-P of the blank and residual IP6-P of the respective sample. Finally, in the last column degraded IP6-P is indicated relative to the phytase having SEQ ID NO:2. In Table 4A the blank and the reference (SEQ ID NO:2) values are averages from a number of independent determinations, whereas the other values are based on single determinations. In Table 4B the blank value is average from a number of independent determinations, wheres the other values are based on single determinations.
Table 4A. In vitro performance of phvtase variant supernatants
Figure imgf000061_0001
Figure imgf000062_0001
Variants 015, 016, 018, 040, 041 , 042, 044, and 050 appear to have an in vitro performance which is at least as good or better than the phytases of SEQ ID NO:2 and 3.
Table 4B: In vitro performance of purified phytase variants
Figure imgf000062_0002
Figure imgf000063_0001
Variants 030, 031 , 037, 056, 072, 085, 087, 089, 090, 095, 098, and 125 appear to perform at least as good in vitro as the phytase of SEQ ID NO:3.
Example 7: Specific activity
The specific activity of a phytase variant is determined on highly purified samples dialysed against 250 mM sodium acetate, pH 5.5. The purity is checked beforehand on an SDS poly acryl amide gel showing the presence of only one component.
The protein concentration is determined by amino acid analysis as follows: An aliquot of the sample is hydrolyzed in 6N HCI, 0.1 % phenol for 16 h at 11 O0C in an evacuated glass tube. The resulting amino acids are quantified using an Applied Biosystems 420A amino acid analysis system operated according to the manufacturer's instructions. From the amounts of the amino acids the total mass - and thus also the concentration - of protein in the hydrolyzed aliquot can be calculated. The phytase activity is determined in the units of FYT as described in Example 1
("Determination of phytase activity"), and the specific activity is calculated as the phytase activity measured in FYT units per mg phytase variant enzyme protein.
The specific activity for the phytase of SEQ ID NO:2 and variant 072 (I362R of SEQ ID NO:2) was determined as described above. The specific activity of variant 072 was 86% of the specific activity of the phytase of SEQ ID NO:2. The uncertainty (standard deviation) is estimated to approximately 10%, which is mainly due to the phytase activity assay based on a complex substrate.
Example 8: Temperature stability A number of variants of SEQ ID NO:2 were prepared as described in Example 1 , and the Bacillus subtilis host strains grown in 100ml PS1 medium (100g/L sucrose, 40g/L Soy flakes, 10g/L Na2HPO4.12H2O, 0.1ml/L Dowfax 63N10 (Dow)) in 500ml shake flasks for four days at 3O0C at 300 rpm.
Two reference phytases were prepared in the same manner, viz. the phytase having SEQ ID NO:3 (corresponding to variant N31 D/Q139K/L197F/N316K of SEQ ID NO:2), and the phytase having SEQ ID NO:4 (corresponding to variant N31 D/N121T/K132T/Q139K of SEQ ID NO:2).
Also the phytase having SEQ ID NO:9 was included for comparison (corresponding to variant Q3P/N31 D/N121T/K132T/Q139K of SEQ ID NO:2). The temperature stability of the variants and the reference phytases was determined as follows:
The supematants were diluted ten times by adding 2OuI (microliter) supernatant to 18OuI 0.1 M Na-acetate buffer, pH5.5 + 0.005% Tween-20. The diluted enzymes were split in two portions, one portion was incubated at 6O0C in plastic containers, and the other portion was stored at 5°C. After 30 minutes incubation at 600C the protein solutions were transferred to an ice-bath. After dilution 1:10 in 0.1 M Na-acetate buffer, pH5.5, and 0.005% Tween-20, the activity of the cooled and heated sample was measured by the phosphatase assay of Example 1 ("Determination of phosphatase activity"), buffer blind subtracted. Table 5 is a list of variants with improved temperature stability as compared to the reference phytases. For each variant, the table also specifies the alterations as compared to SEQ ID NO:2. The enzyme activity (in absorption units (AU)) after incubation for 30 minutes at 5°C and 6O0C, respectively, was determined, and the residual activity (RA) calculated as the activity of the heat-treated sample (60°C incubation) divided by the activity of the cooled sample (50C incubation). Next, the residual activity results were normalized to the residual activity of the phytase of SEQ ID NO:2, having been expressed and treated in the same manner. The resulting Improvement Factor (IF) Is shown in Table 5. For the phytase of SEQ ID NO:2 the IF is 1.0, whereas the two reference phytases of SEQ ID NO:3 and 4 were less thermostable than the phytase of SEQ ID NO:2, which is apparent from the fact that the IF for these two phytases was only 0.1 and 0.3, respectively.
Figure imgf000064_0001
Figure imgf000065_0001
A number of purified variants of SEQ ID NO:2 were prepared as generally described in Example 1. Two reference phytases were prepared in the same manner, viz. the phytase having SEQ ID NO:3 (corresponding to variant N31D/Q139K/L197F/N316K of SEQ ID NO:2), and the phytase having SEQ ID NO:4 (corresponding to variant N31D/N121T/K132T/Q139K of SEQ ID NO:2). Also the phytase having SEQ ID NO:9 was included for comparison (corresponding to variant Q3P/N31D/N121T/K132T/Q139K of SEQ ID NO:2).
Aliquots of the protein samples were dialysed against 2 x 500ml 2OmM Na-acetate, pH4.0 at 4°C in a 2-3h step followed by an over night step. Each sample was 0.45um filtered and diluted with buffer to approx. 2 A2so units. The exact absorbance values measured are given in the results table. DSC was performed on a MicroCal VP-DSC at 90°C/h scan rate from 20-900C in 20 mM Na-acetate buffer, pH 4.0.
The resulting denaturation temperatures (Td) are shown in Table 6 below, which summarizes the results of three different experiments.
Table 6: Td measurements by DSC
Figure imgf000066_0001
