Phytase polypeptides

FIELD OF INVENTION

[0002] The present invention relates to isolated polypeptides having phytase activity, the corresponding cloned DNA sequences, a method of producing such polypeptides, and the use thereof for a number of industrial applications. In particular, the invention relates to phytases derived from the phyllum Basidiomycota, phytases of certain consensus sequences and fungal 6-phytases.

BACKGROUND OF THE INVENTION

[0003] Phytic acid or myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate (or for short myo-inositol hexakisphosphate) is the primary source of inositol and the primary storage form of phosphate in plant seeds. In fact, it is naturally formed during the maturation of seeds and cereal grains. In the seeds of legumes it accounts for about 70% of the phosphate content and is structurally integrated with the protein bodies as phytin, a mixed potassium, magnesium and calcium salt of inositol. Seeds, cereal grains and legumes are important components of food and feed preparations, in particular of animal feed preparations. But also in human food cereals and legumes are becoming increasingly important.

[0004] The phosphate moieties of phytic acid chelates divalent and trivalent cations such as metal ions, i.a. the nutritionally essential ions of calcium, iron, zinc and magnesium as well as the trace minerals mangane, copper and molybdenum.

[0005] Besides, the phytic acid also to a certain extent binds proteins by electrostatic interaction. At a pH below the isoelectric point, pI, of the protein, the positively charged protein binds directly with phytate. At a pH above pI, the negatively charged protein binds via metal ions to phytate.

[0006] Phytic acid and its salts, phytates, are often not metabolized, since they are not absorbable from the gastro intestinal system, i.e. neither the phosphorous thereof, nor the chelated metal ions, nor the bound proteins are nutritionally available.

[0007] Accordingly, since phosphorus is an essential element for the growth of all organisms, food and feed preparations need to be supplemented with inorganic phosphate. Quite often also the nutritionally essential ions such as iron and calcium, must be supplemented. And, besides, the nutritional value of a given diet decreases, because of the binding of proteins by phytic acid. Accordingly, phytic acid is often termed an anti-nutritional factor.

[0008] Still further, since phytic acid is not metabolized, the phytate phosphorus passes through the gastrointestinal tract of such animals and is excreted with the manure, resulting in an undesirable phosphate pollution of the environment resulting e.g. in eutrophication of the water environment and extensive growth of algae.

[0009] Phytic acid or phytates, said terms being, unless otherwise indicated, in the present context used synonymously or at random, are degradable by phytases.

[0010] In most of those plant seeds which contain phytic acid, endogenous phytase enzymes are also found. These enzymes are formed during the germination of the seed and serve the purpose of liberating phosphate and, as the final product, free myo-inositol for use during the plant growth.

[0011] When ingested, the food or feed component phytates are in theory hydrolyzable by the endogenous plant phytases of the seed in question, by phytases stemming from the microbial flora in the gut and by intestinal mucosal phytases. In practice, however the hydrolyzing capability of the endogenous plant phytases and the intestinal mucosal phytases, if existing, is far from sufficient for increasing significantly the bioavailibility of the bound or constituent components of phytates. However, when the process of preparing the food or feed involve germination, fermentation or soaking, the endogenous phytase might contribute to a greater extent to the degradation of phytate.

[0012] In ruminant or polygastric animals such as horses and cows the gastro intestinal system hosts microorganisms capable of degrading phytic acid. However, this is not so in monogastric animals such as human beings, poultry and swine. Therefore, the problems indicated above are primarily of importance as regards such monogastric animals.

[0013] The production of phytases by plants as well as by microorganisms has been reported. Amongst the microorganisms, phytase producing bacteria as well as phytase producing fungi are known.

[0014] From the plant kingdom, e.g. a wheat-bran phytase is known (Thomlinson et al, Biochemistry, 1 (1962), 166-171). An alkaline phytase from lilly pollen has been described by Barrientos et al, Plant. Physiol., 106 (1994), 1489-1495.

[0015] Amongst the bacteria, phytases have been described which are derived from Bacillus subtilis (Paver and Jagannathan, 1982, Journal of Bacteriology 151:1102-1108) and Pseudomonas (Cosgrove, 1970, Australian Journal of Biological Sciences 23:1207-1220). Still further, a phytase from E. coli has been purified and characterized by Greiner et al, Arch. Biochem. Biophys., 303, 107-113, 1993). However, this enzyme is probably an acid phosphatase.

[0016] Phytase producing yeasts are also described, such as Saccharomyces cerevisiae (Nayini et al, 1984, Lebensmittel Wissenschaft und Technologie 17:24-26. However, this enzyme is probably a myo-inositol monophosphatase (Wodzinski et al, Adv. Appl. Microbiol., 42, 263-303). AU-A-24840/95 describes the cloning and expression of a phytase of the yeast Schwanniomyces occidentalis.

[0017] There are several descriptions of phytase producing filamentous fungi, however only belonging to the fungal phyllum of Ascomycota (ascomycetes). In particular, there are several references to phytase producing ascomycetes of the Aspergillus genus such as Aspergillus terreus (Yamada et al., 1986, Agric. Biol. Chem. 322:1275-1282). Also, the cloning and expression of the phytase gene from Aspergillus niger var. awamori has been described (Piddington et al., 1993, Gene 133:55-62). EP 0 420 358 describes the cloning and expression of a phytase of Aspergillus ficuum (niger). EP 0 684 313 describes the cloning and expression of phytases of the ascomycetes Myceliophthora thermophila and Aspergillus terreus.

[0018] Nomenclature and Position Specificity of Phytases

[0019] In the present context a phytase is 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 following, for short, the above compounds are sometimes referred to as IP6, I, IP1, IP2, IP3, IP4, IP5 and P, respectively. This means that by action of a phytase, IP6 is degraded into P+one or more of the components IP5, IP4, IP3, IP2, IP1 and I. Alternatively, myo-inositol carrying in total n phosphate groups attached to positions p, q, r, . . . is denoted Ins(p,q,r, . . . ) Pn. For convenience Ins(1,2,3,4,5,6)P6 (phytic acid) is abbreviated PA.

[0020] According to the Enzyme nomenclature database ExPASy (a repository of information relative to the nomenclature of enzymes primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) describing each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided), two different types of phytases are known: A so-called 3-phytase (myo-inositol hexaphosphate 3-phosphohydrolase, EC 3.1.3.8) and a so-called 6-phytase (myo-inositol hexaphosphate 6-phosphohydrolase, EC 3.1.3.26). The 3-phytase hydrolyses first the ester bond at the 3-position, whereas the 6-phytase hydrolyzes first the ester bond at the 6-position.

[0021] Inositolphosphate Nomenclature

[0022] Considering the primary hydrolysis products of a phytase acting on phytic acid, some of the resulting esters are diastereomers and some are enantiomers. Generally, it is easier to discriminate between diastereomers, since they have different physical properties, whereas it is much more difficult to discriminate between enantiomers which are mirror images of each other.

[0023] Thus, Ins(1,2,4,5,6)P5 (3-phosphate removed) and Ins(1,2,3,4,5)P5 (6-phosphate removed) are diastereomers and easy to discriminate, whereas Ins (1,2,4,5,6) P5 (3-phosphate removed) and Ins(2,3,4,5,6)P5 (1-phosphate removed) are enantiomers. The same holds true for the pair Ins(1,2,3,4,5)P5 (6-phosphate removed) and Ins(1,2,3,5,6)P5 (4-phosphate removed). Accordingly, of the 6 penta-phosphate esters resulting from the first step of the phytase catalyzed hydrolysis of phytic acid, you can only discriminate easily between those esters in which the 2-, 3-, 5- and 6-phosphate has been removed, i.e. you have four diastereomers only, each of the remaining two esters being an enantiomer of one each of these compounds (4- and 6- are enantiomers, as are 1- and 3-).

[0024] Use of Lowest-locant Rule

[0025] It should be noted here, that when using the notations Ins(2,3,4,5,6)P5 and Ins(1,2,3,5,6)P5, a relaxation of the previous recommendations on the numbering of the atoms of myo-inositol has been applied. This relaxation of the lowest-locant rule is recommended by the Nomenclature Committee of the International Union of Biochemistry (Biochem. J. (1989) 258, 1-2) whenever authors wish to bring out structural relationships.

[0026] In this lowest-locant rule, the L- and D-nomenclature is recommended: Inositolphosphate, phosphate esters of myo-inositol, are generally designated 1D- (or 1L-) -Ins(r,s,t,u,w,x)Pn, n indicating the numer of phosphate groups and the locants r,s,t,u,w and x, their positions. The positions are numbered according to the Nomenclature Committee of the International Union of Biochemistry (NC-IUB) cited above (and the references herein), and 1D or 1L is used so as to make a substituent have the lowest possible locant or number (“lowest-locant rule”). Accordingly, 1L-myo-inositol-1-phosphate (1L-Ins(1)P, an intermediary product in the biosynthesis of inositol) and 1D-myo-inositol-1-phosphate (1D-Ins(1)P, a component of phospholipids), are numbered as it is apparent from FIG. 38.

[0027] Phytase Specificity

[0028] As said above, phytases are divided according to their specificity in the initial hydrolysis step, viz. according to which phosphate-ester group is hydrolyzed first.

[0029] As regards the specificity of known phytases, plant phytases are generally said to be 6-phytases. However the lilly pollen phytase is said to be a 5-phytase. The microorganism derived phytases are mainly said to be 3-phytases. E.g. the ExPASy database mentioned above refers for 3-phytases to four phytases of Aspergillus awamori (strain ALK0243) and Aspergillus niger (strain NRRL 3135) (Gene 133:55-62 (1993) and Gene 127:87-94 (1993)).

[0030] Using now the D-/L-notation (in which the D- and L-configuration refer to the 1-position), the wheat-bran phytase hydrolyzes first the phosphate ester group in the L-6 position, whereas the 3-phytases hydrolyzes first the phosphate ester group in position D-3.

[0031] The specificity can be examined in several ways, e.g by HPLC or by NMR spectroscopy. These methods, however, do not immediately allow the discrimination between hydrolysis of e.g. the phosphate-ester groups in positions D-6 and L-6, since the products of the hydrolysis, D-Ins(1,2,3,4,5)P5 and L-Ins(1,2,3,4,5)P5, are enantiomers (mirror images), and therefore have identical NMR spectres.

[0032] In other words, in the present context a 6-phytase means either of a L-6- or a D-6-phytase or both, viz. a phytase being a L-6-phytase, a D-6-phytase or a ((D-6-)+(L-6-))-phytase (having both activities). The latter is sometimes also designated D/L-6-phytase.

SUMMARY OF THE INVENTION

[0033] It is an object of the present invention to provide alternative phytases, in particular with superior properties such as increased heat stability or faster release of phosphate from phytate, and which can be produced in commercially useful quantities.

[0034] Is The present inventors have surprisingly found a whole sub-family of fungal phytases of interesting properties. This sub-family of phytases is characterized by having a high degree of conserved regions or partial sequences in common (consensus sequences). Representatives of this sub-family have turned up to be advantageous as compared to known phytases as regards various enzyme properties, such as e.g. position specificity and specific activity.

[0035] It is presently contemplated that the phytase consensus sequences of the present invention are common to all basidiomycete phytases.

[0036] In the present context a basidiomycete means a microorganism of the phyllum Basidiomycota. This phyllum of Basidiomycota is comprised in the fungal kingdom together with e.g. the phyllum Ascomycota (“ascomycetes”). Reference can be had to Jülich, 1981, Higher Taxa of Basidiomycetes; Ainsworth & Bisby, 1995, Dictionary of the Fungi; Hansen & Knudsen (Eds.), Nordic Macromycetes, vol. 2 (1992) and 3 (1997). Alternatively, a fungal taxonomy data base (NIH Data Base (Entrez)) is available via the internet on World Wide Web at the following address: http://www3.ncbi.nlm.nih.gov/Taxonomy/tax.html.

[0037] A method of screening for such phytases using PCR is also given, as are general procedures for isolating and purifying these phytase enzyme using recombinant DNA technology.

[0038] In a first aspect, the invention relates to an isolated polypeptide having phytase activity and being derived from the phyllum Basidiomycota.

[0039] In a second aspect, the invention relates to an isolated polypeptide having phytase activity and comprising at least one of several consensus sequences.

[0040] In a third aspect, the invention relates to an isolated polypeptide having phytase activity and being encoded by a DNA sequence which hybridizes under medium to high stringency with the product of a PCR reaction using a suitable set of primers derived from alignments disclosed herein and a target sequence, e.g. a DNA library.

[0041] In a fourth aspect, the invention relates to an isolated polypeptide having 6-phytase activity and being derived from a fungus.

[0042] In a fifth aspect, the invention relates to isolated polypeptides having phytase activity and being homologous to five specific sequences.

[0043] In further aspects, the invention provides cloned DNA sequences encoding the above polypeptides, as well as vectors and host cells comprising these cloned DNA sequences.

[0044] Within the scope of the invention, in a still further aspect, is the use of the phytase of the invention for liberating inorganic phosphate from phytic acid, as well as some more specific uses, and compositions, in particular food and feed preparations and additives comprising the phytase of the invention.

[0045] Generally, terms and expressions as used herein are to be interpreted as is usual in the art. In cases of doubt, however, the definitions of the present description might be useful.

[0046] General Definitions

[0047] By the expression “an isolated polypeptide/enzyme having/exhibiting phytase activity” or “an isolated phytase” is meant any peptide or protein having phytase activity (vide below) and which is essentially free of other non-phytase polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE. Sometimes such polypeptide is alternatively referred to as a “purified” phytase.

[0048] The definition of “an isolated polypeptide” also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding another polypeptide to a nucleic acid sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, and include, ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.

[0049] The expression “polypeptide or enzyme exhibiting phytase activity” or “phytase” is intended to cover any enzyme capable of effecting the liberation of inorganic phosphate or phosphorous from various myo-inositol phosphates. Examples of such myo-inositol phosphates (phytase substrates) are phytic acid and any salt thereof, e.g. sodium phytate or potassium phytate or mixed salts. Also any stereoisomer of the mono-, di-, tri-, tetra- or penta-phosphates of myo-inositol might serve as a phytase substrate.

[0050] In accordance with the above definition, the phytase activity can be determined using any assay in which one of these substrates is used. In the present context (unless otherwise specified) the phytase activity is determined in the unit of FYT, one FYT being the amount of enzyme that liberates 1 μmol inorganic ortho-phosphate per min. under the following conditions: pH 5.5; temperature 37° C.; substrate: sodium phytate (C6H6O24P6Na12)in a concentration of 0.0050 mol/l. Suitable phytase assays are described in the experimental part. “Polypeptide homology” or “amino acid homology” is determined as the degree of identity between two sequences. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG version 8 program package (Program Manual for the Wisconsin Package, Version 8, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453. Using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

[0051] In the present context a “6-phytase”means a phytase which hydrolyzes first the 6-position in phytic acid or has a preference for these positions (plural is used since this term covers two positions). In particular, more than 50% of the hydrolysis product of the first step is Ins(1,2,3,4,5)P5 and/or Ins(1,2,3,5,6)P5. Preferably these two compounds comprise at least 60%, more preferably at least 70%, still more preferably at least 80%, especially at least 90% and mostly preferred more than 95% of the product of the initial hydrolysis step of PA.

[0052] The other specificity terms such as e.g. “3-phytase,” “(3+6)-phytase ” “6D-phytase” and “6L-phytase” are to be interpreted correspondingly, including the same preferred embodiments.

[0053] The terms “a phytase encoding part of a DNA sequence cloned into a plasmid present in a deposited E. coli strain” and “a phytase encoding part of the corresponding DNA sequence presented in the sequence listing” are presently believed to be identical, and accordingly they may be used interchangeably.

[0054] Primarily, the term “a phytase encoding part” used in connection with a DNA sequence means that region of the DNA sequence which is translated into a polypeptide sequence having phytase activity. Often this is the region between a first “ATG” start codon (“AUG” codon in MRNA) and a stop codon (“TAA”, “TAG” or “TGA”) first to follow.

[0055] However, the polypeptide translated as described above often comprises, in addition to a mature sequence exhibiting phytase activity, an N-terminal signal sequence and/or a pro-peptide sequence. Generally, the signal sequence guides the secretion of the polypeptide and the pro-peptide guides the folding of the polypeptide. For further information see Egnell, P. et al. Molecular Microbiol. 6(9):1115-19 (1992) or Stryer, L., “Biochemistry” W. H., Freeman and Company/New York, ISBN 0-7167-1920-7. Therefore, the term “phytase encoding part” is also intended to cover the DNA sequence corresponding to the mature part of the translated polypeptide or to each of such mature parts, if several exist.

[0056] Still further, any fragment of such sequence encoding a polypeptide fragment, which still retains some phytase activity, is to be included in this definition.

[0057] An isolated DNA molecule or, alternatively, a “cloned DNA sequence” “a DNA construct,” “a DNA segment” or “an isolated DNA sequence” refers to a DNA molecule or sequence which can be cloned in accordance with standard cloning procedures used in genetic engineering to relocate the DNA segment from its natural location to a different site where it will be replicated. The term refers generally 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 about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by agarose gel electrophoresis. 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.

[0058] The degree of identity or “homology” between two nucleic acid sequences may be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1996, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453). Using GAP with the following settings for DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

[0059] Suitable experimental conditions for determining whether a given DNA or RNA sequence “hybridizes” to a specified nucleotide or oligonucleotide probe involves presoaking of the filter containing the DNA fragments or RNA to examine for hybridization in 5×SSC (Sodium chloride/Sodium citrate), (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.) for 10 min, and prehybridization of the filter in a solution of 5×SSC, 5×Denhardt's solution (Sambrook et al. 1989), 0.5% SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a concentration of 10 ng/ml of a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13), 32P-dCTP-labeled (specific activity >1×109 cpm/pg) probe for 12 hours at approximately 45° C.

[0060] The filter is then washed twice for 30 minutes in 2×SSC, 0.5% SDS at at least 55° C. (low stringency), at at least 60° C. (medium stringency), at at least 65° C. (medium/high stringency), at at least 70° C. (high stringency), or at at least 75° C. (very high stringency).

[0061] Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using an x-ray film.

[0062] It has been found that it is possible to theoretically predict whether or not two given DNA sequences will hybridize under certain specified conditions.

[0063] Accordingly, as an alternative to the above described experimental method the determination whether or not an analogous DNA sequence will hybridize to the nucleotide probe described above, can be based on a theoretical calculation of the Tm (melting temperature) at which two heterologous DNA sequences with known sequences will hybridize under specified conditions (e.g. with respect to cation concentration and temperature).

[0064] In order to determine the melting temperature for heterologous DNA sequences (Tm(hetero)) it is necessary first to determine the melting temperature (Tm(homo)) for homologous DNA sequences.

[0065] The melting temperature (Tm(homo)) between two fully complementary DNA strands (homoduplex formation) may be determined by use of the following formula,

Tm(homo)=81.5° C.+16.6(log M)+0.41(% GC)-0.61 (% form)-500/L

[0066] (“Current protocols in Molecular Biology”. John Wiley and Sons, 1995), wherein

[0067] “M” is the molar cation concentration in wash buffer,

[0068] “% GC” is % Guanine (G) and Cytosine (C) of total number of bases in the DNA sequence,

[0069] “% form” is % formamide in the wash buffer, and

[0070] “L” is the length of the DNA sequence.

[0071] The Tm determined by the above formula is the Tm of a homoduplex formation (Tm(homo)) between two fully complementary DNA sequences. In order to adapt the Tm value to that of two heterologous DNA sequences, it is assumed that a 1% difference in nucleotide sequence between the two heterologous sequences equals a 1° C. decrease in Tm (“Current protocols in Molecular Biology”. John Wiley and Sons, 1995). Therefore, the Tm(hetero) for the heteroduplex formation is found by subtracting the homology % difference between the analogous sequence in question and the nucleotide probe described above from the Tm(homo). The DNA homology percentage to be subtracted is calculated as described herein (vide supra).

[0072] The term “vector” is intended to include such terms/objects as “nucleic acid constructs,” “DNA constructs, expression vectors” or “recombinant vectors.”

[0073] The nucleic acid construct comprises a nucleic acid sequence of the present invention operably linked to one or more control sequences capable of directing the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0074] “Nucleic acid construct” is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature.

[0075] The term nucleic acid construct may be synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention.

[0076] The term “coding sequence” as defined herein primarily comprises a sequence which is transcribed into MRNA and translated into a polypeptide of the present invention when placed under the control of the above mentioned control sequences. The boundaries of the coding sequence are generally determined by a translation start codon ATG at the 5′-terminus and a translation stop codon at the 3′-terminus. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0077] The term “control sequences” is defined herein to include all components which are necessary or advantageous for expression of the coding sequence of the nucleic acid sequence. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a 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 nucleic acid sequence encoding a polypeptide.

[0078] In the expression vector, the DNA sequence encoding the phytase should be operably connected to a suitable promoter and terminator sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins which are either homologous or heterologous to the host cell. The procedures used to ligate the DNA sequences coding for the phytase, the promoter and the terminator and to insert them into suitable vectors are well known to persons skilled in the art (cf. e.g. Sambrook et al., (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, N.Y.).

[0079] The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence.

[0080] More than one copy of a nucleic acid sequence encoding a polypeptide of the present invention may be inserted into the host cell to amplify expression of the nucleic acid sequence. Stable amplification of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome using methods well known in the art and selecting for transformants.

[0081] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

[0082] A “host cell” or “recombinant host cell” encompasses any progeny of a parent cell which is not identical to the parent cell due to mutations that occur during replication.

[0083] The cell is preferably transformed with a vector comprising a nucleic acid sequence of the invention followed by integration of the vector into the host chromosome.

[0084] “Transformation” means introducing a vector comprising a nucleic acid sequence of the present invention into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. Integration is generally considered to be an advantage as the nucleic acid sequence is more likely to be stably maintained in the cell. Integration of the vector into the host chromosome may occur by homologous or non-homologous recombination as described above.

[0085] The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Examples of a eukaryote cell is a mammalian cell, an insect cell, a plant cell or a fungal cell. Useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection.

[0086] In a preferred embodiment, 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).

[0087] 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.

[0088] The present invention also relates to a transgenic plant, plant part, such as a plant seed, or plant cell, which has been transformed with a DNA sequence encoding the phytase of the invention so as to express or produce this enzyme. Also compositions and uses of such plant or plant part are within the scope of the invention, especially its use as feed and food or additives therefore, along the lines of the present use and food/feed claims.

[0089] The transgenic plant can be dicotyledonous or monocotyledonous, for short a dicot or a monocot. Of primary interest are such plants which are potential food or feed components and which comprise phytic acid. A normal phytic acid level of feed components is 0.1-100 g/kg, or more usually 0.5-50 g/kg, most usually 0.5-20 g/kg. 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 and maize (corn).

[0090] Examples of dicot plants are legumes, such as lupins, pea, bean and soybean, and cruciferous (family Brassicaceae), such as cauliflower, oil seed rape and the closely related model organism Arabidopsis thaliana.

[0091] Such transgenic plant etc. is capable of degrading its own phytic acid, and accordingly the need for adding such enzymes to food or feed comprising such plants is alleviated. Preferably, the plant or plant part, e.g. the seeds, are ground or milled, and possibly also soaked before being added to the food or feed or before the use, e.g. intake, thereof, with a view to adapting the speed of the enzymatic degradation to the actual use.

[0092] If desired, the plant produced enzyme can also be recovered from the plant. In certain cases the recovery from the plant is to be preferred with a view to securing a heat stable formulation in a potential subsequent pelleting process.

[0093] Examples of plant parts are stem, callus, leaves, root, fruits, seeds, tubers etc. But also any plant tissue is included in this definition.

[0094] Any plant cell, whatever the tissue origin, is included in the definition of plant cells above.

[0095] Also included within the scope of the invention are the progeny of such plants, plant parts and plant cells.

[0096] The skilled man will known how to construct a DNA expression construct for insertion into the plant in question, paying regard i.a. to whether the enzyme should be excreted in a tissue specific way. Of relevance for this evaluation is the stability (pH-stability, degradability by endogenous proteases etc.) of the phytase in the expression compartments of the plant. He will also be able to select appropriate regulatory sequences such as promoter and terminator sequences, and signal or transit sequences if required (Tague et al, Plant, Phys., 86, 506, 1988).

[0097] The plant, plant part etc. can be transformed with this DNA construct using any known method. An example of such method is the transformation by a viral or bacterial vector such as bacterial species of the genus Agrobacterium genetically engineered to comprise the gene encoding the phytase of the invention. Also methods of directly introducing the phytase DNA into the plant cell or plant tissue are known in the art, e.g. micro injection and electroporation (Gasser et al, Science, 244, 1293; Potrykus, Bio/Techn. 8, 535, 1990; Shimamoto et al, Nature, 338, 274, 1989).

[0098] Following the transformation, the transformants are screened using any method known to the skilled man, following which they are regenerated into whole plants.

[0099] These plants etc. as well as their progeny then carry the phytase encoding DNA as a part of their genetic equipment.

[0100] In general, reference is had to WO 9114782A and WO 9114772A.

[0101]

Agrobacterium tumefaciens
mediated gene transfer is the method of choice for generating transgenic dicots (for review Hooykas & Schilperoort, 1992. Plant Mol. Biol. 19: 15-38). Due to host range limitations it is generally not possible to transform monocots with the help of A. tumefaciens. Here, other methods have to be employed. The method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992. Plant J. 2: 275-281; Shimamoto, 1994. Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992. Bio/Technology 10: 667-674).

[0102] Also other systems for the delivery of free DNA into these plants, including viral vectors (Joshi & Joshi, 1991. FEBS Lett. 281: 1-8), protoplast transformation via polyethylene glycol or electroporation (for review see Potyrkus, 1991. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 205-225), microinjection of DNA into mesophyll protoplasts (Crossway et al., 1986. Mol. Gen. Genet. 202: 79-85), and macroinjection of DNA into young floral tillers of cereal plants (de la Pena et al., 1987. Nature 325: 274-276) are preferred methods.

[0103] In general, the cDNA or gene encoding the phytase of the invention is placed in an expression cassette (e.g. Pietrzak et al., 1986. Nucleic Acids Res. 14: 5857-5868) consisting of a suitable promoter active in the target plant and a suitable terminator (termination of transcription). This cassette (of course including a suitable selection marker, see below) will be transformed into the plant as such in case of monocots via particle bombardment. In case of dicots the expression cassette is placed first into a suitable vector providing the T-DNA borders and a suitable selection marker which in turn are transformed into Agrobacterium tumefaciens. Dicots will be transformed via the Agrobacterium harbouring the expression cassette and selection marker flanked by T-DNA following standard protocols (e.g. Akama et al., 1992. Plant Cell Reports 12: 7-11). The transfer of T-DNA from Agrobacterium to the Plant cell has been recently reviewed (Zupan & Zambryski, 1995. Plant Physiol. 107: 1041-1047). Vectors for plant transformation via Agrobacterium are commercially available or can be obtained from many labs that construct such vectors (e.g. Deblaere et al., 1985. Nucleic Acids Res. 13: 4777-4788; for review see Klee et al., 1987. Annu. Rev. Plant Physiol. 38: 467-486).

[0104] Available plant promoters: Depending on the process under manipulation, organ- and/or cell-specific expression as well as appropriate developmental and environmental control may be required. For instance, it is desirable to express a phytase cDNA in maize endosperm etc. The most commonly used promoter has been the constitutive 35S-CaMV promoter Franck et al., 1980. Cell 21: 285-294). Expression will be more or less equal throughout the whole plant. This promoter has been used successfully to engineer herbicide- and pathogen-resistant plants (for review see Stitt & Sonnewald, 1995. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46: 341-368). Organ-specific promoters have been reported for storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990. Annu. Rev. Genet. 24: 275-303), and for metabolic sink tissues such as meristems (Ito et al., 1994. Plant Mol. Biol. 24: 863-878).

[0105] The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question. The expressed phytase may conveniently be secreted into the culture medium and may be recovered therefrom by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

[0106] Preferred host cells are a strain of Fusarium, Trichoderma or Aspergillus, in particular a strain of Fusarium graminearum, Fusarium venenatum, Fusarium cerealis, Fusarium sp. having the identifying characteristic of Fusarium ATCC 20334, as further described in PCT/US/95/07743, Trichoderma harzianum or Trichoderma reesei, Aspergillus niger or Aspergillus oryzae.

BRIEF DESCRIPTION OF THE DRAWINGS

[0107] In the detailed description of the invention below, reference is had to the drawings, of which

[0108]
FIG. 1 shows the nucleotide sequence of the phyA cDNA and the deduced primary struture of PHYA phytase from Peniophora lycii (the signal peptide is boxed and the restriction sites used for cDNA cloning are underlined);

[0109]
FIG. 2 shows the nucleotide sequence of a phytase from Agrocybe pediades, as in FIG. 1;

[0110]
FIG. 3 shows the nucleotide sequence of a first phytase, PHYA1, from Paxillus involtus, as in FIG. 1, except for the box referring to the SignalP V1.1 prediction of the signal peptide;

[0111]
FIG. 4 shows the nucleotide sequence of a second phytase, PHYA2, from Paxillus involtus, as in FIG. 3;

[0112]
FIG. 5 shows the nucleotide sequence of a phytase from Trametes pubescens, as in FIG. 3;

[0113]
FIG. 6 shows an alignment of the deduced amino acid sequences of the encoded phytases of FIGS. 1-5, identical resides in at least three of the sequences being indicated by a grey box;

[0114]
FIG. 7 shows an alignment of the five phytases of FIG. 6 together with five known phytases which all belong to the fungal phyllum of Ascomycota, identical residues in at least seven of the sequences being indicated by a grey box;

[0115]
FIG. 8 shows a pH-activity curve of the Peniophora phytase;

[0116]
FIG. 9 shows a pH-stability curve thereof;

[0117]
FIG. 10 shows a temperature-activity curve thereof;

[0118]
FIG. 11 shows a temperature-stability curve thereof;

[0119]
FIG. 12 shows a Differential Scanning Calorimetry (DSC) curve thereof;

[0120] FIGS. 13-14 show NMR spectra, stacked plots (up to 24 h), showing the product profiling of an Aspergillus niger phytase and the Peniophora phytase, respectively;

[0121] FIGS. 15-16 show NMR spectra as above, but stacked plots up to 4.5 h;

[0122] FIGS. 17-19 show NMR profiles observed after 20 minutes (at pH 5.5), 24 hours (at pH 5.5) and 20 minutes (at pH 3.5), respectively;

[0123]
FIG. 20 shows curves showing concentration versus time of Ins(1,2)P2 and Ins(2)P, respectively;

[0124] FIGS. 21-22 show curves showing the release of inorganic phosphate versus time from corn at pH 5.5 and pH 3.5, respectively;

[0125]
FIG. 23 shows a pH-activity curve of the Agrocybe phytase;

[0126]
FIG. 24 shows a pH-stability curve thereof;

[0127]
FIG. 25 shows a temperature-activity curve thereof;

[0128]
FIG. 26 shows a temperature-stability curve thereof;

[0129]
FIG. 27 shows a Differential Scanning Calorimetry (DSC) curve thereof;

[0130] FIGS. 28-29 show NMR spectra, stacked plots (up to 24 h), showing the product profiling of an Aspergillus niger phytase and the Agrocybe phytase, respectively;

[0131] FIGS. 30-31 show NMR spectra as above, but stacked plots up to 4.5 h;

[0132] FIGS. 32-33 show NMR profiles observed after 20 minutes and 24 hours, respectively;

[0133] FIGS. 34-35 show curves showing concentration versus time of Ins(1,2)P2 and Ins(2)P, respectively;

[0134] FIGS. 36-37 show curves showing the release of inorganic phosphate versus time from corn at pH 5.5 and pH 3.5, respectively; and

[0135]
FIG. 38 show the structure of 1D- and 1L-myo-inositol-1-phosphate (P=−OPO3−2).

DEPOSITIONS

[0136] Isolates of the strains of Peniophora lycii, Agrocybe pediades, Paxillus involtus, and Trametes pubescens from which phytases of the invention were obtained have been deposited according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG Baarn, The Netherlands, (CBS), as follows:

1Deposit date4th of December 1996Depositor's ref.NN006113CBS No.Peniophora lycii CBS No. 686.96Deposit date4th of December 1996Depositor's ref.NN009289CBS No.Agrocybe pediades CBS No. 900.96Deposit date28th of November 1997Depositor's ref.NN005693CBS No.Paxillus involtus CBS No. 100231Deposit date28th of November 1997Depositor's ref.NN009343CBS No.Trametes pubescens CBS No. 100232

[0137] Still further, the expression plasmids (shuttle vector) pYES 2.0 comprising the full length cDNA sequences encoding these phytases of the invention have been transformed into strains of Escherichia coli which were deposited according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH., Mascheroder Weg lb, D-38124 Braunschweig, Germany, (DSM), as follows, respectively (two phytases of Paxillus involtus):

2Deposit date2nd of December 1996Depositor's ref.NN 049282DSM No.Escherichia coli DSM No. 11312Deposit date2nd of December 1996Depositor's ref.NN 049283DSM No.Escherichia coli DSM No. 11313Deposit date12th of November 1997Depositor's ref.NN 049342DSM No.Escherichia coli DSM No. 11842Deposit date12th of November 1997Depositor's ref.NK 049343DSM No.Escherichia coli DSM No. 11843Deposit date12th of November 1997Depositor's ref.NN 049344DSM No.Escherichia coli DSM No. 11844

DETAILED DESCRIPTION OF THE INVENTION

[0138] The phytases of the invention derived from basidiomycetes are preferably derived from th e classes Gasteromycetes, Hymenomycetes, Urediniomnycetes, Ustilaginomycetes or from unclassified Basidiomycota, more preferably from the class Hymenomycetes.