Example 10: Purification and temperature profile The phytase variants and reference and comparative phytases used herein were purified as follows: The fermentation supernatant with the phytase was first centrifuged at 7200rpm and 50C for one hour and filtered through a sandwich of four Whatman glass microfibre filters (2.7, 1.6, 1.2 and 0.7 micrometer). Following this the solution was sterile filtered (either through a Fast PES Bottle top filter with a 0.22μm cut-off or through a Seitz- EKS depth filter using pressure). The solution was added solid ammonium sulfate giving a final concentration of 1.5M and the pH was adjusted to 6.0 using 6M HCI.
The phytase-containing solution was applied to a butyl-sepharose column, approximately 50ml in a XK26 column, using as buffer A 25mM bis-tris (Bis-(2- hydroxyethyl)imino-tris(hydroxymethyl)methan)) + 1.5M ammonium sulfate pHδ.O, and as buffer B 25mM bis-tris pHδ.O. The fractions from the column were analyzed for activity using the phosphatase assay (see Example 1 , "Determination of phosphatase activity") and fractions with activity were pooled. The pooled fractions were dialyzed extensively against 1OmM sodium acetate pH4.5. Following this the phytase-containing solution was purified by chromatography on S Sepharose, approximately 75ml in a XK26 column, using as buffer A 5OmM sodium acetate pH4.5, and as buffer B 5OmM sodium acetate + 1M NaCI pH4.5. Again the fractions from the column were analyzed for activity and fractions with activity were pooled. Finally, the solution containing the purified phytase was concentrated using an Amicon ultra-15 filtering device with a 1OkDa cut-off membrane.
The molecular weight, as estimated from SDS-PAGE, was approximately 4OkDa for all phytases and the purity was in all cases > 95%.
The temperature profile (phytase activity as a function of temperature) of the variants was determined in the temperature range of 20-90°C essentially as described in Example 1 ("Determination of phytase activity"), however, the enzymatic reactions (100 microliter phytase-containing enzyme solution + 100 microliter substrate) were performed in PCR tubes instead of microtiter plates. After a 15 minute reaction period at desired temperature the tubes were cooled to 200C for 20 seconds and 150 microliter of the reaction mixture was transferred to a microtiter plate. 75 microliter stop reagent was added and the absorbance at 405 nm was measured in a microtiter plate spectrophotometer. The results are summarized in Table 7 below. The numbers given for each temperature (20-90 0C in 100C steps) are relative activity (in %) normalized to the value at optimum.
Table 7: Temperature profiles
Figure imgf000067_0001
Figure imgf000068_0001
Variants 030, 031 , 037, 044, 056, 062, 072, 083, and 093 have a higher relative activity at 7O0C as compared to the reference phytases 026 and 102.
Example 11 : pH profile
The pH profiles (phytase activity as a function of pH) of a number of variants and the same reference and comparative phytases as used in the previous examples were determined at 370C in the pH range of 2.0 to 7.5 (in 0.5 pH-unit steps) as described in Example 1 ("Determination of phytase activity"), except that a buffer cocktail (5OmM glycine, 5OmM acetic acid and 5OmM Bis-Tris was used instead of the 0.25M sodium acetate pH5.5 buffer. The results are summarized in Table 8 below. The numbers given for each pH (2.0-7.5) are relative activity (in %) normalized to the value at optimum.
Table 8: pH profiles
Figure imgf000069_0001
For YC062 and YC091 the pH curve (relative activity as a function of pH) seems to have shifted 0.5 pH unit towards higher pH.
Furthermore, while for most of the Table 8 phytases (including the reference phytases 026 and 102) the optimum is at pH3.5-pH4.0, an optimum pH of 3.5 is observed for no. 062, 085, and 089, and an optimum pH of 4.0 is observed for no. 091 and 093.
Example 12: Temperature stability
The temperature stability of a number of purified variants and the same reference and comparative phytases as in the previous examples was determined by measuring residual phytase activity after incubation at 700C and pH 4.0 (0.1 M sodium acetate). The phytases were incubated and samples were withdrawn after 0, 10, 30 and 60 minutes and cooled on ice. The residual activity at pH 5.5 was determined using the method described in Example 1 ("Determination of phytase activity"). The results, normalized to the activity found at 0 minutes, are shown in Table 9 below.
Table 9: Temperature stability
Figure imgf000070_0001
The above results indicate that Nos. 044, 062, 072, and 083 may be more stable under these conditions (7O0C and pH 4) than the reference phytases (although in this experiment a big variation was observed for No. 000).
Example 13: Calculating percentage of identity and identifying corresponding positions
SEQ ID NO:9 was aligned with SEQ ID NO:2 using the Needle program from the
EMBOSS package version 2.8.0. The substitution matrix used was BLOSUM62, the gap opening penalty was 10.0, and the gap extension penalty was 0.5.
The resulting alignment is shown in Fig. 2. The degree of identity between SEQ ID NO:9 and SEQ ID NO:2 is calculated as follows: The number of exact matches is 406 (all those with a vertical stroke). The length of the shortest sequence is 411 (SEQ ID NO:2). The percentage of identity is 406/411 x 100% =
98.8%.
The alignment of Fig. 2 is also used for deriving corresponding positions as follows: Amino acids on top of each other in this alignment are in corresponding positions. E.g. amino acid Q in position 3 of SEQ ID NO:2 corresponds to amino acid P in position number 25 of
SEQ ID NO:9. For the present purposes we refer to the position number of SEQ ID NO:2.
Therefore, SEQ ID NO:9 may be considered a variant of SEQ ID NO:2 which comprises the substitution Q3P. Other differences in the form of substitutions within the overlap of the alignment are found in positions 31 , 121 , 132, and 139, viz. N31D, N121T, K132T, and Q139K. Additional differences are found in the N-terminus, where SEQ ID NO:9 has an extension of 22 amino acids as compared to SEQ ID NO:2.
Overall, SEQ ID NO:9 may therefore be considered the following variant of SEQ ID NO:2:
*0aM/*0bS/*0cT/*0dFrOeirOfl/*0gR/^h^OiL/*0jF/*0kF/*0mS/*0nL/*0oL/*0pC/*0qGrOrS/*0sF /*0tS/*0ul/*0vH/*0wA/Q3P/N31 D/N121 T/K132T/Q139K.