[0139] The phytases derived from the class Hyeniomycetes are preferably derived from strains of the orders Agaricales, Aphyllophorales, Auriculariales, Boletales, Cantharellales, Ceratobasidiales, Dacrymycetales, Echinodontiaceae, Hericiales, Stereales, Thelephorales, Tremellales, Tulasnellales or from the class of mitosporic Hymenomycetes.

[0140] Other preferred orders are Coriolales, Hyphodermatales, Schizophyllales, Hymenochaetales and Phanerochaetales.

[0141] Below, preferred families of some of these orders are listed, and examples of preferred genera within each family are added in parentheses behind each family.

[0142] Preferred families of the order Aphyllophorales are Polyporaceae (e.g. genus Trametes, Bjerkandera, Irpex, Oxyporus, Trichaptum, Daedalea, Fomes), Coniophoraceae (e.g. genus Coniophora), Corticiaceae (e.g. genus Hyphoderma, Trechispora, Steccherinum, Merulius, Peniophora), Schizophyllaceae (e.g. genus Schizophyllum).

[0143] Preferred families of the order Agaricales are Bolbitiaceae (e.g. genus Agrocybe, Conocybe, Bolbitius), Coprinaceae (e.g. genus Coprinus, Panaeolus), Pluteaceae (e.g. genus Volvariella), Podaxaceae (e.g. genus Podaxis), Tricholomataceae (e.g. genus Marasmiellus, Strobilurus, Lyophyllum, Cystoderma, Merismodes), Strophariaceae (e.g. genus Stropharia, Hypholoma).

[0144] A preferred family of the order Auriculariales is Exidiaceae (e.g. genus Exidia).

[0145] A preferred family of the order Dacrymetales is Dacrymycetaceae (e.g. genus Femsjonia).

[0146] Preferred families of the order Stereales are Hyphodermataceae (e.g. genus Hyphodontia) and Stereaceae (e.g. genus Amylostereum and Stereum).

[0147] A preferred family of the order Doletales is Paxillaceae (e.g. genus Paxillus and Hygrophoropsis).

[0148] A preferred family of the order Thelophorales is Thelephoraceae (e.g. genus Typhula).

[0149] Some examples of preferred strains of the above genera are Stropharia cubensis (in particular ATCC 13966), Agrocybe pediades (in particular CBS 900.96), Bjerkandera adusta (in particular CBS 580.95), Trametes zonatella, Trametes pubescens (in particular CBS 100232), Paxillus involtus (in particular CBS 100231), Trametes hirsuta (in particular DSM 2987), Peniophora quercina, Hyphoderma argillaceum, Scizophyllum sp. (in particular CBS 443.97), Peniophora lycii (in particular CBS 686.96), Amylostereum chailletii, Oxyporus sp. (in particular CBS 422.97). Further examples of preferred strains are listed in Example 5, Table 6.

[0150] The phytases of claim 2 have at least one of 14 partial amino acid sequences in common, the so-called consensus sequences, which are entered in the sequence listing as SEQ ID NOS: 1-14.

[0151] In the sequence listing, the amino acid three-letter-code is used, and some of the amino acids are denoted Xaa, which generally means “any amino acid” interpreted as below. In case of some of these Xaa-positions, however, a note is entered in the sequence listing to the effect that for instance X in position NN means any of two amino acids, cf. below.

[0152] In the claims, the amino acid one-letter-code is used in these sequences, and “X” denotes any amino acid including the non-naturally occuring ones and including any structurally or functionally similar variants thereof. Denotations like “[A/E]” mean any of the amino acids A and E. Accordingly, if in a partial sequence of a given formula two of such multiple choices exist, the number of sequences covered by the formula is 22. Likewise, if there are “N” such multiple choices in a given formula, the number of sequences covered by this formula is 2N.

[0153] SEQ ID NOS: 1 to 9 are listed in the order of N-terminal to C-terminal end of the polypeptides. SEQ ID NOS: 10 to 14 are the amino acid sequences corresponding to the PCR probes specifically listed in Example 5 (viz. corresponding to the 522-sense and 540-anti-sense; 537-sense; 538-sense; 525-anti-sense and 539-anti-sense primers, respectively).

[0154] In preferred embodiments, the isolated polypeptide comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or all fourteen sequences of SEQ ID NOS: 1-14, preferably at the positions indicated in claim 3. Claim 3 could also refer to the alignment in FIG. 6.

[0155] The sets of amino acid sequences of claim 4 reflect the PCR experiments of Examples 5 and 6 (viz. those primer-sets which are proposed).

[0156] Also some deletions seem to be characteristic for this phytase sub-family. Some regions of specific deletions are listed in claim 5. This claim could also refer to the alignment of FIG. 6.

[0157] These partial sequences have been identified i.a. on the basis of the alignment shown in FIG. 7. In this alignment the five phytases at the top, viz. phyA1_P.involtus, phyA2_P.involtus, phyA_T.pubescens, phyA_A.pediades and phyA_P.lycii, are all derived from basidiomycetes and have been cloned as reported in the experimental section hereof. The five phytases listed at the bottom of the alignment are all derived from ascomycetes and their sequences are known from the prior art mentioned previously.

[0158] Please refer to Example 4 hereof for further details and explanations with respect to the alignments, and please refer to Example 5 for one way of carrying out screening for phytases of this sub-family. Examples 8-18 describe the purification and characterization of five phytases of the invention, viz. the phytases of Peniophora lycii, Agrocybe pediades, Paxillus involtus and Trametes pubescens.

[0159] Claims 6-8 are related to the experiments of Examples 5, 6 and 7. The conditions corresponding to the term “medium to high stringency” are found in the general definition part above, viz. the washing conditions are 2×SSC and a temperature of 65° C. Preferably, the washing temperature is 65° C. or even higher, e.g. 70, 75, 80 or even 85° C., coresponding to high stringency, very high stringency or exceptionally high stringency.

[0160] Preferably, the PCR reaction is performed with a template or a target sequence, e.g. a nucleotide sequence, which can be e.g. genomic DNA or cDNA. However, for instance mRNA can also be used as a template. Genomic DNA need not be isolated, the PCR reaction can also be conducted directly on for instance fungal mycelium.

[0161] Alternatively, the PCR reaction is performed with the wild-type gene of any PE variant thereof. In particular, at least one PCR band is obtained using at least one primer set on the wild type gene.

[0162] Some specific primers for the PCR reaction are suggested in the experimental part. However, the skilled man is certainly able to propose also other specific primers from the alignment at FIG. 6 (basidiomycete phytases) which primers seem characteristic for basidiomycete phytases, i.e. he has to consider also the alignment at FIG. 7 (showing basidiomycete phytases as well as known ascomycete phytases). Therefore, claim 6 refers in general to such primers, as the skilled man would suggest be specific for basidiomycete phytases, based on his common general knowledge and the alignments at FIGS. 6 and 7.

[0163] As regards 6-phytases, the invention relates to such phytases derived from any fungus. “Fungal” is defined as described above and this term includes i.a. basidiomycetes. How to interpret the specificity is explained in the general definitions part hereof. It is noted, that in the present context, the concept of “a 6-phytase” means any of a D6-, L6- or D/L-6-phytase. Surprisingly, it has turned up that such fungal 6-phytases are of a superior performance as compared to known fungal 3-phytases, reference being had to Examples 10-12 hereof revealing the Peniophora phytase as a 6-phytase and of a highly superior performance as compared to the known Aspergillus phytases. In particular, e.g. the initial rate of hydrolysis of PA is increased and a very plausible explanation of this fact could be that this phytase is a 6-phytase, since these positions (4- and 6- in PA) are less sterically hindered than any of the other positions.

[0164] Preferably, the fungal 6-phytase is a basidiomycete phytase, still more preferably of the class, sub-class, orders, genera and strains as described at the beginning of this section headed “Detailed description of the invention.” In another preferred embodiment the phytase is a D6-phytase. In another preferred embodiment the phytase is a L6-phytase.

[0165] As is apparent from claims 32-36 this application also relates to e.g. the use of a fungal 6-phytase in feed and food, compositions comprising such fungal 6-phytase, in particular food and feed additives, and the use of such fungal 6-phytase for liberating inorganic phosphate from phytic acid or phytate.

[0166] In a preferred embodiment of claims 1-8, the phytase is a (3+6)-phytase, viz. any of a D3-/D6-; L3-/L6-; D3-/L6-; L3-/D6-phytase, reference being had in particular to Examples 15-17 herein regarding the Agrocybe phytase which is also of superior performance as compared to the known Aspergillus phytase.

[0167] Preferably, the (3+6)-phytase is a basidiomycete phytase, still more preferably of the class, sub-class, orders, genera and strains as described at the beginning of this section headed “Detailed description of the invention.”

[0168] Still more preferably, the (3+6)-phytase has a slight preference for one of these positions, in particular the 6-position.

[0169] The phytases of claims 11, 14, 17, 19 and 21 are all more than 50% homologous to the isolated phytases of Agrocybe pediades, Peniophora lycii, Paxillus involtus (phyAl and phyA2) and Trametes pubescens, respectively, corresponding to SEQ ID NOS: 22, 24, 26, 28 and 30, respectively (or the mature polypeptides thereof or any fragment thereof still retaining phytase activity). For a definition of “homologous”, please refer to the section headed “General definitions.” The homology to known phytases appear i.a. from Table 1 in Example 4. Preferably, the number of amino acids in the fragments referred to above is at least 50%, more preferably at least 60%, still more preferably at least 70%, even more preferably at least 80%, in particular at least 90% of the number of amino acids of the sequences listed in the sequence listing. This is so also for any polypeptide fragment disclosed herein.

[0170] Preferably, all amino acid homologies of the present application are at least 55%, or at least 60%, or at least 65%, especially at least 70%. Preferably, the degree of homology is at least 80%, more preferably at least 90%, still more preferably at least 95%, especially at least 97%.

[0171] Claim 12 relates to certain fragments of the amino acid sequence of SEQ ID NO: 22 derived from Agrocybe, these fragments, however, still exhibiting phytase activity. As described in more detail in the experimental part below (Examples 13-15), when expressed in yeast approximately 80% of the Agrocybe phytase enzyme has the N-terminal amino acid of Val (amino acid no. 27 in SEQ ID NO: 22), whereas approximately 20% has the N-terminal amino acid of Thr (amino acid no. 25 in SEQ ID NO: 22). When expressed in Aspergillus approximately 2/3 has the N-terminal amino acid of Phe (amino acid no.31 in SEQ ID NO: 22), whereas approximately 1/3 has the N-terminal amino acid of Gln (amino acid no. 28 in SEQ ID NO: 22). Accordingly, there are at least these four possible mature amino acid sequences.

[0172] Analogously, claim 15 relates to a fragment of the amino acid sequence of SEQ ID NO: 24 derived from Peniophora, this fragment, however, still exhibiting phytase activity. As described in more detail in the experimental part below, when expressed in Aspergillus, the Peniophora phytase has an N-terminal amino acid sequence of Leu-Pro-Ile-Pro-Ala-Gln-Asn- (amino acids no. 31-37 in SEQ ID NO: 24). Accordingly the sequence of amino acids nos. 31-439 of SEQ ID NO: 24 is presently believed to be a mature phytase sequence.

[0173] Claims 13, 16, 18, 20 and 22 are drawn to phytases homologous to the isolated phytases of Agrocybe pediades, Peniophora lycii, Paxillus involtus (phyAl and phyA2) and Trametes pubescens, respectively.

[0174] These phytases are here defined as being encoded by a phytase encoding part of

[0175] (i) the DNA sequences listed in the sequence listing as SEQ ID NOS: 21, 23, 25, 27 and 29, respectively (phyA1 and phyA2 of Paxillus involtus); or

[0176] (ii) the DNA sequences cloned into plasmid pYES 2.0 present in E. coli DSM 11313, 11312, 11842, 11843 and 11844, respectively; or analogues or derivatives thereof which are at least 50% homologous thereto.

[0177] For the definition of a “phytase encoding part” please refer to the general definitions section.

[0178] The five DNA sequences are obtainable directly from the deposited parent strains or from the deposited E. coli strains by extraction of plasmid DNA by methods known in the art (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.).

[0179] Claims 23-26 relate to nucleotide sequences encoding the phytases of the invention, in particular to DNA molecules.

[0180] The DNA molecule of claim 24 comprises at least one of the specific primers suggested herein. In preferred embodiments, it comprises at least two, three, four, five or six of these sequences.

[0181] The DNA molecule of claim 25 is defined by encoding a phytase, and being selected from:

[0182] (a) the specific nucleotide sequences of the Agrocybe, Peniophora, Paxillus and Trametes phytases (phyA1 and phyA2 of Paxillus), e.g. DNA as shown in the sequence listings having the SEQ ID NOS: indicated (or their complementary strands);

[0183] (b) the same sequences as under (a) but expressed via the deposited plasmid clones;

[0184] (c) sequences which are at least 55% homologous to these sequences;

[0185] (d) sequences hybridizing under low stringency with the sequences of (a) or (b);

[0186] (e) sequences which do not hybridize because of the degeneracy of the genetic code but encode the specific polypeptides or phytase active fragments thereof.

[0187] For a definition of “hybridization,” please refer to the section headed “General definitions,” which also lists some preferred hybridization conditions.

[0188] With respect to the homology part in feature (c), the degree of homology to the nucleic acid sequence set forth under heading (a) and (b) is at least about 55%, (still encoding an polypeptide exhibiting phytase activity). In particular, the homology is at least 60%, or at least 65%, especially at least 70%, preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 97%. In particular, the degree of homology is based on a comparison with the entire sequences listed or their complementary strand or any of the sub-sequences thereof corresponding to a “mature” phytase.

[0189] The homology of the DNA of selected phytases of the invention to known phytases appear i.a. from Table 1 in Example 4.

[0190] Nucleotide claim 26 is related to polypeptide claims 6-8, and corresponding preferred embodiments mentioned with respect to these claims are hereby incorporated also with respect to this DNA claim (reference to Examples 5, 6 and 7; “medium to high stringency” means the washing conditions are 2×SSC and a temperature of 65° C., preferably 65° C. or even higher, e.g. 70, 75, 80 or even 85° C.; the PCR reaction is performed with a target nucleotide sequence, e.g. DNA, for instance genomic DNA or cDNA (or mRNA); wild-type gene of any PE variant thereof).

[0191] The DNA sequences of the invention can also be cloned by any general method involving

[0192] (a) cloning, in suitable vectors, a cDNA library from any organism expected to produce the phytase of interest,

[0193] (b) transforming suitable yeast host cells with said vectors,

[0194] (c) culturing the host cells under suitable conditions to express any enzyme of interest encoded by a clone in the cDNA library,

[0195] (d) screening for positive clones by determining any phytase activity of the enzyme produced by such clones, and

[0196] (e) isolating the enzyme encoding DNA from such clones.

[0197] A general isolation method has been disclosed in WO 93/11249 and WO 94/14953, the contents of which are hereby incorporated by reference. A detailed description of the screening methods is given in the experimental part.

[0198] Claim 27 relates to a primer, probe, oligonucleotide molecule/DNA molecule which can be derived from the alignment of FIG. 6. Only primers specific or unique for the novel phytases of the invention are of course included herein, viz. one has to consider also the alignment at FIG. 7 (cf. remarks to claims 6-8 above).

[0199] A method of identifying further phytase producing cells, in particular microorganisms, is disclosed in claim 31. In particular, this is also a method of selecting or screening for phytase producing microorganisms. The concept of “cell” is here generally to be defined as the term “host cell” in the general definitions part hereof). Preferred cells are microorganism cells, in particular fungal cells, preferably of the phyllum Basidiomycota. More preferred basidiomycete cells are listed in the very beginning of the detailed description of the invention.

[0200] Any source, in particular any microorganism, can be selected to provide a template or a target sequence, usually in the form of genomic DNA, cDNA or mRNA.

[0201] General references to the PCR reaction, including standard reaction conditions, are found in the experimental part. As regards the selection of suitable primers, reference is had to the remarks under claims 6-8 above.

[0202] Preferably, it has to be verified that the amplified PCR fragment derived from the template is specific.

[0203] Some examples of suitable verification procedures are:

[0204] (a) running an electrophoresis in agarose gels revealing the existence of a PCR band corresponding to the amplified PCR fragment; and, if desired, also

[0205] (b) controlling that the size of the amplified PCR-fragment is as expected; and, if desired, also

[0206] (c) isolating and sequencing the PCR fragment or band to show a high degree of homology to the parent sequences, from which the primers were derived.