Claims

Claims
1. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one alteration as compared to SEQ ID NO:2 in at least one position selected from the following: 1 , 2, 3, 4, 5, 31 , 41 , 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91 , 99, 100, 104, 105, 107, 109, 111 , 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 136, 137, 141 , 154, 161 , 162, 164, 167, 171 , 176, 177, 179, 180, 181 , 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316, 324, 331 , 339, 351 , 355, 362, 379, 385, 406, 409, 410, and 411 ; with the proviso that the phytase is not SEQ ID NO:3, not SEQ ID NO:4, and not SEQ ID NO:6.
2. The phytase of claim 1 which is not SEQ ID NO:9 and the variants thereof listed in Fig.1.
3. The phytase of any one of claims 1 and 2, which comprises at least one of the following alterations: 1*, 2*, 3*, 4P, 5P, 31C1T, 41 P, 46C1D1E, 52C.E, 53V1Q1 55D1I1 57Y1 59C1 74A, 76G, 82E, 84Y, 91C.P, 99C, 100C1 104A, 105F, 107D,E,G, 109A.G, 111P, 114H1NJ1 115Q, 116A1E1PJ1Q1 117D1E1K 118I1L1MJ1 119G1K1R1S, 12OK1SJ1Q, 121 A1D1M1P J, V, 122D, 123P1S, 124L1T1V, 136P, 137P, 141C, 154P, 161P, 162C, 164D1E, 167Q1 171T1 176C1 177C1 179G1I1K1N1Q, 18OA1E1GJ, 181D1G1I1K, 182H1K1S1Q, 183A1L1P1S1V1Q, 184*, 185*, 186*, 196Q1 199C1 200K.R, 202N1 203T1 218Q, 223E, 239Q1 240P1 241Q1 247C, 273L,Q, 276K1R1 281 H, 282P1 283P, 284P, 285G1N1R, 286K.Q, 289P, 294T, 299L, 308A, 314G1N1 316D, 324N1 331K1 339D, 351Y1 355P1 362K1R1 379K1R, 385D, 406A1 409D,E, 41OD1E, and/or 411R1K.
4. The phytase of claim 3, wherein the amino acids in position 179, 180, 181 , 182, 183, 184, 185, and 186 have been replaced by QADKP1 GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV, or KTDKL.
5. A phytase which has at least 74% identity to SEQ ID NO:2 and which comprises at least one of the following alterations: 1 H1K1R, 60P1 105E, 106A1G1 155F1 157F1 173P, 175L, 188P, 205P, 215M, 231 P, 254Y, 280P, 330D, and/or 371 P; with the proviso that the phytase is not SEQ ID NO:3, not SEQ ID NO:4, not SEQ ID NO:6, and not SEQ ID NO:9 and the variants thereof listed in Fig. 1.
6. The phytase of any one of claims 1-4, which comprises an alteration selected from the following: 52C, 141C1 162C, 31C1 52C1 99C, 59C, 100C, 141 C/199C, 4P1 5P, 111P1 137P, 161 P, 52E, 57Y1 76G1 107D, 107G1 109A1 1*, 1*/2*, 1*/2*/3*, 121T1 273L, 285G1 286Q1 299L1 362K, 331K/55D, 107E, 46E, 82E1 119R1 119K1 164E, 223E, 276R, 276K1 362R1 379R1 379K1
385D, 410D, 410E1 411R1 411K1 53V, 121 D, 167Q1 196Q, 200K1 202N1 218Q1 241 Q, 285N, 314N1 314G, 406A,
179K/180E/181 K/182H/183Q/184*/185*/186*, 179K/180E/181 K/182Q/183Q/184*/185*/186*, 179K/180E/181 K/182K/183V/184*/1857186*, 179K/180T/181 D/182K/183L/184*/185*/186*. 111P/241Q, 1K,
114T/115Q/116A/117D/118T/119S/120S/121 P/122D/123P/124L, and 114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L
7. The phytase of any one of claims 1-6 which is a variant of the phytase of SEQ ID NO:2.
8. The phytase of any one of claims 1-6 which is a variant of the phytase of SEQ ID NO:3.
9. The phytase of any one of claims 1-6 which is a variant of the phytase of SEQ ID NO:4.
10. The phytase of any one of claims 1-6 which is a variant of the phytase of SEQ ID NO:6.
11. The phytase of any one of claims 1-6 which is a variant of the phytase of SEQ ID NO:9.
12. The phytase of any one of claims 1-6 which is a variant of any one of the phytase variants related to SEQ ID NO:9 and listed in Fig.1.
13. The phytase of any one of claims 1-12 which furthermore comprises a substitution or a combination of substitutions selected from amongst the substitutions and combinations of substitutions listed in each row of Fig.1.
14. The phytase of any one of claims 1-13, which has an improved thermostability, an improved pH profile, an improved specific activity, an amended glycosylation pattern, an improved temperature profile, an improved performance in animal feed, and/or which incorporates a change of a potential protease cleavage site.
15. An isolated nucleic acid sequence comprising a nucleic acid sequence which encodes the phytase of any of claims 1-14.
16. A nucleic acid construct comprising the nucleic acid sequence of claim 15 operably linked to one or more control sequences that direct the production of the phytase in a suitable expression host.
17. A recombinant expression vector comprising the nucleic acid construct of claim 16.
18. A recombinant host cell comprising the nucleic acid construct of claim 16 and/or the expression vector of claim 17.
19. A method for producing the phytase of any one of claims 1-14, comprising (a) cultivating the host cell of claim 18 to produce a supernatant comprising the phytase; and (b) recovering the phytase.
20. A transgenic plant, or plant part, capable of expressing a phytase of any one of claims 1-14.
21. A transgenic, non-human animal, or products, or elements thereof, being capable of expressing a phytase of any one of claims 1-14.
22. A composition comprising at least one phytase of any one of claims 1-14, and (a) at least one fat soluble vitamin;
(b) at least one water soluble vitamin; and/or
(c) at least one trace mineral.
23. The composition of claim 22 further comprising at least one enzyme selected from the following group of enzymes: amylase, phytase, phosphatase, xylanase, galactanase, alpha- galactosidase, protease, phospholipase, and/or beta-glucanase.
24. The composition of any one of claims 22-23 which is an animal feed additive.
25. An animal feed composition having a crude protein content of 50 to 800 g/kg and comprising the phytase of any one of claims 1-14 or the composition of any one of claims 22- 24.
26. A method for improving the nutritional value of an animal feed, wherein the phytase of any one of claims 1-14 or the composition of any one of claims 22-24 is added to the feed.
27. A process for reducing phytate levels in animal manure comprising feeding an animal with an effective amount of the feed of claim 25.
28. A method for the treatment of vegetable proteins, comprising the step of adding the phytase of any one of claims 1-14 or the composition of any one of claims 22-24 to at least one vegetable protein or protein source.
29. Use of the phytase of any one of claims 1-14 or the composition of any one of claims 22-24 in animal feed; in the preparation of animal feed; for improving the nutritional value of animal feed; for reducing phytate levels in animal manure; for the treatment of vegetable proteins; or for liberating phosphorous from a phytase substrate.
PCT/DK2007/000135 2006-04-04 2007-03-19 Phytase variants WO2007112739A1 (en)

Priority Applications (12)

Application Number Priority Date Filing Date Title
CA2647476A CA2647476C (en) 2006-04-04 2007-03-19 Phytase variants
CN200780020771.5A CN101460612B (en) 2006-04-04 2007-03-19 Phytase variants
US12/294,526 US8460656B2 (en) 2006-04-04 2007-03-19 Phytase variants
EP07711278A EP2001999B1 (en) 2006-04-04 2007-03-19 Phytase variants
MX2008012632A MX2008012632A (en) 2006-04-04 2007-03-19 Phytase variants.
JP2009503408A JP5221516B2 (en) 2006-04-04 2007-03-19 Phytase mutant
BRPI0709732A BRPI0709732B1 (en) 2006-04-04 2007-03-19 phytase, isolated nucleic acid sequence, nucleic acid construct, recombinant expression vector, recombinant microorganism, method for producing phytase, composition, method for improving the nutritional value of an animal feed, process for reducing phytate levels in manure method for the treatment of vegetable protein and use of phytase or composition in animal feed
AU2007234177A AU2007234177B2 (en) 2006-04-04 2007-03-19 Phytase variants
ES07711278T ES2387203T3 (en) 2006-04-04 2007-03-19 Phytase variants
US13/874,954 US8877471B2 (en) 2006-04-04 2013-05-01 Phytase variants
US14/504,517 US9451783B2 (en) 2006-04-04 2014-10-02 Phytase variants
US15/249,856 US10041052B2 (en) 2006-04-04 2016-08-29 Phytase variants

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DKPA200600484 2006-04-04
DKPA200600484 2006-04-04
DKPA200600581 2006-04-25
DKPA200600581 2006-04-25

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US12/294,526 A-371-Of-International US8460656B2 (en) 2006-04-04 2007-03-19 Phytase variants
US13/874,954 Division US8877471B2 (en) 2006-04-04 2013-05-01 Phytase variants

Publications (1)

Publication Number Publication Date
WO2007112739A1 true WO2007112739A1 (en) 2007-10-11

Family

ID=38123737

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2007/000135 WO2007112739A1 (en) 2006-04-04 2007-03-19 Phytase variants

Country Status (11)

Country Link
US (4) US8460656B2 (en)
EP (2) EP2001999B1 (en)
JP (1) JP5221516B2 (en)
AU (1) AU2007234177B2 (en)
BR (1) BRPI0709732B1 (en)
CA (1) CA2647476C (en)
DK (1) DK2365064T3 (en)
ES (2) ES2387203T3 (en)
MX (1) MX2008012632A (en)
PL (1) PL2365064T3 (en)
WO (1) WO2007112739A1 (en)

Cited By (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010034835A2 (en) 2008-09-26 2010-04-01 Novozymes A/S Hafnia phytase variants
WO2010106170A1 (en) 2009-03-20 2010-09-23 Novozymes A/S A nutritional beverage and a method of making the same
WO2010135588A2 (en) 2009-05-21 2010-11-25 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
WO2011048046A2 (en) 2009-10-22 2011-04-28 Basf Se Synthetic phytase variants
WO2011117397A1 (en) * 2010-03-26 2011-09-29 Novozymes A/S Thermostable phytase variants
WO2011117396A3 (en) * 2010-03-26 2011-11-24 Novozymes A/S Thermostable phytase variants
WO2012110776A2 (en) 2011-02-18 2012-08-23 Dupont Nutrition Biosciences Aps Feed additive composition
WO2012110778A2 (en) 2011-02-18 2012-08-23 Dupont Nutrition Biosciences Aps Feed additive composition
WO2012110777A2 (en) 2011-02-18 2012-08-23 Dupont Nutrition Biosciences Aps Feed additive composition
WO2012143861A1 (en) 2011-04-21 2012-10-26 Basf Se Synthetic phytase variants
WO2013005003A1 (en) 2011-07-07 2013-01-10 Dupont Nutrition Biosciences Aps Assay
US20130017185A1 (en) * 2010-03-26 2013-01-17 Novozymes A/S Thermostable Phytase Variants
RU2472855C2 (en) * 2009-12-15 2013-01-20 Федеральное государственное унитарное предприятие "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов" (ФГУП ГосНИИгенетика) MUTANT RECOMBINANT THERMALLY STABLE PHYTASE (VERSIONS), DNA FRAGMENT CODING SAID PHYTASE (VERSIONS) Pichia pastoris STRAIN - PRODUCER OF SAID PHYTASE (VERSIONS)
US8455232B2 (en) 1998-06-25 2013-06-04 Cornell Research Foundation, Inc. Overexpression of phytase genes in yeast systems
US8540984B2 (en) 2006-08-03 2013-09-24 Cornell Research Foundation, Inc. Phytases with improved thermal stability
US8557555B2 (en) 2011-04-21 2013-10-15 Basf Se Synthetic phytase variants
US8877478B2 (en) 2006-09-21 2014-11-04 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
WO2015035914A1 (en) 2013-09-11 2015-03-19 Novozymes A/S Processes for producing fermentation products
US9179693B2 (en) 2012-08-03 2015-11-10 Dupont Nutrition Biosciences Aps Feed additive composition
WO2015197871A1 (en) 2014-06-27 2015-12-30 Dsm Ip Assets B.V. A method for improving the nutritional value of animal feed
US9273295B2 (en) 2004-10-04 2016-03-01 Dupont Nutrition Biosciences Aps Mutant citrobacter freundii phytase polypeptide
AU2013204082B2 (en) * 2009-05-21 2016-03-31 Basf Enzymes Llc Phytases, nucleic acids encoding them and methods for making and using them
WO2017112540A1 (en) 2015-12-22 2017-06-29 Novozymes A/S Processes for producing fermentation products
TWI607088B (en) * 2016-02-18 2017-12-01 基酵生物科技股份有限公司 Phytase having improved thermostability
TWI615471B (en) * 2016-02-18 2018-02-21 基酵生物科技股份有限公司 Phytase having improved thermostability
TWI616532B (en) * 2016-02-18 2018-03-01 基酵生物科技股份有限公司 Phytase having improved thermostability
US10011822B2 (en) 2016-02-18 2018-07-03 Dongguan APAC Biotechnology Co., Ltd. Phytase having improved thermostability
CN104684408B (en) * 2012-08-03 2018-08-31 杜邦营养生物科学有限公司 Feed addictive composition
WO2019055455A1 (en) 2017-09-15 2019-03-21 Novozymes A/S Enzyme blends and processes for improving the nutritional quality of animal feed
WO2019083831A1 (en) 2017-10-23 2019-05-02 Novozymes A/S Processes for reducing lactic acid in a biofuel fermentation system
EP3527667A2 (en) 2013-03-08 2019-08-21 Biogrammatics, Inc. Yeast promoters for protein expression
RU2706086C1 (en) * 2018-10-29 2019-11-13 Акционерное Общество "Биоамид" Mutant recombinant thermostable phytase
WO2019231944A2 (en) 2018-05-31 2019-12-05 Novozymes A/S Processes for enhancing yeast growth and productivity
EP3594351A1 (en) 2013-03-08 2020-01-15 Keck Graduate Institute of Applied Life Sciences Yeast promoters from pichia pastoris
WO2020160126A1 (en) 2019-01-31 2020-08-06 Novozymes A/S Polypeptides having xylanase activity and use thereof for improving the nutritional quality of animal feed
WO2021007379A1 (en) 2019-07-09 2021-01-14 Dupont Nutrition Biosciences Aps Fat coated particulate enzyme compositions
WO2021026201A1 (en) 2019-08-05 2021-02-11 Novozymes A/S Enzyme blends and processes for producing a high protein feed ingredient from a whole stillage byproduct
WO2021034660A1 (en) 2019-08-16 2021-02-25 Dupont Nutrition Biosciences Aps Compositions for gut health comprising combinations of lactobacillus strains
WO2021046073A1 (en) 2019-09-05 2021-03-11 Dupont Nutrition Biosciences Aps Feed composition
WO2021080864A2 (en) 2019-10-21 2021-04-29 Dupont Nutrition Biosciences Aps Compositions for gut health
WO2021126966A1 (en) 2019-12-16 2021-06-24 Novozymes A/S Processes for producing fermentation products
WO2021158927A1 (en) 2020-02-07 2021-08-12 Dupont Nutrition Biosciences Aps Feed compositions for animal health
WO2022034211A1 (en) 2020-08-13 2022-02-17 Novozymes A/S Phytase variants and polynucleotides encoding same
WO2022081947A1 (en) 2020-10-16 2022-04-21 Dupont Nutrition Biosciences Feed compositions for animal health
WO2022169933A2 (en) 2021-02-03 2022-08-11 Dupont Nutrition Biosciences Aps Compositions for gut health
WO2023049488A1 (en) 2021-09-27 2023-03-30 Dupont Nutrition Biosciences Aps Feed additive compositions and methods for using the same
WO2024137248A1 (en) 2022-12-19 2024-06-27 Novozymes A/S Compositions comprising arabinofuranosidases and a xylanase, and use thereof for increasing hemicellulosic fiber solubilization
WO2024137252A1 (en) 2022-12-19 2024-06-27 Novozymes A/S Process for reducing syrup viscosity in the backend of a process for producing a fermentation product
WO2024137250A1 (en) 2022-12-19 2024-06-27 Novozymes A/S Carbohydrate esterase family 3 (ce3) polypeptides having acetyl xylan esterase activity and polynucleotides encoding same
WO2024137704A2 (en) 2022-12-19 2024-06-27 Novozymes A/S Processes for producing fermentation products using fiber-degrading enzymes with engineered yeast
WO2024137246A1 (en) 2022-12-19 2024-06-27 Novozymes A/S Carbohydrate esterase family 1 (ce1) polypeptides having ferulic acid esterase and/or acetyl xylan esterase activity and polynucleotides encoding same

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2387203T3 (en) 2006-04-04 2012-09-17 Novozymes A/S Phytase variants
EP3970506A1 (en) * 2015-07-02 2022-03-23 Novozymes A/S Animal feed compositions comprising gh25 lysozyme and ec 3.1.3.26 phytase and uses thereof
BR112020016068A2 (en) 2018-02-08 2020-12-08 Danisco Us Inc. THERMALLY RESISTANT MATRIX WAX PARTICLES FOR ENZYME ENCAPSULATION
KR20210005665A (en) 2018-04-26 2021-01-14 다니스코 유에스 인크. Method for increasing the stability of phytase in solid composition and granule composition comprising phosphate and phytase
WO2020106796A1 (en) 2018-11-20 2020-05-28 Dupont Nutrition Biosciences Aps ENGINEERED ROBUST HIGH Tm-PHYTASE CLADE POLYPEPTIDES AND FRAGMENTS THEREOF
TWI725754B (en) 2020-02-27 2021-04-21 金全益股份有限公司 RGV feeding system
US20230240334A1 (en) 2020-02-28 2023-08-03 Dupont Nutrition Biosciences Aps Feed compositions