[0207] Ad b): The potential presence of introns, (an example: 50 bases out of 300), is one of several reasons for allowing a deviation from exact size match. Preferably, the size of the amplified PCR-fragment (including introns) as measured for instance by the number of bases is within the ranges of 50-150%, 60-140%, 70-130%, 80-120%, 85-115%, 90-110%, 95-105% of the number of bases/nucleotides inbetween the primers in the parent sequences (FIG. 6), from which the primers were derived. In another preferred embodiment (excluding introns), the size of the amplified PCR-fragment is within the range of 80-120%, 85-115%, 90-110%, 95-105% of the number of bases inbetween the primers in the parent sequences (FIG. 6), from which the primers were derived.

[0208] Ad c): Preferably the degree of homology is more than 50, 60, 70, 75, 80, 85, 90, 95% homology (determined as described above). Alternatively, it is checked if the amplified PCR-fragment comprises at least one of the conserved regions inbetween the primers used, said conserved regions being shown in grey shading at FIG. 6. Preferably, the fragment comprises at least two, three, four, five etc. or all of such conserved regions. Another way of checking homology is by checking presence of areas of deletions characteristic for the parent sequences of FIG. 6. A further way of checking homology is checking characteristic distances between conserved regions, vide e.g. claim 5.

[0209] Claim 34 relates to a process for preparing the phytase polypeptide, said process logically following from this screening method of claim 31.

[0210] Steps a)+d) of claim 34 relate to the preparation of the phytase from the wild-type cell, in particular microorganism strain.

[0211] Steps b)+d) relate to the cloning of the entire phytase encoding gene from the under a) identified phytase producing cell, in particular microorganism, and transferring this gene into a heterologous or homologous host cell using any general recombinant DNA technic, e.g. as hereinbefore described and referring generally to e.g. Maniatis (cited above).

[0212] Steps c)+d) relate to the use of the amplified PCR fragment as a hybridization probe to identify and isolate phytase encoding DNA molecules (phytase encoding genes or phytase encoding parts of genes). Such DNA molecules may be isolated from any source (target sequence, template) which comprises polynucleotides, such as genomic DNA, cDNA, mRNA.

[0213] Hybridization experiments and in particular hybridization conditions, including preferred conditions, and isolation procedures are as generally hereinbefore described.

[0214] Once isolated, the DNA molecule is transferred to a host cell, as is also generally hereinbefore described.

[0215] Finally, the invention also relates generally to the use of the polypeptide according to any of claims 1-22 for liberating (or catalyzing the liberation of) phosphorous from any phytase substrate, in particular inorganic phosphate from phytate or phytic acid; alternatively for converting phytate to inorganic phosphate and (myo-inositol and/or mono-, di-, tri-, tetra-, penta-phosphate esters thereof). This claim encompasses any process wherein the phytase of the invention excerts its phytase activity as previously defined.

[0216] More specific uses according to the invention are in human food or animal feed preparations or in additives for such preparations, wherein the phytase i.a. serves the purposes of

[0217] (i) reducing the phytate level of manure,

[0218] (ii) improving the digestibility, promoting the growth, or improving the food and feed utilization or its conversion efficiency, i.a. by making available proteins, which would otherwise have been bound by phytate,

[0219] (iii)preventing malnutrition or diseases such as anemia caused by essential ions and phosphate lacking, i.e. improving the bioavailibility of minerals or increasing the absorption thereof, eliminating the need for adding supplemental phosphate and ions etc.

[0220] In particular, the phytases of the invention can also be used in chicken food to improve egg shell quality (reduction of losses due to breaking), cf. e.g. The Merck Veterinary Manual, (Seventh edition, Merck & CO., Inc., Rahway, N.J., U.S.A., 1991; p.1268); Jeroch et al; Bodenkultur Vol. 45, No. 4 pp. 361-368 (1994); Poultry Science, Vol. 75, No. 1 pp. 62-68 (1996); Canadian Journal of Animal Science Vol. 75 , No. 3 pp. 439-444 (1995); Poultry Science Vol. 74 , No. 5 pp. 784-787 (1995) and Poultry Science Vol. 73 , No. 10 pp. 1590-1596 (1994).

[0221] A “feed” and a “food,” respectively, means any natural or artificial diet, meal or the like or components of such meals intended or suitable for being eaten, taken in, digested, by an animal and a human being, respectively.

[0222] The phytase may exert its effect in vitro or in viva, i.e. before intake or in the stomach of the individual, respectively. Also a combined action is possible.

[0223] A phytase composition according to the invention always comprises at least one phytase of the invention.

[0224] Generally, phytase compositions are liquid or dry.

[0225] Liquid compositions need not contain anything more than the phytase enzyme, preferably in a highly purified form. Usually, however, a stabilizer such as glycerol, sorbitol or mono propylen glycol is also added. The liquid composition may also comprise other additives, such as salts, sugars, preservatives, pH-adjusting agents, proteins, phytate (a phytase substrate). Typical liquid compositions are aqueous or oil-based slurries. The liquid compositions can be added to a food or feed after an optional pelleting thereof.

[0226] Dry compositions may be spraydried compositions, in which case the composition need not contain anything more than the enzyme in a dry form. Usually, however, dry compositions are so-called granulates which may readily be mixed with e.g. food or feed components, or more preferably, form a component of a pre-mix. The particle size of the enzyme granulates preferably is compatible with that of the other components of the mixture. This provides a safe and convenient mean of incorporating enzymes into e.g. animal feed.

[0227] Agglomeration granulates are prepared using agglomeration technique in a high shear mixer (e.g. Lödige) during which a filler material and the enzyme are co-agglomerated to form granules. Absorption granulates are prepared by having cores of a carrier material to absorp/be coated by the enzyme.

[0228] Typical filler materials are salts such as disodium sulphate. Other fillers are kaolin, talc, magnesium aluminium silicate and cellulose fibres. Optionally, binders such as dextrins are also included in agglomeration granulates.

[0229] Typical carrier materials are starch, e.g. in the form of cassava, corn, potato, rice and wheat. Salts may also be used.

[0230] Optionally, the granulates are coated with a coating mixture. Such mixture comprises coating agents, preferably hydrophobic coating agents, such as hydrogenated palm oil and beef tallow, and if desired other additives, such as calcium carbonate or kaolin.

[0231] Additionally, phytase compositions may contain other substituents such as colouring agents, aroma compounds, stabilizers, vitamins, minerals, other feed or food enhancing enzymes etc. This is so in particular for the so-called pre-mixes.

[0232] A “food or feed additive” is an essentially pure compound or a multi component composition intended for or suitable for being added to food or feed. In particular it is a substance which by its intended use is becoming a component of a food or feed product or affects any characteristics of a food or feed product. It is composed as indicated for phytase compositions above. A typical additive usually comprises one or more compounds such as vitamins, minerals or feed enhancing enzymes and suitable carriers and/or excipients.

[0233] In a preferred embodiment, the phytase compositions of the invention additionally comprises an effective amount of one or more feed enhancing enzymes, in particular feed enhancing enzymes selected from the group consisting of α-galactosidases, β-galactosidases, in particular lactases, other phytases, β-glucanases, in particular endo-β-1,4-glucanases and endo-β-1,3(4)-glucanases, cellulases, xylosidases, galactanases, in particular arabinogalactan endo-1,4-β-galactosidases and arabinogalactan endo-1,3-β-galactosidases, endoglucanases, in particular endo-1,2-β-glucanase, endo-1,3-α-glucanase, and endo-1,3-β-glucanase, pectin degrading enzymes, in particular pectinases, pectinesterases, pectin lyases, polygalacturonases, arabinanases, rhamnogalacturonases, rhamnogalacturonan acetyl esterases, rhamnogalacturonan-α-rhamnosidase, pectate lyases, and α-galacturonisidases, mannanases, β-mannosidases, mannan acetyl esterases, xylan acetyl esterases, proteases, xylanases, arabinoxylanases and lipolytic enzymes such as lipases, phospholipases and cutinases.

[0234] The animal feed additive of the invention is supplemented to the mono-gastric animal before or simultaneously with the diet. Preferably, the animal feed additive of the invention is supplemented to the mono-gastric animal simultaneously with the diet. In a more preferred embodiment, the animal feed additive is added to the diet in the form of a granulate or a stabilized liquid.

[0235] An effective amount of phytase in food or feed is from about 10-20.000; preferably from about 10 to 15.000, more preferably from about 10 to 10.000, in particular from about 100 to 5.000, especially from about 100 to about 2.000 FYT/kg feed or food.

[0236] Examples of other specific uses of the phytase of the invention is in soy processing and in the manufacture of inositol or derivatives thereof.

[0237] The invention also relates to a method for reducing phytate levels in animal manure, wherein the animal is fed a feed comprising an effective amount of the phytase of the invention. As stated in the beginning of the present application one important effect thereof is to reduce the phosphate pollution of the environment.

[0238] Also within the scope of the invention is the use of a phytase of the invention during the preparation of food or feed preparations or additives, i.e. the phytase excerts its phytase activity during the manufacture only and is not active in the final food or feed product. This aspect is relevant for instance in dough making and baking.

[0239] The invention also relates to substantially pure biological cultures of the deposited microorganisms and to strains comprising, as a part of their genetic equipment, a DNA sequence encoding a phytase of the invention. Included within the definition of a substantially pure biological culture is any mutant of said strains having retained the phytase encoding capability.

[0240] The invention is described in further detail in the following examples which are not in any way intended to limit the scope of the invention.

EXAMPLES

[0241] Materials and Methods Media

[0242] Phytate Replication Plates

[0243] Add to 200 ml of melted SC agar

[0244] 20 ml 20% galactose

[0245] 800 μl 5% threonine

[0246] 25 ml solution A

[0247] 25 ml solution B

[0248] 200 μl Trace element solution (DSM Catalogue 141)

[0249] Solution A

[0250] 6 g CaCl2, 2H2O

[0251] 8 g MgCl2, 6H2O

[0252] add ddH2O to 1l

[0253] pH=6.5

[0254] Solution B

[0255] 35.12 g Na-phytate

[0256] add H2O to 1l

[0257] pH=6.5

3Medium A:Yeast Nitrogen Base w/o Amino acids (Difco 0919)7.5g/lSuccinic acid (Merck 822260)11.3g/lNaOH (Merck 6498)6.8g/lCasamino acid w/o vitamin (Difco 0288)5.6g/ltryptophan (Merck 8374)0.1g/lThreonine1.0g/lNa-phytate (35.12 g/l pH 6.5)125ml/lGalactose20.0g/lTrace metal (DSM 141)1.0ml/lad 1 l with H2OTrace metal solution:Nitrilotriacetic acid1.50gMgSO4, 7 H2O3.00gMnSO4.2H2O0.50gNaCl1.00gFeSO4, 7H2O0.10gCoSO4.7H2O0.18gCaCl2, 2H2O0.10gZnSO4, 7H2O0.18gCuSO4, 5H2O0.01gKAl (SO4)2, 12H2O0.02gH3BO30.01gNa2MoO4, 2H2O0.01gNiCl2, 6H2O0.025gNa2Se3O, 5H2O0.30gDistilled water1lpH 7.0

[0258] First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Final pH 7.0 (with KOH).

[0259] Medium B

[0260] Similar to medium A except for glucose is added as a C-source instead of galactose.

[0261] YPD

[0262] 10 g yeast extract, 20 g peptone, H2O to 900 ml. Autoclaved, 100 ml 20% glucose (sterile filtered) added.

[0263] YPM

[0264] 10 g yeast extract, 20 g peptone, H2O to 900 ml. Autoclaved, 100 ml 20% maltodextrin (sterile filtered) added.

[0265] 10×Basal salt

[0266] 75 g yeast nitrogen base, 113 g succinic acid, 68 g NaOH, H2O ad 1000 ml, sterile filtered.

[0267] SC-URA

[0268] 100 ml 10×Basal salt, 28 ml 20% casamino acids without vitamins, 10 ml 1% tryptophan, H2O ad 900 ml, autoclaved, 3.6 ml 5% threonine and 100 ml 20% glucose or 20% galactose added.

[0269] SC-agar

[0270] SC-URA, 20 g/l agar added.

[0271] SC-variant agar

[0272] 20 g agar, 20 ml 10×Basal salt, H2O ad 900 ml, autoclaved

[0273] Phytase Activity Assay

[0274] The phytase activity can be measured using the following assay:

[0275] 10 μl diluted enzyme samples (diluted in 0.1 M sodium acetate, 0.01% Tween20, pH 5.5) were added into 250 μl 5 mM sodium phytate (Sigma) in 0.1 M sodium acetate, 0.01% Tween20, pH 5.5 (pH adjusted after dissolving the sodium phytate; the substrate was preheated) and incubated for 30 minutes at 37° C. The reaction was stopped by adding 250 μl 10% TCA and free phosphate was measured by adding 500 μl 7.3 g FeSO4 in 100 ml molybdate reagent (2.5 g (NH4)6Mo7024.4H20 in 8 ml H2SO4 diluted to 250 ml The absorbance at 750 nm was measured on 200 μl samples in 96 well microtiter plates. Substrate and enzyme blanks were included. A phosphate standard curve was also included (0-2 mM phosphate). 1 FYT equals the amount of enzyme that releases 1 μmol phosphate/min at the given conditions.

[0276] General Molecular Biology Methods

[0277] Unless otherwise mentioned the DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al. (eds.) “Current protocols in Molecular Biology”. John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) “Molecular Biological Methods for Bacillus”. John Wiley and Sons, 1990).

[0278] Unless otherwise specified all enzymes for DNA manipulations, such as e.g. restriction endonucleases, ligases etc., were obtained from New England Biolabs, Inc. The enzymes were used according to the specifications of the suppliers.

Example 1

Cloning and Expression of a Phytase from Peniophora Lycii CBS No. 686.96

[0279] Deposited Organisms

[0280]

Peniophora lycii
CBS No. 686.96 comprises a phytase encoding Ah DNA sequence of the invention.

[0281]

Escherichia coli
DSM NO 11312 contains the plasmid comprising the full length cDNA sequence, coding for a phytase of the invention, in the shuttle vector PYES 2.0.

[0282] Other Strains

[0283] Yeast strain: The Saccharomyces cerevisiae strain used was W3124 (van den Hazel, H. B; Kielland-Brandt, M. C.; Winther, J. R. in Eur. J. Biochem., 207, 277-283, 1992; (MATa; ura 3-52; leu 2-3, 112; his 3-D200; pep 4-1137; prcl::HIS3; prbl:: LEU2; cir+).

[0284]

E. coli strain:
DH1OB (Life Technologies)

[0285] Plasmids

[0286] The Aspergillus expression vector pHD414 is a derivative of the plasmid p775 (described in EP 238 023). The construction of pHD414 is further described in WO 93/11249.

[0287] pYES 2.0 (Invitrogen)

[0288] pA2phy2 (See example 1)

[0289] Expression Cloning in Yeast

[0290] Expression cloning in yeast was done as described by H. Dalboege et al. (H. Dalboege et al Mol. Gen. Genet (1994) 243:253-260.; WO 93/11249; WO 94/14953), which are hereby incorporated as reference. All individual steps of Extraction of total RNA, cDNA synthesis, Mung bean nuclease treatment, Blunt-ending with T4 DNA polymerase, and Construction of libraries was done according to the references mentioned above.

[0291] Fermentation Procedure of Peniophora Lycii CBS No. 686.96 for mRNA Isolation

[0292]

Peniophora lycii
CBS 686.96 was inoculated from a plate with outgrown mycelium into a shake flask containing 100 ml medium B (soya 30 g/l, malto dextrin 15 g/l, bacto peptone 5 g/l, pluronic 0.2 g/l). The culture was incubated stationary at 26° C. for 15 days. The resulting culture broth was filtered through miracloth and the mycelium was frozen down in liquid nitrogen.

[0293] mRNA was isolated from mycelium from this culture as described in (H. Dalboege et al Mol. Gen. Genet (1994) 243:253-260.; WO 93/11249; WO 94/14953).

[0294] Extraction of total RNA was performed with guanidinium thiocyanate followed by ultracentrifugation through a 5.7 M CsCl cushion, and isolation of poly(A)+RNA was carried out by oligo(dT)-cellulose affinity chromatography using the procedures described in WO 94/14953.

[0295] cDNA Synthesis

[0296] Double-stranded cDNA was synthesized from 5 mg poly(A)+RNA by the RNase H method (Gubler and Hoffman (1983) Gene 25:263-269, Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY). The poly(A)+RNA (5 μg in 5 μl of DEPC-treated water) was heated at 70° C. for 8 min. in a pre-siliconized, RNase-free Eppendorph tube, quenched on ice and combined in a final volume of 50 μl with reverse transcriptase buffer (50 mM Tris-Cl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, Bethesda Research Laboratories) containing 1 mM of dATP, dGTP and dTTP and 0.5 mM 5-methyl-dCTP (Pharmacia), 40 units human placental ribonuclease inhibitor (RNasin, Promega), 1.45 μg of oligo(dT)18-Not I primer (Pharmacia) and 1000 units SuperScript II RNase H reverse transcriptase (Bethesda Research Laboratories). First-strand cDNA was synthesized by incubating the reaction mixture at 45° C. for 1 hour. After synthesis, the mRNA:cDNA hybrid mixture was gelfiltrated through a MicroSpin S-400 HR (Pharmacia) spin column according to the manufacturer's instructions.

[0297] After the gelfiltration, the hybrids were diluted in 250 μl second strand buffer (20 mM Tris-Cl, pH 7.4, 90 mM KCl, 4.6 mM MgCl2, 10 mM (NH4)2SO4, 0.16 mM bNAD+) containing 200 μl of each dNTP, 60 units E. coli DNA polymerase I (Pharmacia), 5.25 units RNase H (Promega) and 15 units E. coli DNA ligase (Boehringer Mannheim). Second strand cDNA synthesis was performed by incubating the reaction tube at 16° C. for 2 hours and additional 15 min. at 25° C. The reaction was stopped by addition of EDTA to a final concentration of 20 mM followed by phenol and chloroform extractions.

[0298] Mung Bean Nuclease Treatment

[0299] The double-stranded cDNA was precipitated at −20° C. for 12 hours by addition of 2 vols 96% EtOH, 0.2 vol 10 M NH4Ac, recovered by centrifugation, washed in 70% EtOH, dried and resuspended in 30 μl Mung bean nuclease buffer (30 mM NaAc, pH 4.6, 300 mM NaCl, 1 mM ZnSO4, 0.35 mM DTT, 2% glycerol) containing 25 units Mung bean nuclease (Pharmacia). The single-stranded hair-pin DNA was clipped by incubating the reaction at 30° C. for 30 min., followed by addition of 70 μl 10 mM Tris-Cl, pH 7.5, 1 mM EDTA, phenol extraction and precipitation with 2 vols of 96% EtOH and 0.1 vol 3 M NaAc, pH 5.2 on ice for 30 min.

[0300] Blunt-ending with T4 DNA Polymerase

[0301] The double-stranded cDNAs were recovered by centrifugation and blunt-ended in 30 ml T4 DNA polymerase buffer (20 mM Tris-acetate, pH 7.9, 10 mM MgAc, 50 mM KAc, 1 mM DTT) containing 0.5 mM of each dNTP and 5 units T4 DNA polymerase (New England Biolabs) by incubating the reaction mixture at 16° C. for 1 hour.

[0302] The reaction was stopped by addition of EDTA to a final concentration of 20 mM, followed by phenol and chloroform extractions, and precipitation for 12 hours at −20° C. by adding 2 vols 96% EtOH and 0.1 vol 3 M NaAc pH 5.2.

[0303] Adaptor Ligation, Not I Digestion and Size Selection

[0304] After the fill-in reaction the cDNAs were recovered by centrifugation, washed in 70% EtOH and dried. The cDNA pellet was resuspended in 25 μl ligation buffer (30 mM Tris-Cl, pH 7.8, 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP) containing 2.5 μg non-palindromic BstXI adaptors (Invitrogen) and 30 units T4 ligase (Promega) and incubated at 16° C. for 12 hours. The reaction was stopped by heating at 65° C. for 20 min. and then cooling on ice for 5 min. The adapted cDNA was digested with Not I restriction enzyme by addition of 20 μl water, 5 μl 10× Not I restriction enzyme buffer (New England Biolabs) and 50 units Not I (New England Biolabs), followed by incubation for 2.5 hours at 37° C. The reaction was stopped by heating at 65° C. for 10 min. The cDNAs were size-fractionated by gel electrophoresis on a 0.8% SeaPlaque GTG low melting temperature agarose gel (FMC) in 1× TBE to separate unligated adaptors and small cDNAs. The cDNA was size-selected with a cut-off at 0.7 kb and rescued from the gel by use of b-Agarase (New England Biolabs) according to the manufacturer's instructions and precipitated for 12 hours at −20° C. by adding 2 vols 96% EtOH and 0.1 vol 3 M NaAc pH 5.2.

[0305] Construction of Libraries

[0306] The directional, size-selected cDNA was recovered by centrifugation, washed in 70% EtOH, dried and resuspended in 30 μl 10 mM Tris-Cl, pH 7.5, 1 mM EDTA. The cDNAs were desalted by gelfiltration through a MicroSpin S-300 HR (Pharmacia) spin column according to the manufacturer's instructions. Three test ligations were carried out in 10 μl ligation buffer (30 mM Tris-Cl, pH 7.8, 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP) containing 5 μl double-stranded cDNA (reaction tubes #1 and #2), 15 units T4 ligase (Promega) and 30 ng (tube #1), 40 ng (tube #2) and 40 ng (tube #3, the vector background control) of BstXI-NotI cleaved pYES 2.0 vector. The ligation reactions were performed by incubation at 16° C. for 12 hours, heating at 70° C. for 20 min. and addition of 10 μl water to each tube. 1 μl of each ligation mixture was electroporated into 40 μl electrocompetent E. coli DH10B cells (Bethesda research Laboratories) as described (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, N.Y.). Using the optimal conditions a library was established in E. coli consisting of pools. Each pool was made by spreading transformed E. coli on LB+ampicillin agar plates giving 15.000-30.000 colonies/plate after incubation at 37° C. for 24 hours. 20 ml LB+ampicillin was added to the plate and the cells were suspended herein. The cell suspension was shaked in a 50 ml tube for 1 hour at 37° C. Plasmid DNA was isolated from the cells according to the manufacturer's instructions using QIAGEN plasmid kit and stored at -20° C.

[0307] 0 1 μl aliquots of purified plasmid DNA (100 ng/ml) from individual pools were transformed into S. cerevisiae W3124 by electroporation (Becker and Guarante (1991) Methods Enzymol. 194:182-187) and the transformants were plated on SC agar containing 2% glucose and incubated at 30° C.

[0308] Identification of Positive Colonies

[0309] After 3-5 days of growth, the agar plates were replica plated onto a set of the phytate replication plates, and incubated for 3-5 days at 30° C. 1% LSB-agarose (Sigma) containing 0.2 M CaCl2 is poured over the plates and after 1-4 days the phytase positive colonies are identified as colonies surrounded by a clearing zone.

[0310] Cells from enzyme-positive colonies were spread for single colony isolation on agar, and an enzyme-producing single colony was selected for each of the phytase-producing colonies identified.

[0311] Isolation of a CDNA Gene for Expression in Aspergillus

[0312] A phytase-producing yeast colony was inoculated into 20 ml YPD broth in a 50 ml glass test tube. The tube was shaken for 2 days at 30° C. The cells were harvested by centrifugation for 10 min. at 3000 rpm.

[0313] DNA was isolated according to WO 94/14953 and dissolved in 50 ml water. The DNA was transformed into E. coli by standard procedures. Plasmid DNA was isolated from E. coli using standard procedures, and analyzed by restriction enzyme analysis.

[0314] The cDNA insert was excised using the restriction enzymes Hind III and Xba I and ligated into the Aspergillus expression vector pHD414 resulting in the plasmid pA2phy2.

[0315] The cDNA inset of Qiagen purified plasmid DNA of pA2phy2 (Qiagen, USA) was sequenced with the Taq deoxy terminal cycle sequencing kit (Perkin Elmer, USA) and synthetic oligonucleotide primers using an Applied Biosystems ABI PRISM™ 377 DNA Sequencer according to the manufacturers instructions.

[0316] Transformation of Aspergillus Oryzae or Aspergillus Nicer

[0317] Protoplasts are prepared as described in WO 95/02043, p. 16, line 21-page 17, line 12.

[0318] 100 μl of protoplast suspension is mixed with 5-25 μg of the appropriate DNA in 10 μl of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH=7.5, 10 mM CaCl2). Protoplasts are mixed with p3SR2 (an A. nidulans amdS gene carrying plasmid) (Tove Christensen et al. Bio/Technology, pp 1419-1422 vol.6, Dec. 1988). The mixture is left at room temperature for 25 minutes. 0.2 ml of 60% PEG 4000 (BDH 29576), 10 mM CaCl2 and 10 mM Tris-HCl, pH 7.5 is added and carefully mixed (twice) and finally 0.85 ml of the same solution is added and carefully mixed. The mixture is left at room temperature for 25 minutes, spun at 2500 g for 15 minutes and the pellet is resuspended in 2 ml of 1.2 M sorbitol. After one more sedimentation the protoplasts are spread on minimal plates (Cove, Biochem. Biophys. Acta 113 (1966) 51-56) containing 1.0 M sucrose, pH 7.0, 10 mM acetamide as nitrogen source and 20 mM CsCl to inhibit background growth. After incubation for 4-7 days at 37° C. spores are picked and spread for single colonies. This procedure is repeated and spores of a single colony after the second reisolation is stored as a defined transformant.

[0319] Test of A. oryzae transformants

[0320] Each of the A. oryzae transformants are inoculated in 10 ml of YPM (cf. below) and propagated. After 2-5 days of incubation at 30° C., the supernatant is removed.

[0321] The phytase activity is identified by applying 20 μl supernatant to 4 mm diameter holes punched out in 1% LSB-agarose plates containing 0.1 M Sodium acetate pH 4.5 and 0.1% Inositol hexaphosphoric acid. The plates are left over night at 37° C. A buffer consisting of 0.1 M CaCl2 and 0.2 M Sodium acetate pH 4.5 is poured over the plates and the plates are left at room temperature for 1 h. Phytase activity is then identified as a clear zone.

[0322] Fed Batch Fermentation

[0323] Fed batch fermentation was performed in a medium comprising maltodextrin as a carbon source, urea as a nitrogen source and yeast extract. The fed batch fermentation was performed by inoculating a shake flask culture of A. oryzae host cells in question into a medium comprising 3.5% of the carbon source and 0.5% of the nitrogen source. After 24 hours of cultivation at pH 7.0 and 34° C. the continuous supply of additional carbon and nitrogen sources were initiated. The carbon source was kept as the limiting factor and it was secured that oxygen was present in excess. The fed batch cultivation was continued for 4 days.

[0324] Isolation of the DNA Sequence shown in SEQ ID NO: 23

[0325] The phytase encoding part of the DNA sequence shown in SEQ ID NO: 23 coding for the phytase of the invention can be obtained from the deposited organism Escherichia coli DSM 11312 by extraction of plasmid DNA by methods known in the art (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, N.Y.).

[0326] Cloning and expression was done by using the Expression cloning in yeast technique as described above.

[0327] mRNA was isolated from Peniophora lycii, CBS No. 686.96, grown as described above.

[0328] Mycelia were harvested after 15 days' growth, immediately frozen in liquid nitrogen and stored at −80° C. A library from Peniophora lycii, CBS No. 686.96, consisting of approx. 9×105 individual clones was constructed in E. coli as described with a vector background of 1%. Plasmid DNA from some of the pools was transformed into yeast, and 50-100 plates containing 250-400 yeast colonies were obtained from each pool.

[0329] Phytase-positive colonies were identified and isolated as described above and inoculated into 20 ml YPD broth in a 50 ml glass test tube. The tube was shaken for 2 days at 30° C. The cells were harvested by centrifugation for 10 min. at 3000 rpm. DNA was isolated according to WO 94/14953 and dissolved in 50 μl water. The DNA was transformed into E. coli by standard procedures.

[0330] Plasmid DNA was isolated from E. coli using standard procedures, and the DNA sequence of the cDNA encoding the phytase was sequenced with the Taq deoxy terminal cycle sequencing kit (Perkin Elmer, USA) and synthetic oligonucleotide primers using an Applied Biosystems ABI PRISM™ 377 DNA Sequencer according to the manufacturers instructions. The DNA sequence of the cDNA encoding the phytase is shown in SEQ ID NO: 23 and the corresponding amino acid sequence is shown in SEQ ID NO: 24. In SEQ ID NO: 23 DNA nucleotides from No 1 to No. 1320 define a phytase encoding region.

[0331] The part of the DNA sequence in SEQ ID NO: 23, which is encoding the mature part of the phytase is position 91 to 1320, which corresponds to amino acid position 31-439 in SEQ ID NO: 24.

[0332] The cDNA is obtainable from the plasmid in DSM 11312.

[0333] Total DNA was isolated from a yeast colony and plasmid DNA was rescued by transformation of E. coli as described above. In order to express the phytase in Aspergillus, the DNA was digested with appropriate restriction enzymes, size fractionated on gel, and a fragment corresponding to the phytase gene was purified. The gene was subsequently ligated to pHD414, digested with appropriate restriction enzymes, resulting in the plasmid pA2phy2.

[0334] After amplification of the DNA in E. coli the plasmid was transformed into Aspergillus oryzae as described above.

[0335] Test of A. Oryzae Transformants

[0336] Each of the transformants were tested for enzyme activity as described above. Some of the transformants had phytase activity which was significantly larger than the Aspergillus oryzae background. This demonstrates efficient expression of the phytase in Aspergillus oryzae.

Example 2

Cloning and Expression of a Phytase from Agrocybe Pediades CBS No. 900.96

[0337] Deposited organisms

[0338]

Agrocybe pediades
CBS No. 900.96 comprises a phytase encoding DNA sequence of the invention.

[0339]

Escherichia coli
DSM NO 11313 contains the plasmid comprising the full length cDNA sequence, coding for a phytase of the invention, in the shuttle vector pYES 2.0.

[0340] Isolation of the DNA Sequence Shown in SEQ ID NO: 21

[0341] The phytase encoding part of the DNA sequence shown in SEQ ID NO: 21 coding for a phytase of the invention can be obtained from the deposited organism Escherichia coli DSM 11313 by extraction of plasmid DNA by methods known in the art (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.).

[0342] Cloning and expression was done by using the Expression cloning in yeast technique as described in Example 1.

[0343] mRNA was isolated from Agrocybe pediades, CBS No. 900.96, grown as described above with agitation to ensure sufficient aeration.

[0344] Mycelia were harvested after 3-5 days' growth, immediately frozen in liquid nitrogen and stored at -80° C. A library from Agrocybe pediades, CBS No. 900.96, consisting of approx. 9×105 individual clones was constructed in E. coli as described with a vector background of 1%. Plasmid DNA from some of the pools was transformed into yeast, and 50-100 plates containing 250-400 yeast colonies were obtained from each pool.

[0345] Phytase-positive colonies were identified and isolated as described above. cDNA inserts were amplified directly from the yeast colonies and characterized as described in the Materials and Methods section above. The DNA sequence of the cDNA encoding the phytase is shown in SEQ ID NO: 21 and the corresponding amino acid sequence is shown in SEQ ID NO: 22. In SEQ ID NO: 21 DNA nucleotides from No 1 to No. 1362 define the phytase encoding region.

[0346] The part of the DNA sequence in SEQ ID NO: 21, which is encoding the mature part of the phytase is position 79 to 1362, which correspond to amino acid position 27-453 in SEQ ID NO: 22.

[0347] The cDNA is obtainable from the plasmid in DSM 11313.

[0348] Total DNA was isolated from a yeast colony and plasmid DNA was rescued by transformation of E. coli as described above. In order to express the phytase in Aspergillus, the DNA was digested with appropriate restriction enzymes, size fractionated on gel, and a fragment corresponding to the phytase gene was purified. The gene was subsequently ligated to pHD414, digested with appropriate restriction enzymes, resulting in the plasmid pA3phy3.

[0349] After amplification of the DNA in E. coli the plasmid was transformed into Aspergillus oryzae as described in Example 1.

[0350] Test of A. Oryzae Transformants

[0351] Each of the transformants was tested for enzyme activity as described in Example 1. Some of the transformants had phytase activity which was significantly larger than the Aspergillus oryzae background. This demonstrates efficient expression of the phytase in Aspergillus oryzae.

Example 3

Cloning and Expression of Phytases from Paxillus Involtus CBS 100231 and Trametes pubescens CBS 100232

[0352] Deposited Organisms

[0353]

Paxillus involtus
CBS No. 100231 comprises two phytase encoding DNA sequences of the invention and Trametes pubescens CBS 100232 comprises a phytase of the invention.

[0354]

Escherichia coli
DSM Nos. 11842, 11843 and 11844 contain the plasmids comprising the full length cDNA sequences, coding for these phytases of the invention, in the shuttle vector pYES 2.0, viz. PhyA1, PhyA2 of Paxillus involtus and the phytase of Trametes pubescens, respectively.