Citations (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0238023A2 (en) 1986-03-17 1987-09-23 Novo Nordisk A/S Process for the production of protein products in Aspergillus oryzae and a promoter for use in Aspergillus
WO1991014772A1 (en) 1990-03-23 1991-10-03 Gist-Brocades N.V. Production of enzymes in seeds and their use
EP0561907A1 (en) 1990-12-05 1993-09-29 Novo Nordisk A/S Proteins with changed epitopes and methods for the production thereof
WO1994001459A1 (en) 1992-07-10 1994-01-20 Novo Nordisk A/S A fungicidally active compound
WO1995022625A1 (en) 1994-02-17 1995-08-24 Affymax Technologies N.V. Dna mutagenesis by random fragmentation and reassembly
WO1996000343A1 (en) 1994-06-24 1996-01-04 Audi Ag Method of controlling the electric heating of a catalytic converter
WO1996000787A1 (en) 1994-06-30 1996-01-11 Novo Nordisk Biotech, Inc. Non-toxic, non-toxigenic, non-pathogenic fusarium expression system and promoters and terminators for use therein
WO1996016177A1 (en) 1994-11-24 1996-05-30 Novo Nordisk A/S A process for producing polypeptides with reduced allergenicity
WO1996017929A1 (en) 1994-12-07 1996-06-13 Novo Nordisk A/S Polypeptide with reduced allergenicity
US5689054A (en) 1994-03-17 1997-11-18 The United States Of America As Represented By The Secretary Of Agriculture Low phytic acid mutants and selection thereof
WO1998030682A1 (en) 1997-01-10 1998-07-16 Novo Nordisk A/S Enzyme coupled with polymeric molecules for skin care
WO1998035026A1 (en) 1997-02-06 1998-08-13 Novo Nordisk A/S Polypeptide-polymer conjugates having added and/or removed attachment groups
WO1999000489A1 (en) 1997-06-25 1999-01-07 Novo Nordisk A/S A modified polypeptide
EP0897985A2 (en) 1997-07-24 1999-02-24 F.Hoffmann-La Roche Ag Consensus phytases
WO1999043835A2 (en) 1998-02-26 1999-09-02 Novo Nordisk Biotech, Inc. Methods for producing a polypeptide in a bacillus cell
WO2000020569A1 (en) 1998-10-02 2000-04-13 Novozymes A/S Solid phytase compositions
WO2000022103A1 (en) 1998-10-13 2000-04-20 Novozymes A/S A modified polypeptide with reduced immune response
WO2000026230A1 (en) 1998-10-30 2000-05-11 Novozymes A/S Low allergenic protein variants
WO2000026354A1 (en) 1998-10-30 2000-05-11 Novozymes A/S Glycosylated proteins having reduced allergenicity
WO2000064247A1 (en) 1999-04-23 2000-11-02 University Of Guelph Transgenic animals expressing salivary proteins
WO2001058275A2 (en) 2000-02-08 2001-08-16 F Hoffmann-La Roche Ag Use of acid-stable subtilisin proteases in animal feed
WO2001083559A2 (en) 2000-04-28 2001-11-08 Novozymes A/S Production and use of protein variants having modified immunogenecity
WO2002090384A2 (en) 2001-05-04 2002-11-14 Novozymes A/S Antimicrobial polypeptide from aspergillus niger
WO2003044049A1 (en) 2001-11-20 2003-05-30 Novozymes A/S Antimicrobial polypeptides from pseudoplectania nigrella
WO2003048148A2 (en) 2001-12-03 2003-06-12 Novozymes A/S Statin-like compounds
WO2004015084A2 (en) * 2002-08-12 2004-02-19 Genencor International, Inc. Mutant e. coli appa phytase enzymes
WO2004085638A1 (en) 2003-03-25 2004-10-07 Republic Of National Fisheries Research And Development Institute Phytase produced from citrobacter braakii
WO2006038128A2 (en) 2004-10-04 2006-04-13 Danisco A/S Citrobacter freundii phytase and homologues
WO2006038062A1 (en) 2004-10-04 2006-04-13 Danisco A/S Microbial phytase as supplement to food or fodder
WO2006037327A2 (en) * 2004-10-04 2006-04-13 Novozymes A/S Polypeptides having phytase activity and polynucleotides encoding same
WO2006037328A1 (en) 2004-10-04 2006-04-13 Novozymes A/S Polypeptides having phytase activity and polynucleotides encoding same
WO2006063588A1 (en) * 2004-12-13 2006-06-22 Novozymes A/S Polypeptides having acid phosphatase activity and polynucleotides encoding same

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0795946B2 (en) * 1993-08-19 1995-10-18 イチビキ株式会社 Phytase and its production method
US5830732A (en) 1994-07-05 1998-11-03 Mitsui Toatsu Chemicals, Inc. Phytase
JP3570784B2 (en) * 1994-07-05 2004-09-29 三井化学株式会社 New phytase
US6720014B1 (en) 1997-08-13 2004-04-13 Diversa Corporation Phytase-containing foodstuffs and methods of making and using them
BR9909010A (en) 1998-03-23 2000-11-28 Novo Nordisk As Phytase variant, phytase polypeptide, DNA construction, recombinant expression vector, host cell, processes for preparing a variant of phytase, feed or food, for preparing a feed or food composition, and for reducing phytate levels in animal manure , use of the phytase variant or composition, and transgenic plant or plant part
KR100790918B1 (en) 1999-11-18 2008-01-03 코넬 리서치 파운데이션 인코포레이티드 Site-directed mutagenesis of escherichia coli phytase
BR0311287A (en) 2002-05-30 2005-03-29 Basf Ag Polypeptide, polynucleotide, vector, host cell, process for producing a polypeptide, and, composition
JP2005137293A (en) * 2003-11-07 2005-06-02 Mitsui Chemicals Inc Method for producing extraneous phytase in plant through combination of codon modification with extracellular secretion
JP2006136314A (en) * 2004-10-12 2006-06-01 Mitsui Chemicals Inc New modified phytase
KR100663112B1 (en) * 2004-10-29 2007-01-02 주식회사 기성기전 Electromagnetic switch
FR2888250B1 (en) 2005-07-08 2007-08-17 Adisseo France Sas Soc Par Act PHYTASE OF DEBARYOMYCES CASTELLII
ES2387203T3 (en) 2006-04-04 2012-09-17 Novozymes A/S Phytase variants

Patent Citations (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0238023A2 (en) 1986-03-17 1987-09-23 Novo Nordisk A/S Process for the production of protein products in Aspergillus oryzae and a promoter for use in Aspergillus
WO1991014772A1 (en) 1990-03-23 1991-10-03 Gist-Brocades N.V. Production of enzymes in seeds and their use
EP0561907A1 (en) 1990-12-05 1993-09-29 Novo Nordisk A/S Proteins with changed epitopes and methods for the production thereof
WO1994001459A1 (en) 1992-07-10 1994-01-20 Novo Nordisk A/S A fungicidally active compound
WO1995022625A1 (en) 1994-02-17 1995-08-24 Affymax Technologies N.V. Dna mutagenesis by random fragmentation and reassembly
US6111168A (en) 1994-03-17 2000-08-29 The United States Of America As Represented By The Secretary Of Agriculture Low phytic acid mutants and selection thereof
US5689054A (en) 1994-03-17 1997-11-18 The United States Of America As Represented By The Secretary Of Agriculture Low phytic acid mutants and selection thereof
WO1996000343A1 (en) 1994-06-24 1996-01-04 Audi Ag Method of controlling the electric heating of a catalytic converter
WO1996000787A1 (en) 1994-06-30 1996-01-11 Novo Nordisk Biotech, Inc. Non-toxic, non-toxigenic, non-pathogenic fusarium expression system and promoters and terminators for use therein
WO1996016177A1 (en) 1994-11-24 1996-05-30 Novo Nordisk A/S A process for producing polypeptides with reduced allergenicity
WO1996017929A1 (en) 1994-12-07 1996-06-13 Novo Nordisk A/S Polypeptide with reduced allergenicity
WO1998030682A1 (en) 1997-01-10 1998-07-16 Novo Nordisk A/S Enzyme coupled with polymeric molecules for skin care
WO1998035026A1 (en) 1997-02-06 1998-08-13 Novo Nordisk A/S Polypeptide-polymer conjugates having added and/or removed attachment groups
WO1999000489A1 (en) 1997-06-25 1999-01-07 Novo Nordisk A/S A modified polypeptide
EP0897985A2 (en) 1997-07-24 1999-02-24 F.Hoffmann-La Roche Ag Consensus phytases
WO1999043835A2 (en) 1998-02-26 1999-09-02 Novo Nordisk Biotech, Inc. Methods for producing a polypeptide in a bacillus cell
WO2000020569A1 (en) 1998-10-02 2000-04-13 Novozymes A/S Solid phytase compositions
WO2000022103A1 (en) 1998-10-13 2000-04-20 Novozymes A/S A modified polypeptide with reduced immune response
WO2000026354A1 (en) 1998-10-30 2000-05-11 Novozymes A/S Glycosylated proteins having reduced allergenicity
WO2000026230A1 (en) 1998-10-30 2000-05-11 Novozymes A/S Low allergenic protein variants
WO2000064247A1 (en) 1999-04-23 2000-11-02 University Of Guelph Transgenic animals expressing salivary proteins
WO2001058275A2 (en) 2000-02-08 2001-08-16 F Hoffmann-La Roche Ag Use of acid-stable subtilisin proteases in animal feed
WO2001083559A2 (en) 2000-04-28 2001-11-08 Novozymes A/S Production and use of protein variants having modified immunogenecity
WO2002090384A2 (en) 2001-05-04 2002-11-14 Novozymes A/S Antimicrobial polypeptide from aspergillus niger
WO2003044049A1 (en) 2001-11-20 2003-05-30 Novozymes A/S Antimicrobial polypeptides from pseudoplectania nigrella
WO2003048148A2 (en) 2001-12-03 2003-06-12 Novozymes A/S Statin-like compounds
WO2004015084A2 (en) * 2002-08-12 2004-02-19 Genencor International, Inc. Mutant e. coli appa phytase enzymes
WO2004085638A1 (en) 2003-03-25 2004-10-07 Republic Of National Fisheries Research And Development Institute Phytase produced from citrobacter braakii
WO2006038128A2 (en) 2004-10-04 2006-04-13 Danisco A/S Citrobacter freundii phytase and homologues
WO2006038062A1 (en) 2004-10-04 2006-04-13 Danisco A/S Microbial phytase as supplement to food or fodder
WO2006037327A2 (en) * 2004-10-04 2006-04-13 Novozymes A/S Polypeptides having phytase activity and polynucleotides encoding same
WO2006037328A1 (en) 2004-10-04 2006-04-13 Novozymes A/S Polypeptides having phytase activity and polynucleotides encoding same
WO2006063588A1 (en) * 2004-12-13 2006-06-22 Novozymes A/S Polypeptides having acid phosphatase activity and polynucleotides encoding same

Non-Patent Citations (53)

* Cited by examiner, † Cited by third party
Title
"Citrobacter freundii phytase (phyA) gene, complete cds", EMBL, 1 September 2004 (2004-09-01), XP002339845 *
"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
"handbook Enzyme Nomenclature from NC-IUBMB", 1992
"subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council,ninth revised edition", 1988, NATIONAL ACADEMY PRESS, pages: 2 - 6
BAIROCH A.: "The ENZYME database", NUCLEIC ACIDS RES, vol. 28, 2000, pages 304 - 305
BECKER, GUARENTE: "Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology", vol. 194, ACADEMIC PRESS, INC., pages: 182 - 187
CENTRAL VEEVOEDERBUREAU: "Veevoedertabel", 1997, ISBN: 9072839137
CHANG, COHEN, MOLECULAR GENERAL GENETICS, vol. 168, 1979, pages 111 - 115
CHEN ET AL., JOURNAL OF CHROMATOGRAPHY A, vol. 1018, 2003, pages 41 - 52
CHEN ET AL., PLANT AND CELL PHYSIOLOGY, vol. 39, 1998, pages 935 - 941
CHRISTOU, PLANT JOURNAL, vol. 2, 1992, pages 275 - 281
CONRAD ET AL., JOURNAL OF PLANT PHYSIOLOGY, vol. 152, 1998, pages 708 - 711
DIDERICHSEN ET AL., J. BACTERIOL., vol. 172, 1990, pages 4315 - 4321
DUBNAU, DAVIDOFF-ABELSON, JOURNAL OF MOLECULAR BIOLOGY, vol. 56, 1971, pages 209 - 221
EDWARDS, CORUZZI, ANN. REV. GENET., vol. 24, 1990, pages 275 - 303
FRANCK ET AL., CELL, vol. 21, 1980, pages 285 - 294
GASSER ET AL., SCIENCE, vol. 244, 1990, pages 1293
HAEFNER S ET AL: "Biotechnological production and applications of phytases", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER VERLAG, BERLIN, DE, vol. 68, no. 5, 23 July 2005 (2005-07-23), pages 588 - 597, XP002398064, ISSN: 0175-7598 *
HAWKSWORTH ET AL.: "Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,", 1995, CAB INTERNATIONAL, UNIVERSITY PRESS
HIGGINS AND HAMES: "handbook Protein Expression: A Practical Approach", 1999, OXFORD UNIVERSITY PRESS
HINNEN ET AL., PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, vol. 75, 1978, pages 1920
HOOYKAS, SCHILPEROORT, PLANT MOLECULAR BIOLOGY, vol. 19, 1992, pages 15 - 38
HORVATH ET AL., PNAS, vol. 97, no. 4, 15 February 2000 (2000-02-15), pages 1914 - 1919
ITO ET AL., JOURNAL OF BACTERIOLOGY, vol. 153, 1983, pages 163
ITO ET AL., PLANT MOL. BIOL., vol. 24, 1994, pages 863 - 878
J.-C. JANSON AND LARS RYDEN,: "Protein Purification", 1989, VCH PUBLISHERS
KAGAYA ET AL., MOLECULAR AND GENERAL GENETICS, vol. 248, 1995, pages 668 - 674
KIM ET AL.: "Molecular cloning of the phytase gene from Citrobacter braakii and its expression in Saccharomyces cerevisiae", BIOTECHNOLOGY LETTERS., vol. 28, no. 1, 2006
KIM YOUNG-OK ET AL: "Molecular cloning of the phytase gene from Citrobacter braakii and its expression in Saccharomyces cerevisiae.", BIOTECHNOLOGY LETTERS. JAN 2006, vol. 28, no. 1, January 2006 (2006-01-01), pages 33 - 38, XP002392154, ISSN: 0141-5492 *
KOEHLER, THORNE, JOURNAL OF BACTERIOLOGY, vol. 169, 1987, pages 5771 - 5278
KYOZUKA ET AL., PLANT PHYSIOLOGY, vol. 102, 1993, pages 991 - 1000
LIM ET AL., NAT. STRUCT. BIOL., vol. 2, 2000, pages 108 - 113
MALARDIER ET AL., GENE, vol. 78, 1989, pages 147 - 156
MEADE, H.M. ET AL.: "Gene expression systems: Using nature for the art of expression", 1999, ACADEMIC PRESS, article "Expression of recombinant proteins in the milk of transgenic animals"
MITRA, HIGGINS, PLANT MOLECULAR BIOLOGY, vol. 26, 1994, pages 85 - 93
NEEDLEMAN, S. B., WUNSCH, C. D., J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
OMIRULLEH ET AL., PLANT MOLECULAR BIOLOGY, vol. 21, 1993, pages 415 - 428
PLANT MO. BIOL., vol. 18, pages 675 - 689
POTRYKUS, BIO/TECHNOLOGY, vol. 8, 1990, pages 535
RODRIGUEZ ET AL., ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, vol. 382, no. 1, 2000, pages 105 - 112
SHIGEKAWA, DOWER, BIOTECHNIQUES, vol. 6, 1988, pages 742 - 751
SHIMAMOTO ET AL., NATURE, vol. 338, 1989, pages 274
SHIMAMOTO, CURRENT OPINION BIOTECHNOLOGY, vol. 5, 1994, pages 158 - 162
SKINNER, F.A., PASSMORE, S.M., DAVENPORT, R.R.: "Soc. App. Bacteriol", 1980, article "Biology and Activities of Yeast"
SKOGLUND ET AL., J. AGRIC. FOOD CHEM., vol. 45, 1997, pages 431 - 436
TAGUE ET AL., PLANT PHYSIOLOGY, vol. 86, 1988, pages 506
TAKAMI ET AL., BIOSCI. BIOTECHNOL. BIOCHEM., vol. 56, 1992, pages 1455
VASIL ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 667 - 674
WU ET AL., PLANT AND CELL PHYSIOLOGY, vol. 39, 1998, pages 885 - 889
XU ET AL., PLANT MOLECULAR BIOLOGY, vol. 22, 1993, pages 573 - 588
YELTON ET AL., PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, vol. 81, 1984, pages 1470 - 1474
YOUNG, SPIZIZIN, JOURNAL OF BACTERIOLOGY, vol. 81, 1961, pages 823 - 829
ZHANG W, MCELROY D., WU R: "Analysis of rice Act1 5' region activity in transgenic rice plants", PLANT CELL, vol. 3, 1991, pages 1155 - 1165