[0355] Isolation of the DNA Sequences Shown in SEQ ID NOS: 25, 27 and 29

[0356] The phytase encoding part of the DNA sequences shown in SEQ ID NOS: 25, 27 and 29 coding for phytases of the invention can be obtained from the deposited organisms Escherichia coli DSM 11842, 11843 and 11844, respectively, by extraction of plasmid DNA by methods known in the art (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.).

[0357] Cloning and expression was done by using the Expression cloning in yeast technique as described in Example 1.

[0358] mRNA was isolated from the respective microorganisms, grown under phytase producing conditions, e.g. as described above with agitation to ensure sufficient aeration.

[0359] Mycelia were harvested after 3-5 days' growth, immediately frozen in liquid nitrogen and stored at -80° C. Libraries, consisting of approx. 9×105 individual clones was constructed in E. coli as described with a vector background of 1%. Plasmid DNA from some of the pools was transformed into yeast, and 50-100 plates containing 250-400 yeast colonies were obtained from each pool.

[0360] Phytase-positive colonies were identified and isolated as described above. cDNA inserts were amplified directly from the yeast colonies and characterized as described in the Materials and Methods section above. The DNA sequences of the cDNA encoding the phytases are shown in SEQ ID NOS: 25, 27 and 29 and the corresponding amino acid sequences are shown in SEQ ID NOS: 26, 28 and 30, respectively.

[0361] The cDNA is obtainable from the plasmids in DSM 11842, 11843 and 11844.

[0362] Total DNA was isolated from a yeast colony and plasmid DNA was rescued by transformation of E. coli as described above. In order to express the phytases in Aspergillus, the DNA was digested with appropriate restriction enzymes, size fractionated on gel, and a fragment corresponding to the phytase gene was purified. The gene was subsequently ligated to pHD414, digested with appropriate restriction enzymes.

[0363] After amplification of the DNA in E. coli the plasmid was transformed into Aspergillus oryzae as described in Example 1.

[0364] Test of A. Oryzae Transformants

[0365] Each of the transformants were tested for enzyme activity as described in Example 1. Some of the transformants had phytase activity which was significantly larger than the Aspergillus oryzae background. This demonstrates efficient expression of the phytases in Aspergillus oryzae.

[0366] The two phytases of Paxillus involtus CBS 100231 (PhyA1P.i. and PhyA2P.i.) and the phytase of Trametes pubescens CBS 100232 (PhyAT.p.) have the following characteristics:

4CalculatedNumber of aminomolecularIsoelectricacidsweight (MW)point (pI)PhyA1 P.i.42346K6.4PhyA2 P.i.42345K4.5PhyA T.p.42646K4.3

Example 4

Expression Cloning and Characterization of Five Phytase (phyA) cDNAs from four Basidiomycetes Agrocybe Pediades, Peniophora Lycii, Paxillus Involtus and Trametes Pubescens.

[0367] Directional CDNA libraries are constructed as described in the previous examples from phytase induced mycelia from the four basidiomycetes A. pediades, P. lycii, P. involtus and T. pubescens, in the yeast expression vector pYES2.0.

[0368] The cDNA libraries are screened for phytase activity, resulting in isolation of five different phytase cDNAs, phyA P. lycii, phyA A. pediades, phyA1 P. involtus, phyA2 P. involtus, and phyA T. pubescens.

[0369] Characterization of the phyA cDNA from these clones reveals conserved regions apparently specific to the basidiomycete phytases. This indicates that the basidiomycete phytases belong to their own subfamily within the group of fungal phytases.

[0370] The five new phytases are transformed and overexpressed in A. oryzae in order to facilitate the purification and characterization of the recombinant enzymes.

[0371] Isolation of PhyA cDNAs by Expression Cloning in Yeast.

[0372] The fungal strains A. pediades, P. lycii, P. involtus and T. pubescens are cultivated stationary on FG-4 medium (30 g/l soy meal, 15 g/l malto dextrine, 5 g/l bacto peptone, 0.2 g/l pluronic).

[0373] The accumulation of total phytase activity in the culture supernatants is monitored on a plate assay as described in the section “Test of A. oryzae transformants” of Example 1.

[0374] Highest levels of phytase activity are detected after five to fifteen days of growth, and therefore poly(A)+RNAs isolated from mycelia harvested according to this, are used to construct four cDNA libraries in the yeast expression vector pYES2.0. Aliquots of the libraries are transformed into S. cerevisiae W3124 and the transformants are plated on SC agar containing 2% glucose and incubated at 30° C.

[0375] Isolation of poly(A)+ RNA and construction of cDNA libraries is performed as described in Example 1 (the section “Fermentation procedure of Peniophora lycii CBS no. 686.96 for mRNA isolation” to the section “Transformation of Aspergillus oryzae or Aspergillus niger.”

[0376] Identification of Positive Colonies

[0377] Positive colonies are identified as described in Example 1 under the same heading.

[0378] Between 20000 and 30000 yeast clones from each library are screened for phytase activity and one to four phytase positive yeast clones are found in each library. The positive colonies correspond to five different phytase genes, phyA P. lycii (pC1phy2), phyA A. pediades (pC1phy3), phyA1 P. involtus (pC1phy5), phyA2 P. involtus (pC1phy7), and phyA T. pubescens (pC1phy6).

[0379] Characterization of the phyA cDNAs

[0380] The primary structure of the phyA cDNA encoding PhyA of Peniophora lycii is shown in FIG. 1. The 1593 bp cDNA from pC1phy2 contains a 1320 bp open reading frame (ORF), coding for a 439 residue polypeptide with a calculated molecular weight of 47560. The phyA cDNA encodes an 30 amino acid signal peptide. The mature protein has a calculated molecular weight of 44473 and an isoelectric point of pI 4.15. The cDNA and amino acid sequences are included in the sequence listing, (SEQ ID NO: 23) and (SEQ ID NO: 24), respectively.

[0381] The phyA cDNA sequence and the deduced sequence of PHYA from A. pediades are presented in FIG. 2. The 1501 bp cDNA from pC1phy3 contains a 1362 ORF coding for a 453 residue polypeptide with a 31 amino acid long signal peptide. The 422 amino acid mature protein has a calculated molecular weight of 46781 and an isoelectric point of pI 4.82. The cDNA and amino acid sequences are included in the sequence listing as (SEQ ID NO: 21) and (SEQ ID NO: 22), respectively.

[0382] The nucleotide sequences of the phyA7 and the phyA2 CDNA cloned from Paxilus involtus, and the deduced sequences of PHY1 and PHY2, are shown in FIG. 3. and FIG. 4. respectively.

[0383] The 1522 bp insert in pC1phy5 (phyA1) contains a 1329 bp ORF coding for a 442 amino acid polypeptide. According to the SignalP V1.1 prediction (Henrik Nielsen, Jacob Engelbrecht, Stren Brunak and Gunnar von Heijne: “Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites,” Protein Engineering 10, 1-6 (1997)), the signal peptide consists of 19 amino acids. The mature protein therefore has a predicted molecular weight of 45932 and a pI of 6.39. The cDNA and amino acid sequences are included in the sequence listing as (SEQ ID NO: 25) and (SEQ ID NO: 26), respectively.

[0384] The plasmid pC1phy7 (phyA2) contains a 1642 bp insert with a 1329 bp ORF coding for a 442 residue polypeptide. The SignalP V1.1 program referred to above predicts a putative signal peptidase cleavage site between Ala-19 and Ala-20 in the phyA2 encoded preprotein and thus the predicted molecular weight of the mature protein is 45466 and the predicted isoelectric point is 4.50. The cDNA and amino acid sequences are included in the sequence listing as (SEQ ID NO: 27) and (SEQ ID NO: 28), respectively.

[0385] In FIG. 5. the phyA cDNA sequence and the deduced sequence of PHYA from T. pubescens are shown. The 1536 bp insert in pC1phy6 contains a 1332 bp ORF coding for a 443 residue polypeptide. According to the SignalP V1.1 prediction referred to above, the signal peptide consists of 17 residues. The mature protein therefore consists of 426 amino acids and has a predicted molecular weight of 45905 and a pI of 4.34. The cDNA and amino acid sequences are included in the sequence listing as (SEQ ID NO: 29) and (SEQ ID NO: 30), respectively.

[0386] Conserved Basidiomycete Phytase Regions

[0387] The overall identities between known phytases of the phyllum Ascomycota and phytases of the invention of the phyllum Basidiomycota and are shown in table 1 below (“X/Y” meaning “DNA/peptide” identity as determined by GAP GCGv8).

[0388] In this table, the first five phytases in the leftmost column are basidiomycete phytases, whereas the rest are ascomycete phytases.

5TABLE 1Homology of ascomycete and basidiomycete phytases (complete cDNA compared)

[0389] In this experiment, the complete cDNA sequences were compared. According to table 1, the DNA-homology for phytases within the basidiomycetes group is in the range of from 81% to 56% identity, and within the ascomycetes group in the range of from about 65% to 55% identity. Accordingly, the internal group homology seems higher within the group of basidiomycetes phytases as compared to ascomycetes phytases.

[0390] The DNA homology of the basidiomycet phytases versus the ascomycet phytases, however, is only in the range of from about 54% to 48%. Accordingly, these two groups as such are more different from each other than the difference observed within each group (and this points towards the discrimination between ascomycete phytases and basidiomycete phytases being legitimate.

[0391] This relationship is also visualized in the alignments in FIG. 6. and FIG. 7.

[0392] For some of the phytases of Table 1, Table 2 below shows the results when comparing cDNA sequences of ORF and peptide sequences of the mature protein (signal peptide cleaved off).

6TABLE 2Homology of selected ascomycete and basidiomycete phytases(ORF cDNA and mature polypeptide compared)

[0393] In this table, peptide homologies are indicated in the upper right half of the table, whereas DNA homologies are indicated in the lower left half (both % identity according to GAP GCGv8).

[0394] From the alignments at FIGS. 6 and 7 it is apparent that several sequence motifs are conserved within the five basidiomycete phytases. Based on this alignment several conserved partial sequences have been derived (SEQ ID NOS: 1-14). Still further, some regions of deletions, which are also conserved in the basidiomycete phytases, have also been derived (see e.g claim 5).

[0395] Some examples of particularly highly conserved sequences are the so-called Consensus Sequences I, II and III below, the corresponding alignments of which are shown in Tables 3 and 4 below. In these tables, identical residues in at least nine of the sequences are indicated by a grey box and identical residues for the phytases from basidiomycetes are indicated by a white box.

[0396] Consensus Sequence I: I-Q-R-H-G-A-R-[F/W]-P-T-S-G-A-X-X-R (SEQ ID NO: 3)

[0397] Consensus Sequence II: N-W-T-[A/E]-G-F-X-X-A-S (SEQ ID NO: 5)

[0398] Consensus Sequence III: F-V-E-S-Q-X- [Y/F]-A-R-X-X-G-X-G-D-F- [E/A] -K-C (SEQ ID NO: 9)

7TABLE 3Partial alignments corresponding to consensus sequences I and IIAA pos. 68-83 in P. lyciiAA pos. 162-171 in P. lyciiphyA1 P. involutusI Q R H G A R F P T S G A T T RN W T A G F A S A - - - - - - - SphyA2 P. involutusI Q R H G A R F P T S G A A T RN W T A G F A S A - - - - - - - SphyA T. pubescensI Q R H G A R F P T S G A A K RN W T A G F A L A - - - - - - - SphyA A. pediadesI Q R H G A R F P T S G A G T RN W T E G F S A A - - - - - - - SphyA P. lyciiI Q R H G A R W P T S G A R S RN W T A G F G D A - - - - - - - SphyA A. fumigatusL S R H G A R Y P T S S K S K KK F I E G F Q Q A K L A D P G A -phyA A. nigerL S R H G A R Y P T D S K G K KK F I E G F Q S T K L K D P R A QphyA A. terreusL A R H G A R S P T H S K V K AK F V E G F Q T A R Q D D H H A NphyA T. lanuginosaL S R H G A R Y P T A H K S E VF F N R G F Q D A K D R D P R S NphyA M. thermophilaL S R H G A R A P T L K R A A SN F T Q G F H S A L L A D R G S T

[0399]

8

TABLE 4

Partial alignments corresponding to consensus

sequence III

AA pos. 415-433 in P. lycii

phyA1 P.
F V E S Q T F A R S D G A G D F E K C

involutus

phyA2 P.
F V E S Q A Y A R S G G A G D F E K C

involutus

phyA T.
F V E S Q A Y A R N D G E G D F E K C

pubescens

phyA A.
F V E S Q K Y A R E D G Q G D F E K C

pediades

phyA P. lycii
F V E S Q T Y A R E N G Q G D F A K C

phyA A.
F V K G L S W A R S G - - G N W G E C

fumigatus

phyA A. niger
F V R G L S F A R S G - - G D W A E C

phyA A.
F V A G L S F A Q A G - - G N W A D C

terreus

phyA T.
W I K G L T F A R Q G - - G H W D R C

lanuginosa

phyA N.
F I E S M A F A R G N - - G K W D L C

thermophila

[0400] Table 5 also shows some of the consensus sequences, viz. SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 9, respectively, in an alignment as in FIG. 7.

9TABLE 5Basidiomycete phytase consensus sequences in alignmentAA pos. P. lycii64 70 75 80phyA1 P. involtusQ V N I I Q R H G A R F P T S G AphyA2 P. involtusQ V N I I Q R H G A R F P T S G AphyA T. pubescensQ V H I I Q R H G A R F P P S G AphyA A. pediadesQ V N I I Q R H G A R F P T S G AphyA P. lyciiQ V N L I Q R H G A R W P T S G AphyA A. fumigatusL V Q V L S R H G A R Y P T S S KphyA A. nigerF A Q V L S R H G A R Y P T D S KphyA A. terreusF V Q V L A R H G A R S P T H S KphyA T. lanuginosaF V Q V L S R H G A R Y P T A H KphyA M. thermophilaF A Q V L S R H G A R A P T L K R162 170 171phyA1 P. involtusN W T A G F A S A - - - - - - - SphyA2 P. involtusN W T A G F A S A - - - - - - - SphyA T. pubescensN W T A G F A L A - - - - - - - SphyA A. pediadesN W T E G F S A A - - - - - - - SphyA P. lyciiN W T A G F G D A - - - - - - - SphyA A. fumigatusK F I E G F Q Q A K L A D P G A -phyA A. nigerK F I E G F Q S T K L K D P R A QphyA A. terreusK F V E G F Q T A R Q D D H H A NphyA P. lanuginosaF F N R G F Q D A K D R D P R S NphyA M. thermophilaN F T Q G F H S A L L A D R G S T415 423 431phyA1 P. involtusF V E S Q T F A R S D G A G D F E K CphyA2 P. involtusF V E S Q A Y A R S G G A G D F E K CphyA T. pubescensF V E S Q A Y A R N D G E G D F E K CphyA A. pediadesF V E S Q K Y A R E D G Q G D F E K CphyA P. lyciiF V E S Q T Y A R E N G Q G D F A K CphyA A. fumigatusF V K G L S W A R S G - - G N W G E CphyA A. nigerF V R G L S F A R S G - - G D W A E CphyA A. terreusF V A G L S F A Q A G - - G N W A D CphyA T. lanuginosaW I K G L T F A R Q C - - G H W D R CphyA M. thermophilaF I E S M A F A R G N - - G K W D L C

[0401] Consensus Sequence I (SEQ ID NO: 3), residue position 68 to 83 with the numbering for PHYA P. lycii in FIG. 7., is around the active site, and all five basidiomycetes phytases have this consensus sequence I-Q-R-H-G-A-R-[F/W]-P-T-S-G-A-X-X-R with thirteen conserved residues. Still further, four of the five phytases have a fourteenth common residue F75. This is in contrast to the ascomycetes phytases which only have eight conserved residues in the same region (Table 3).

[0402] When Consensus Sequence II (SEQ ID NO: 5), AA position 162 -171 in P. lycii, is compared to the ascomycete phytases it can be seen that the basidiomycete phytases lack six to seven residues between P. lycii F167 (A. niger F177) to P. lycii P177 (A. niger P194) (See FIG. 7) and that the basidiomycetes phytases overall have a much larger degree of conservation with seven identical residues out of ten (Table 3). The ascomycetes phytases have only three conserved residues out of seventeen in the same region.

[0403] Consensus Sequence III (SEQ ID NO: 9), AA pos. 415-433 in p. lycli, consists of nineteen residues with thirteen residues conserved in the basidiomycetes phytases. There are three residues in the consensus sequence that are conserved through all the fungal phytases. In the P. lycii sequence the residues are A422, G428, and C433 and for A. niger they are A454, G458, and C463. All the basidiomycete phytases have five residues between the conserved alanine and glycine while all the ascomycete phytases only have three (Table 4).

[0404] Expression of PHYA in Asperfillus Oryzae

[0405] In order to obtain high level expression of the PHYA phytases in Aspergillus oryzae for further purification and characterization of the protein, the five phyA cDNAs from A. pediades, P. lycii, P. involtus, and T. pubescens were subcloned into pHD414, a fungal expression vector. The phyA cDNA is here inserted 3′ to the TAKA-amylase promoter sequence and 5′ to the polyA and terminator sequence from the A. niger glucoamylase gene. The pHD414 phyA constructs were transformed into A. oryzae by co-transformation with the amdS selection plasmid (see the section “Transformation of Aspergillus oryzae or Aspergillus niger” in Example 1). The transformants were screened for phytase activity in the supernatants, and the highest yielding transformants were selected for fermentation.

[0406] Conclusion

[0407] The high degree of conserved regions within this group of basidiomycete phytases indicate that they belong to their own subfamily within the group of fungal phytases.

[0408] Based on these regions PCR-primers specific for molecular screening of related phytases can be designed (Example 5).

Example 5

Molecular screening (primerset 522/538)

[0409] The following degenerate oligonucleotide primers coding for highly conserved regions within the five basidiomycete phytases have been designed for molecular screening:

[0410] 522 sense primer:

[0411] 5′-CCC AAG CTT AAY TGG ACN GMN GGN TT-3′ (SEQ ID NO: 15) corresponds to amino acids N-W-T-[A,E,D]-G-[F,L] with a CCC and HindIII site 5′ tail;

[0412] 537 sense primer:

[0413] 5′-CCC AAG CTT GAY AAR TWY GGN AC-3′ (SEQ ID NO: 16) corresponds to amino acids D-K-[F,Y]-Y-G-T with a CCC and HindIII site 5′ tail;

[0414] 538 anti-sense primer:

[0415] 5′-GCT CTA GAC RTA RWA YTT RTC NAR RTC-3′ (SEQ ID NO: 17) corresponds to amino acids D-[F,L]-D-K-[F,Y]-Y-G with a GC and XbaI site 5′ tail;

[0416] 525 anti-sense primer:

[0417] 5′-GCT CTA GAC AYT TNK CRA ART CNC C-3′ (SEQ ID NO: 18) corresponds to amino acids G-D-F-[A,D,E]-K with a GC and XbaI site 5′ tail;

[0418] 539 sense primer: 5′-CCC AAG CTT CAR GTN MAY MTN ATH CA-3′ (SEQ ID NO: 19) corresponds to amino acids Q-V-[N,H]-[I,L,M]-I-[Q,H] with a CCC and HindIII site 5′ tail (SEQ ID NO: 15);

[0419] 540 anti-sense primer:

[0420] 5′-GCT CTA GAC RAA NCC NKC NGT CCA RTT-3′ (SEQ ID NO: 20) corresponds to amino acids N-W-T-[A,D,E]-G-F with a GC and XbaI site 5′ tail;

[0421] wherein N=A, C, G or T; R=A or G; Y=C or T; M=A or C; W=A or T.

[0422] The design of the primers is based on the alignment in FIG. 7.

[0423] For a general reference to the PCR reaction, reference can be had to e.g. Sambrook et al, Molecular Cloning, a Laboratory Manual, 2nd edition; or Lubert Stryer: Biochemistry, 4th edition, Freeman and Company, New York, 1995, e.g. pp. 132-134.

[0424] First, the 522/538 primer set is tested on genomic DNA from selected ascomycetes and basidiomycetes shown in Table 6 below.

[0425] The genomic DNA is isolated according to the following procedure:

[0426] Procedure for Isolation of Fungal Genomic DNA.

[0427] 1. Grind mycelia in liquid N2 in a morter

[0428] 2. Transfer mycelia to a 2.0 ml Eppendorf tube up to the 0.5 ml mark

[0429] 3. Add 1.0 ml lysis buffer and mix

[0430] 4. Add 10 μl 4 mg/ml DNase free RNase A (New England Biolabs)

[0431] 5. Incubate for 30 min. at 37° C.

[0432] 6. Add 40 μl 16 mg/ml Protease K (New England Biolabs)

[0433] 7. Incubate for 1 h. at 50° C. with gently shaking

[0434] 8. Centrifuge for 15 minutes full speed in a microcentrifuge

[0435] 9. Apply supernatant to a QIAprep-spin column. Spin for 1 min. and discard filtrate

[0436] 10. Wash with 0.5 ml buffer PB, spin for 1 min. and discard filtrate

[0437] 11. Wash with 0.75 ml buffer PE, spin for 1 min. and discard filtrate

[0438] 12. Drain any existing PE buffer with a quick spin and let dry completely

[0439] 13. Place spin column in a clean microfuge tube and elute by adding 125 μl H20. Let sit for 5 min. and then spin for 3 min

[0440] Lysis Buffer

[0441] 100 mM EDTA

[0442] 10 mM Tris pH. 8

[0443] 1% Triton X-100

[0444]

200
mM NaCl

[0445] 500 mM Guanidine-HCl

[0446] For further information on QIAprep spin column, PB buffer and PE buffer please refer to the QIAprep™ Plasmid Handbook from QIAGEN GmbH.

[0447] Experimental Procedure

[0448] Approximately 100 to 200 ng genomic DNA or 10-20 ng doublestranded cDNA is used as template for PCR amplification in PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl) containing 200 μM of each dNTP, 3.5 mM MgCl2, 2.5 Units AmpliTaq Gold™, and 100 pmol of each of the degenerate primers 522 and 538. The total volume is 50 μl. The PCR reaction is caried out in a Perkin-Elmer GeneAmp PCR System 2400. The PCR reaction is performed using a cycle profile of:

[0449] 94° C.-10 min; 1 cycle

[0450] 94° C.-1 min, 60° C.-1 min, 72° C.-30 sec; 2 cycles

[0451] 94° C.-1 min, 59° C.-1 min, 72° C.-30 sec; 2 cycles

[0452] 94° C.-1 min, 58° C.-1 min, 72° C.-30 sec; 2 cycles

[0453] 94° C.-1 min, 52° C.-1 min, 72° C.-30 sec; 2 cycles

[0454] 94° C.-1 min, 50° C.-1 min, 72° C.-30 sec; 14 cycles

[0455] 72° C.-7 min; 1 cycles

[0456] 5 μl aliquots of the amplification products are analyzed by electrophoresis in 1.5% agarose gels.

[0457] Table 6 below shows the results of the test of this primerset, viz. whether a specific PCR band was detected or not.

10TABLE 6Test of primerset 522/538 on genomic DNA from asco- andbasidiomycetesStraincollectionPCR bandMicroorganismPhyllumnumberdetectedCladorinum sp.AscomycotaCBS 427.97NoCytospora sp.AscomycotaCBS 424.97NoCytospora sp.AscomycotaCBS 425.97NoGelasinospora sp.AscomycotaNN 040455NoAgrocybe pediadesBasidiomycotaCBS 900.96YesAmylostereumBasidiomycotaNN strainYesChailletiicollectionBjerkandera adustaBasidiomycotaCBS 580.95YesBjerkandera sp.BasidiomycotaNN strainYescollectionBolbitius aleuritusBasidiomycotadoYesCerrena unicolorBasidiomycotaDoYesConiophora aridaBasidiomycotadoYesConocybe sp.BasidiomycotadoYesCoprinus cinereusBasidiomycotaIFO 30116YesCystoderma carchariasBasidiomycotaNN strainYescollectionDaedalea quercinaBasidiomycotaNN005877YesExidia glandulosaBasidiomycotaCBS 277.96YesFemsjonia sp.BasidiomycotaNN strainYescollectionFomes fomentariusBasidiomycotaCBS 276.96YesHygrophoropsisBasidiomycotaNN strainYespallidacollectionHyphodermaBasidiomycotadoYesargillaceumHyphodontia pallidulaBasidiomycotadoYesHypholoma fasciculareBasidiomycotadoYesIrpex lacteusBasidiomycotadoYesLaetisaria arvalisBasidiomycotadoYesLyophyllum sp.BasidiomycotadoYesMarasmiellus ramealisBasidiomycotadoYesMerismodes sp.BasidiomycotaDoYesMerulius tremellosusBasidiomycotadoYesOxyporus corticolaBasidiomycotadoYesOxyporus sp.BasidiomycotaCBS 422.97YesPanaeolus semiovatusBasidiomycotaCBS 819.95YesPaxillus involtusBasidiomycotaCBS 100231YesPeniophora cinereaBasidiomycotaNN007373YesPeniophora lyciiBasidiomycotaCBS 686.96YesPeniophora quercinaBasidiomycotaNN 009335YesPodaxis pistillarisBasidiomycotaATCC 38868YesScizophyllum communeBasidiomycotaNN strainYescollectionScizophyllum sp.BasidiomycotaCBS 443.97YesSkeletocutis sp.BasidiomycotaNN strainYescollectionSteccherinumBasidiomycotaDoYesochraceumStereum subtomentosumBasidiomycotaDoYesStrobilurusBasidiomycotaDoYestenacellusStropharia cubensisBasidiomycotaATCC 13966YesTrametes hirsutaBasidiomycotaDSM 2987YesTrametes pubescensBasidiomycotaCBS 100232YesTrametes zonatellaBasidiomycotaNN strainYescollectionTrechisporaBasidiomycotaDoYesfarinaceaeTrichaptumBasidiomycotaDoYesfuscoviolaceumTyphula setipesBasidiomycotaDoYesVolvariella speciosaBasidiomycotaDoYes

Example 6

Molecular Screening (other Primersets)

[0458] Primer sets 522/525, 539/540, 539/538, 539/525 and 537/525 are tested as described in Example 5 above, using 100 pmol of each of the sense and anti-sense degenerate primers. Touchdown PCR is used for amplification (ref: R.H.Don et al. (1991), Nucleic Acid Research, Vol. 19, No. 14) modified for the AmpliTaq Gold (TM). The PCR reaction is performed using a cycle profile of:

[0459] 94° C.-10 min; 1 cycle

[0460] 94° C.-1 min, 60° C.-1 min, 72° C.-1.5 min; 2 cycles

[0461] 94° C.-1 min, 59° C.-1 min, 72° C.-1.5 min; 2 cycles

[0462] 94° C.-1 min, 58° C.-1 min, 72° C.-1.5 min; 2 cycles

[0463] 94° C.-1 min, 52° C.-1 min, 72° C.-1.5 min; 2 cycles

[0464] 94° C.-1 min, 50° C.-1 min, 72° C.-1.5 min; 14 cycles

[0465] 72° C.-7 min; 1 cycle.

Example 7

Purification and Sequencing of PCR Bands

[0466] The PCR fragments can be purified and sequenced using the Jet sorb Gel extraction Kit (Genomed GmbH, Germany) according to the manufacturer's instructions. The nucleotide sequences of the amplified PCR fragments are determined directly on the purified PCR products using 200-300 ng as template, the Taq deoxy-terminal cycle sequencing kit (Perkin-Elmer, USA), flourescent labeled terminators and 5 pmol sequence primer on a ABI PRISMt 377 DNA Sequencer, Perkin Elmer.

[0467] The PCR fragments generated with primer set 522/538, and with approximately 10-20 ng of doublestranded cDNA from Schizophyllum sp. CBS 443.97 as template, was purified and sequenced as described above and the DNA sequence was deduced to (5′- to 3′-):

11TCTGCCGCATCTGACGGTGTCTATAACCCCGTCCTCAACCTGATTATATCAGAAGAGCTTAACGAC(SEQ ID NO:31)ACCCTCGATGATGCGATGTGCCCGAACGTCGGCGAATCGGACGCCCAAACGGACGAATGGACGTCTATTTACGCAGCGCCCATCGCTGAGCGTCTGAACAACAACGCCGTGGGCGCTAACCTGACCACCACGAACGTTTACAACCTCATOTCTTTATOCCCCTTCGACACGCTTGCGAAGGAGACGCCGAGCCCCTTCTGCGATCTCTTT

[0468] and translated into amino acid sequence:

12SAASDGVYNPVLNLIISEELNDTLDDAMCPNVGESDAQTDEWTSIYAAPIAERLNNNAVGANLTTT(SEQ ID NO:32)NVYNLMSLCPFDTLAKETPSPFCDLF.

[0469] The double stranded cDNA was synthesized as described in Example 1.

Example 8

Purification and Characterization of the Phytase from Peniophora Lycii Expressed in Aspergillus Oryzae

[0470] The Peniophora lycii phytase was expressed in and excreted from Aspergillus oryzae IFO 4177.

[0471] Filter aid was added to the culture broth which was filtered through a filtration cloth. This solution was further filtered through a Seitz depth filter plate resulting in a clear solution. The filtrate was concentrated by ultrafiltration on 3kDa cut-off polyethersulphone membranes followed by diafiltration with distilled water to reduce the conductivity. The pH of the concentrated enzyme was adjusted to pH 7.5. The conductivity of the concentrated enzyme was 1.2 mS/cm.

[0472] The phytase was applied to a Q-sepharose FF column equilibrated in 20 mM Tris/CH3COOH, pH 7.5 and the enzyme was eluted with an increasing linear NaCl gradient (0→0.5 M). The phytase activity eluted as a single peak. This peak was pooled and (NH4)2SO4 was added to 1.5 M final concentration. A Phenyl Toyopearl 650S column was equilibrated in 1.5 M (NH4)2SO4, 10 mM succinic acid/NaOH, pH 6.0 and the phytase was applied to this column and eluted with a decreasing linear (NH4)2SO4 gradient (1.5→0 M). Phytase containing fractions were pooled and the buffer was exchanged for 20 mM Tris/CH3COOH, pH 7.5 on a Sephadex G25 column. The G25 filtrate was applied to a Q-sepharose FF column equilibrated in 20 mM Tris/CH3COOH, pH 7.5. After washing the column extensively with the equilibration buffer, the phytase was eluted with an increasing linear NaCl gradient (0→0.5 M). The phytase activity was pooled and the buffer was exchanged for 20 mM Tris/CH3COOH, pH 7.5 by dialysis. The dialysed phytase was applied to a SOURCE 30Q column equilibrated in 20 mM Tris/CH3COOH, pH 7.5. After washing the column thoroughly with the equilibration buffer a phytase was eluted with an increasing linear NaCl gradient (0→0.3 M). Fractions from the SOURCE 30Q column were analyzed by SDS-PAGE and pure phytase fractions were pooled.

[0473] The Peniophora phytase migrates in the gel as a band with Mr =67 kDa. N-terminal amino acid sequencing of the 67 kDa component was carried out following SDS-PAGE and electroblotting onto a PVDF-membrane. The following N-terminal amino acid sequence could be deduced:

[0474] Leu-Pro-Ile-Pro-Ala-Gln-Asn-

[0475] The sequence corresponds to amino acid residues 31-37 in the cDNA derived amino acid sequence.

[0476] Accordingly a mature amino acid sequence of the phytase when expressed in Aspergillus is supposed to be no. 31-439 of SEQ ID NO: 24.

Example 9

Further Characterization of the Purified Phytase of Peniophora Lycii

[0477] The phytase of Peniophora lycii was expressed in Aspergillus and purified as described in Example 8.

[0478] The phytase activity is measured using the following assay: 10 μl diluted enzyme samples (diluted in 0.1 M sodium acetate, 0.01% Tween20, pH 5.5) were added into 250 μl 5 mM sodium phytate (Sigma) in 0.1 M sodium acetate, 0.01% Tween20, pH 5.5 (pH adjusted after dissolving the sodium phytate; the substrate was preheated) and incubated for 30 minutes at 37° C. The reaction was stopped by adding 250 μl 10% TCA and free phosphate was measured by adding 500 μl 7.3 g FeSO4 in 100 ml molybdate reagent (2.5 g (NH4)6Mo7024.4H20 in 8 ml H2SO4 diluted to 250 ml). The absorbance at 750 nm was measured on 200 μl samples in 96 well microtiter plates. Substrate and enzyme blanks were included. A phosphate standard curve was also included (0-2 mM phosphate). 1 FYT equals the amount of enzyme that releases 1 μmol phosphate/min at the given conditions.

[0479] Temperature profiles were obtained by running the assay at various temperatures (preheating the substrate).

[0480] Temperature stability was investigated by preincubating the phytases in 0.1 M sodium phosphate, pH 5.5 at various temperatures before measuring the residual activity.

[0481] The pH-stability was measured by incubating the enzyme at pH 3 (25 mM glycine-HCl), pH 4-5 (25 mM sodium acetate), pH 6 (25 mM MES), pH 7-9 (25 mM Tris-HCl) for 1 hour at 40° C., before measuring the residual activity.

[0482] The pH-profiles were obtained by running the assay at the various pH using the same buffer-systems (50 mM, pH was re-adjusted when dissolving the substrate).