Cited By (87)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8455232B2 (en) 1998-06-25 2013-06-04 Cornell Research Foundation, Inc. Overexpression of phytase genes in yeast systems
US8993300B2 (en) 1998-06-25 2015-03-31 Cornell Research Foundation, Inc. Overexpression of phytase genes in yeast systems
US9273295B2 (en) 2004-10-04 2016-03-01 Dupont Nutrition Biosciences Aps Mutant citrobacter freundii phytase polypeptide
US8540984B2 (en) 2006-08-03 2013-09-24 Cornell Research Foundation, Inc. Phytases with improved thermal stability
US8936924B2 (en) 2006-09-21 2015-01-20 Verenium Corporation Methods for using a thermostable phytase in ethanol production
US8877478B2 (en) 2006-09-21 2014-11-04 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
US10196617B2 (en) 2006-09-21 2019-02-05 Basf Enzymes Llc Phytases, nucleic acids encoding them and methods for making and using them
EP2933329A1 (en) 2008-09-26 2015-10-21 Novozymes A/S Hafnia phytase variants
WO2010034835A2 (en) 2008-09-26 2010-04-01 Novozymes A/S Hafnia phytase variants
EP2650364A1 (en) 2008-09-26 2013-10-16 Novozymes A/S Hafnia phytase variants
WO2010106170A1 (en) 2009-03-20 2010-09-23 Novozymes A/S A nutritional beverage and a method of making the same
EP2698374A1 (en) * 2009-05-21 2014-02-19 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
AU2013204082B2 (en) * 2009-05-21 2016-03-31 Basf Enzymes Llc Phytases, nucleic acids encoding them and methods for making and using them
AU2010249500B2 (en) * 2009-05-21 2016-03-24 Basf Enzymes Llc Phytases, nucleic acids encoding them and methods for making and using them
JP2016039823A (en) * 2009-05-21 2016-03-24 ビーエーエスエフ エンザイムズ エルエルシー Phytases, nucleic acids encoding phytases, and methods for making and using them
WO2010135588A2 (en) 2009-05-21 2010-11-25 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
EP2432797A4 (en) * 2009-05-21 2012-11-14 Verenium Corp Phytases, nucleic acids encoding them and methods for making and using them
EP2432797A2 (en) * 2009-05-21 2012-03-28 Verenium Corporation Phytases, nucleic acids encoding them and methods for making and using them
US9695403B2 (en) 2009-05-21 2017-07-04 Syngenta Participations Ag Phytases, nucleic acids encoding them and methods for making and using them
US9969992B2 (en) 2009-05-21 2018-05-15 Syngenta Participations Ag Phytases, nucleic acids encoding them and methods for making and using them
JP2016127823A (en) * 2009-05-21 2016-07-14 ビーエーエスエフ エンザイムズ エルエルシー Phytases, nucleic acids encoding them and methods for making and using them
US10428340B2 (en) 2009-05-21 2019-10-01 Syngenta Participations Ag Phytases, nucleic acids encoding them and methods for making and using them
CN102459303A (en) * 2009-05-21 2012-05-16 维莱尼姆公司 Phytases, nucleic acids encoding them and methods for making and using them
WO2011048046A2 (en) 2009-10-22 2011-04-28 Basf Se Synthetic phytase variants
RU2472855C2 (en) * 2009-12-15 2013-01-20 Федеральное государственное унитарное предприятие "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов" (ФГУП ГосНИИгенетика) MUTANT RECOMBINANT THERMALLY STABLE PHYTASE (VERSIONS), DNA FRAGMENT CODING SAID PHYTASE (VERSIONS) Pichia pastoris STRAIN - PRODUCER OF SAID PHYTASE (VERSIONS)
US20160289655A1 (en) * 2010-03-26 2016-10-06 Novozymes A/S Thermostable Phytase Variants
EP3101126A1 (en) * 2010-03-26 2016-12-07 Novozymes A/S Thermostable phytase variants
WO2011117396A3 (en) * 2010-03-26 2011-11-24 Novozymes A/S Thermostable phytase variants
WO2011117397A1 (en) * 2010-03-26 2011-09-29 Novozymes A/S Thermostable phytase variants
CN102906254A (en) * 2010-03-26 2013-01-30 诺维信公司 Thermostable phytase variants
AU2011231562B2 (en) * 2010-03-26 2015-10-22 Novozymes A/S Thermostable phytase variants
AU2011231572B2 (en) * 2010-03-26 2015-10-22 Novozymes A/S Thermostable phytase variants
US9173421B2 (en) 2010-03-26 2015-11-03 Novozymes A/S Thermostable phytase variants
US20130017185A1 (en) * 2010-03-26 2013-01-17 Novozymes A/S Thermostable Phytase Variants
US9611469B2 (en) 2010-03-26 2017-04-04 Novozymes A/S Thermostable phytase variants
CN103429093A (en) * 2011-02-18 2013-12-04 杜邦营养生物科学有限公司 Feed additive composition
US11172693B2 (en) 2011-02-18 2021-11-16 Dupont Nutrition Biosciences Aps Feed additive composition
US10695384B2 (en) 2011-02-18 2020-06-30 Dupont Nutrition Biosciences Aps Feed additive composition
WO2012110776A2 (en) 2011-02-18 2012-08-23 Dupont Nutrition Biosciences Aps Feed additive composition
US20130330308A1 (en) * 2011-02-18 2013-12-12 Dupont Nutrition Biosciences Aps Feed additive composition
WO2012110776A3 (en) * 2011-02-18 2013-04-18 Dupont Nutrition Biosciences Aps Feed additive composition
WO2012110778A2 (en) 2011-02-18 2012-08-23 Dupont Nutrition Biosciences Aps Feed additive composition
WO2012110777A2 (en) 2011-02-18 2012-08-23 Dupont Nutrition Biosciences Aps Feed additive composition
US8557555B2 (en) 2011-04-21 2013-10-15 Basf Se Synthetic phytase variants
WO2012143861A1 (en) 2011-04-21 2012-10-26 Basf Se Synthetic phytase variants
WO2013005003A1 (en) 2011-07-07 2013-01-10 Dupont Nutrition Biosciences Aps Assay
EP3486656A1 (en) 2011-07-07 2019-05-22 DuPont Nutrition Biosciences ApS Assay
US10149891B2 (en) 2012-08-03 2018-12-11 Dupont Nutrition Biosciences Aps Feed additive composition
US9179693B2 (en) 2012-08-03 2015-11-10 Dupont Nutrition Biosciences Aps Feed additive composition
US11235035B2 (en) 2012-08-03 2022-02-01 Dupont Nutrition Biosciences Aps Feed additive composition
CN104684408B (en) * 2012-08-03 2018-08-31 杜邦营养生物科学有限公司 Feed addictive composition
EP3839055A2 (en) 2013-03-08 2021-06-23 Biogrammatics, Inc. Yeast promoters for protein expression
US10619164B2 (en) 2013-03-08 2020-04-14 Keck Graduate Institute Of Applied Life Sciences Yeast promoters from Pichia pastoris
EP3527667A2 (en) 2013-03-08 2019-08-21 Biogrammatics, Inc. Yeast promoters for protein expression
US10676750B2 (en) 2013-03-08 2020-06-09 Biogrammatics, Inc. Yeast promoters for protein expression
EP3594351A1 (en) 2013-03-08 2020-01-15 Keck Graduate Institute of Applied Life Sciences Yeast promoters from pichia pastoris
EP3712274A1 (en) 2013-09-11 2020-09-23 Novozymes A/S Processes for producing fermentation products
WO2015035914A1 (en) 2013-09-11 2015-03-19 Novozymes A/S Processes for producing fermentation products
US10980249B2 (en) 2014-06-27 2021-04-20 Dsm Ip Assets B.V. Method for improving the nutritional value of animal feed
WO2015197871A1 (en) 2014-06-27 2015-12-30 Dsm Ip Assets B.V. A method for improving the nutritional value of animal feed
WO2017112540A1 (en) 2015-12-22 2017-06-29 Novozymes A/S Processes for producing fermentation products
TWI616532B (en) * 2016-02-18 2018-03-01 基酵生物科技股份有限公司 Phytase having improved thermostability
US10011822B2 (en) 2016-02-18 2018-07-03 Dongguan APAC Biotechnology Co., Ltd. Phytase having improved thermostability
TWI607088B (en) * 2016-02-18 2017-12-01 基酵生物科技股份有限公司 Phytase having improved thermostability
TWI615471B (en) * 2016-02-18 2018-02-21 基酵生物科技股份有限公司 Phytase having improved thermostability
WO2019055455A1 (en) 2017-09-15 2019-03-21 Novozymes A/S Enzyme blends and processes for improving the nutritional quality of animal feed
WO2019083831A1 (en) 2017-10-23 2019-05-02 Novozymes A/S Processes for reducing lactic acid in a biofuel fermentation system
WO2019231944A2 (en) 2018-05-31 2019-12-05 Novozymes A/S Processes for enhancing yeast growth and productivity
RU2706086C1 (en) * 2018-10-29 2019-11-13 Акционерное Общество "Биоамид" Mutant recombinant thermostable phytase
WO2020160126A1 (en) 2019-01-31 2020-08-06 Novozymes A/S Polypeptides having xylanase activity and use thereof for improving the nutritional quality of animal feed
WO2021007379A1 (en) 2019-07-09 2021-01-14 Dupont Nutrition Biosciences Aps Fat coated particulate enzyme compositions
WO2021026201A1 (en) 2019-08-05 2021-02-11 Novozymes A/S Enzyme blends and processes for producing a high protein feed ingredient from a whole stillage byproduct
WO2021034660A1 (en) 2019-08-16 2021-02-25 Dupont Nutrition Biosciences Aps Compositions for gut health comprising combinations of lactobacillus strains
WO2021046073A1 (en) 2019-09-05 2021-03-11 Dupont Nutrition Biosciences Aps Feed composition
WO2021080864A2 (en) 2019-10-21 2021-04-29 Dupont Nutrition Biosciences Aps Compositions for gut health
WO2021126966A1 (en) 2019-12-16 2021-06-24 Novozymes A/S Processes for producing fermentation products
WO2021158927A1 (en) 2020-02-07 2021-08-12 Dupont Nutrition Biosciences Aps Feed compositions for animal health
WO2022034211A1 (en) 2020-08-13 2022-02-17 Novozymes A/S Phytase variants and polynucleotides encoding same
US12037613B2 (en) 2020-08-13 2024-07-16 Novozymes A/S Phytase variants and polynucleotides encoding same
WO2022081947A1 (en) 2020-10-16 2022-04-21 Dupont Nutrition Biosciences Feed compositions for animal health
WO2022169933A2 (en) 2021-02-03 2022-08-11 Dupont Nutrition Biosciences Aps Compositions for gut health
WO2023049488A1 (en) 2021-09-27 2023-03-30 Dupont Nutrition Biosciences Aps Feed additive compositions and methods for using the same
WO2024137248A1 (en) 2022-12-19 2024-06-27 Novozymes A/S Compositions comprising arabinofuranosidases and a xylanase, and use thereof for increasing hemicellulosic fiber solubilization
WO2024137252A1 (en) 2022-12-19 2024-06-27 Novozymes A/S Process for reducing syrup viscosity in the backend of a process for producing a fermentation product
WO2024137250A1 (en) 2022-12-19 2024-06-27 Novozymes A/S Carbohydrate esterase family 3 (ce3) polypeptides having acetyl xylan esterase activity and polynucleotides encoding same
WO2024137704A2 (en) 2022-12-19 2024-06-27 Novozymes A/S Processes for producing fermentation products using fiber-degrading enzymes with engineered yeast
WO2024137246A1 (en) 2022-12-19 2024-06-27 Novozymes A/S Carbohydrate esterase family 1 (ce1) polypeptides having ferulic acid esterase and/or acetyl xylan esterase activity and polynucleotides encoding same

Also Published As

Publication number Publication date
BRPI0709732B1 (en) 2017-06-06
US20130224332A1 (en) 2013-08-29
MX2008012632A (en) 2008-10-13
US20110258714A1 (en) 2011-10-20
JP5221516B2 (en) 2013-06-26
US8877471B2 (en) 2014-11-04
EP2001999A1 (en) 2008-12-17
ES2387203T3 (en) 2012-09-17
US20150024084A1 (en) 2015-01-22
AU2007234177B2 (en) 2012-11-08
JP2009532043A (en) 2009-09-10
EP2365064B1 (en) 2014-12-24
EP2365064A1 (en) 2011-09-14
US9451783B2 (en) 2016-09-27
CA2647476C (en) 2016-08-16
AU2007234177A1 (en) 2007-10-11
ES2531434T3 (en) 2015-03-16
DK2365064T3 (en) 2015-03-30
US10041052B2 (en) 2018-08-07
US8460656B2 (en) 2013-06-11
EP2001999B1 (en) 2012-05-16
BRPI0709732A2 (en) 2011-07-26
CA2647476A1 (en) 2007-10-11
US20160362666A1 (en) 2016-12-15
PL2365064T3 (en) 2015-05-29

Similar Documents

Publication Publication Date Title
US10041052B2 (en) Phytase variants
US8507240B2 (en) Hafnia phytase variants
US9611469B2 (en) Thermostable phytase variants
AU2011231572B2 (en) Thermostable phytase variants

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200780020771.5

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07711278

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2007234177

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2007711278

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2647476

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: MX/a/2008/012632

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 2009503408

Country of ref document: JP

Ref document number: 5326/CHENP/2008

Country of ref document: IN

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2007234177

Country of ref document: AU

Date of ref document: 20070319

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 12294526

Country of ref document: US

ENP Entry into the national phase

Ref document number: PI0709732

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20081003