[0483] The results of the above pH-profile, pH-stability, temperature-profile and temperature stability studies are shown in FIGS. 8, 9, 10 and 11, respectively. From FIG. 9 it appears that the phytase of Peniophora lycii is very stable (i.e. more than 80% of the maximum activity retained) for 1 hour at 40° C. in the whole range of pH 3-9. And as regards the temperature stability results shown at FIG. 11, it appears that at 60-80° C. some 50-60% of the residual activity still remains. This fact is contemplated to be due to the enzyme being surprisingly capable of refolding following its thermal denaturation. The degree of refolding will depend on the exact conditions (pH, enzyme concentration).

[0484]
FIG. 12 shows the result of differential scanning calorimetry (DSC) measurements on the Peniophora phytase. In DSC the heat consumed to keep a constant temperature increase in the sample-cell is measured relative to a reference cell. A constant heating rate is kept (e.g. 90° C./hour). An endothermal process (heat consuming process-e.g. the unfolding of an enzyme/protein) is observed as an increase in the heat transferred to the cell in order to keep the constant temperature increase. DSC was performed using the MC2-apparatus from MicroCal. Cells were equilibrated 20 minutes at 20° C. before scanning to 90° C. at a scan rate of 90°/h.

[0485] Samples of around 2.5 mg/ml Peniophora phytase in 0.1 M sodium acetate, pH 5.5 were loaded.

Example 10

Determination of the Specific Activity of the Peniophora Phytase

[0486] The specific activity is determined on a highly purified sample of the phytase (the purity was checked beforehand on an SDS poly acryl amide gel showing the presence of only one component).

[0487] The protein concentration in the phytase sample was determined by amino acid analysis as follows: An aliquot of the phytase sample was hydrolyzed in 6 N HCl, 0.1% phenol for 16 h at 110° C. in an evacuated glass tube. The resulting amino acids were quantified using an Applied Biosystems 420A amino acid analysis system operated according to the manufacturers 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.

[0488] The activity is determined in the units of FYT. One FYT equals the amount of enzyme that liberates 1 micromol inorganic phosphate from phytate (5 mM phytate) per minute at pH 5.5, 37° C.; assay described e.g. in example 11.

[0489] The specific activity is calculated to 987 FYT/mg enzyme protein.

Example 11

Time-resolved product-profiling of phytase-catalyzed hydrolysis of phytic acid by 1H NMR spectroscopy

[0490] The hydrolysis of phytic acid (PA) catalyzed by the Peni ophora phytase and by a commercial Aspergillus niger phytase (Phytase Novo®) was investigated (27 mM phytate, 1 FYT/ml, pH 5.5 and 3.5, and 27° C.) by 1H NMR profiling the product mixture in the course of 24 hours.

[0491] In the following (Ins(p,q,r, . . . )Pn denotes myo-inositol carrying in total n phosphate groups attached to positions p, q, r, . . . For convenience Ins(1,2,3,4,5,6)P6 (phytic acid) is abbreviated PA. Please refer, however, to the section “Nomenclature and position specificity of phytases” in the general part of this application.

[0492] The technique provide specific information about initial points of attack by the enzyme on the PA molecule, as well as information about the identity of the end product. On the other side the evolving patterns of peaks reflecting the composition of the intermediate product mixtures, provide a qualitative measure, a finger print, suitable for identification of similarities and differences between individual enzymes.

[0493] NMR, like most other analytical methods, can distinguish between stereo-isomers which are not mirror images (diastereomers), but not between a set of isomers, which are mirror-images (enantiomers), since they exhibit identical NMR spectra.

[0494] Thus, Ins(1,2,4,5,6)P5 (3-phosphate removed) exhibits a NMR spectrum different from Ins(1,2,3,4,5)P5 (6-phosphate removed) because the isomers are diastereomers.

[0495] However, the NMR spectra of Ins(1,2,4,5,6) P5 and Ins(2,3,4,5,6)P5 (1-phosphate removed) are identical because the isomers are enantiomers. The same holds for the pair Ins(1,2,3,4,5)P5 and Ins(1,2,3,5,6)P5 (4-phosphate removed).

[0496] Thus, by NMR it is not possible to distinguish between a 3- and a 1-phytase, and it is not possible to distinguish between a 6- and a 4-phytase (or a L-6- and a D-6-phytase using the lowest-locant rule).

[0497] Biased by the description of 3- and 6-phytases in the literature, we have used the terms 3- and 6-phytases for our enzymes, but, though unlikely, we do not actually know if we have a 1- and a 4-phytase instead.

[0498] Experimental

[0499] NMR spectra were recorded at 300 K (27° C.) on a Bruker DRX400 instrument equipped with a 5 mm selective inverse probe head. 16 scans preceded by 4 dummy scans were accumulated using a sweep width of 2003 Hz (5 ppm) covered by 8 K data points. Attenuation of the residual HOD resonance was achieved by a 3 seconds presaturation period. The spectra were referenced to the HOD signal (δ 4.70).

[0500] PA samples for NMR analysis were prepared as follows: PA (100 mg, Phytic acid dipotassium salt, Sigma P-5681) was dissolved in deionized water (4.0 ml) and pH adjusted to 5.5 or 3.5 by addition of aqueous NaOH (4 N). Deionized water was added (ad 5 ml) and 1 ml portions, each corresponding to 20 mg of phytic acid, were transferred to screw-cap vials and the solvent evaporated (vacuum centrifuge). The dry samples were dissolved in deuterium oxide (2 ml, Merck 99.5% D) and again evaporated to dryness (stored at −18° C. until use).

[0501] For NMR analysis one 20 mg phytic acid sample was dissolved in deuterium oxide (1.0 ml, Merck 99.95% D). The solution was transferred to a NMR tube and the 1H NMR spectrum recorded. Enzyme solution (1 FTU, dissolved in/diluted, as appropriate, with deuterium oxide) was added followed by thorough mixing (1 minute). 1H NMR spectra were recorded immediately after addition of enzyme (t=0), then after 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105, 120, 135 150, 165, 180, 195, 210 minutes (=3.5 hours), 4.5, 5.5 6.5, 7.5, 8.5, 9.5, 11.5, 13.5, 15.5, 17.5, 19.5, 21.5, and 23.5 hours. The pH in the NMR tube was measured. Additional spectra were acquired after 48 and 120 hours (5 days), where a portion of substrate (PA, 6 mg) was added to probe if the enzyme retained its catalytic activity.

[0502] By means of 2D NMR analysis of inositol phosphate mixtures obtained by partial digestion of PA, in conjunction with published NMR data (Scholz, P.; Bergmann, G., and Mayr, G. W.: Methods in Inositide Research (Ed. Irvine, R. F.), pp. 65-82, Raven Press, Ltd., New York (1990)), characteristic 1H NMR signals attributable to Ins(1,2,3,4,5,6)P6 (PA), Ins(1,2,4,5,6)P5, Ins(1,2,3,4,5)P5, Ins(1,2,5,6) P4, Ins(1,2,6) P3, Ins(1,2)P2, and Ins(2)P, were identified and permitted relative quantification of these species during the course of the reaction.

[0503] Stacked plots of product profiles for the Aspergillus phytase and the Peniophora phytase covering 24 hours of reaction time at pH 5.5 is presented in FIG. 13 and FIG. 14, respectively.

[0504] The signal at δ 3.25(t) represents H-5 in Ins(1,2)P2 whereas the signal at δ 3.18(t) represents H-5 in Ins(2)P. Ins(1,2)P2 starts accumulating after about 4 hours of reaction time with the Aspergillus phytase and after about 1 hours of reaction time with the Peniophora phytase. Ins(2)P is observed after about 10 hours of reaction with the Aspergillus phytase and after about 3 hours of reaction with the Peniophora phytase. After 24 hours of reaction the amount or level of Ins(1,2)P2 is very low for both phytases, whereas the amount of Ins(2)P is maximum for both phytases after 24 hours.

[0505] Accordingly, the profiles observed after 24 hours of reaction time demonstrate that both phytases degrade PA to Ins(2)P.

[0506] For both enzymes the reaction mixture at 24 h comprised in addition to Ins(2)P minor amounts of Ins(1,2)P2. Prolonged reaction times (several days) resulted in disappearance of the residual Ins(1,2)P2, but the fully dephosphorylated species, inositol (Ins), was not observed at all. The observation is not explained by irreversible inhibition/denaturation of the enzyme, since the enzymes retained their catalytic activities for prolonged periods, as demonstrated by their ability to digest fresh portions of PA added to the NMR tubes after keeping them 5 days at room temperature.

[0507] Turning now to FIGS. 15 and 16, these depict in more detail the profiles evolving at pH 5.5 during the initial 4.5 hours. It is inferred from FIG. 10 that H-3 in Ins(1,2,4,5,6)P5 (designated A) shows a signal at δ 3.66(dd), H-6 in Ins(1,2,3,4,5)P5 (B) a signal at δ 3.87(t) and H-3 in Ins(1,2,5,6)P4 (C) a signal at 6 3.56(dd). Now, compound A corresponds to phosphate in position 3 having been hydrolyzed, B position 6 and C position 3 and 4.

[0508] It is apparent from FIG. 15 that compound A appears as the major primary product (t=5 min) using the Aspergillus phytase, whereas compound B does not appear. Compound C appears after 20-25 minutes.

[0509] From FIG. 16 (the Peniophora phytase) one infers that compound B appears as the major primary product (t=5 min) using the Peniophora phytase.

[0510] The signals at δ 4.82(dt, H-2), 4.38 (q, H-4/H-6), 4.13(q, H-5) and 4.11(dt,H1/H3) are attributable to the substrate, phytic acid, PA. Comparing FIGS. 15 and 16 it is apparent, that these peaks diminish faster with the Peniophora phytase than with the Aspergillus phytase.

[0511] These differences are highlighted in FIG. 17, which present the profiles observed after 20 min at pH 5.5 with the above indicated diagnostic signals (A,B,C) labelled.

[0512]
FIG. 18 shows the final result (under these conditions) of the hydrolysis of phytic acid at pH 5.5 (i.e. corresponding to the upper line of FIGS. 13 and 14). All signals labelled at the upper Peniophora embodiment represent the compound Ins(2)P, viz. the protons thereof, from the right to the left: H-5, H1 and H3, H4 and H6 and finally H-2. Relative intensity: 1:2:2:1. The corresponding signals are found in the bottom embodiment of Aspergillus. This means that the end product is in both embodiments Ins(2)P. However, a minor amount of Ins(1,2)P2 is also detected in both embodiments, the corresponding peaks being indicated at the Aspergillus embodiment only.

[0513] Marked differences are observed:

[0514] Aspergillus: The initial major product was identified as Ins(1,2,4,5,6)P5 (A) followed by appearance of Ins(1,2,5,6)P4(C), and Ins(1,2,6)P3 (D) (H-3 at δ 3.49(dd) after 1½ hours) corresponding to consecutive removal of the phosphate groups in the 3-, 4- and 5-positions. The concentration of Ins(1,2)P2 (E) builds up slowly starting at 4 hours and decreases very steeply between 12 and 14 hours with a concomitant rapid increase of the Ins(2)P (F) level. This is visualized in FIG. 11 representing the time dependent concentration of Ins(1,2)P2 and Ins(2)P, respectively, determined by measuring the area under the signals corresponding to H-5 in Ins(1,2)P2 (δ 3.25(t)) and Ins(2)P (δ 3.18 (t)), respectively, relative to the area under the signals corresponding to the substrates (t=0).

[0515] Peniophora: At pH 5.5 only the 6-position is initially attacked. A characteristic feature is that PA is digested at a faster rate compared to the Aspergillus phytase. Additional characteristic features are that the end product, Ins(2)P (F) appears very early (3 hours) and builds up slowly, in contrast to the very steep increase in the Ins (2) P-level towards the end of the reaction observed for the Aspergillus phytase.

[0516]
FIG. 19 is a plot similar to FIG. 17, but at pH 3.5. Surprisingly, at this pH the Peniophora phytase turns up to have high initial affinity to the 6- as well as the 3-position of PA (B as well as A are observed), probably with a slight preference for the 6-position.

[0517] The data generated permit i.a. the following conclusions:

[0518] At pH 5.5 as well as 3.5 the Aspergillus phytase attacks with a high degree of selectivity PA in the 3-position, whereas the Peniophora phytase at pH 5.5 with a high degree of selectivity attacks PA in the 6-position, at pH 3.5 however it seems to hydrolyze the phosphate groups at the 3- and 6-positions at comparable rates.

[0519] At pH 5.5, the Peniophora phytase digests PA at a faster rate compared to the Aspergillus phytase.

[0520] The end-product is, at pH 3.5 as well as 5.5, under the conditions applied, Ins(2)P (F).

[0521] The overall reaction rates (PA→Ins(2)P) were comparable, approximately 20 hours (FIG. 20; pH 5.5).

[0522] Accordingly, the Aspergillus phytase prove to be an essentially clean 3-phytase, whereas the Peniophora phytase at pH 5.5 appear to be an essentially clean 6-phytase and at pH 3.5 a phytase of a hitherto unknown type, viz a 3+6-phytase.

[0523] The exact configuration of myo-inositol tetrakisphosphate produced by partial hydrolysis af phytic acid with the Peniophora phytase could be determined as outlined below, also allowing us to conclude whether the Peniophora phytase is a D-, L- or a D/L-6-phytase.

[0524] Other ways of determining the exact specificity is by determining optical rotation or using a chiral HPLC column.

[0525] HPLC-isolation of myo-inositol tetrakisphosphate produced by partial degradation of phytic acid with the Peniophora phytase. Desalting (ion exchange, dialysis, (2), (4) and (9) and references herein).

[0526] NMR analysis to check purity (i), determine whether several diastereomer tetrakisphosphates are produced (ii), and determine which of these are produced (iii)

[0527] Synthesis of relevant polyols using reduction by boronhydrid (BH) of the corresponding carbonhydrates (10)

[0528] Disintegration using periodate, reduction by boronhydrid and dephosphorylation following (2). Identification of polyol using HPLC

[0529] Oxidation of polyol using L-iditol dehydrogenase and final identification of carbonhydrid using HPLC.

[0530] References

[0531] (2) Van der Kaay et al, Biochem. J., 312 (1995), 907-910

[0532] (4) Irving et al, J. Bacteriology, 112 (1972), 434-438

[0533] (9) Stevens, L. R. in “Methods in Inositide Research”(Irvine, R. F. Ed.), 9-30 (1990), Raven Press, Ltd., New York.

[0534] (10) Stephens, L. et al, Biochem. J., 249 (1988), 271-282

Example 12

Comparative assay, Aspergillus and Peniophora phytase Release of inorganic phosphate from corn

[0535] The present example gives a simple assay for the phytase catalyzed liberation of phosphorous from corn at pH 3.5 and 5.5. Two parameters have been focused on—velocity and level of P-liberation.

[0536] Materials and Methods

[0537] Corn was obtained from North Carolina State University (sample No. R27), and ground at a mill (Bühler Universal) at point 6.8.

[0538] A corn-suspension (16.7% w/w) was prepared by weighing 20 g of ground corn into a 250 ml blue cap bottle and adding 100 ml of buffer.

[0539] The following buffer was used:

[0540] pH 5.5: 0.22 M acetate-buffer

[0541] The pH value of 3.5 was adjusted by 8N HCl/NaOH.

[0542] Enzymes tested: Two phytases was tested: A commercial phytase of Aspergillus niger (Phytase Novo®) and a Peniophora phytase of the invention, purified as described in example 2.

[0543] Dosage: All enzymes were applied at 25 FYT/20 g corn (correspond to 1250 FYT/kg).

[0544] The bottles with the corn suspension were closed by caps, and immediately placed in a water bath at 37° C. and subjected to constant stirring. pH was measured at this stage and again after 24 hours. After 30 min of stirring a sample of 5 ml was collected.

[0545] Then the phytase enzymes were added at a dosage of 25 FYT/20 g of corn.

[0546] Samples were then collected 5, 10, 15, 20, 25, 30, 40, 50, 60 and 120 min after the addition of the phytases, and the content of released P determined as follows:

[0547] Phytase containing samples were diluted 1+4 in buffer. Then the samples were centrifuged at 3000 rpm for 5 min, and 1.0 ml of the supernatant was collected. 2.0 ml buffer and 2.0 ml MoV stop solution (cfr. the FYT assay of Example 6) was added. The samples were placed in a refrigerator at 3-5° C. until all samples could be measured at the spectrophotometer at 415 nm.

[0548] pH was measured at time 0 and 20 hours.

[0549] For the determinations a phosphate standard or stock solution of 50 mM was used prepared. 0.5, 1.0, 1.5 and 2.0 ml stock solution is diluted to a total volume of 50 ml using buffer. 3.0 ml of each solution is added 2.0 ml MoV stop solution.

[0550] Two experiments were conducted: at pH 5.5 and at pH 3.5. The analysis results are shown at FIGS. 21 and 22 (pH 5.5 and 3.5, respectively). At these figures, symbol “u” represents the control experiment, “s” the Peniophora phytase and “g” the Aspergillus phytase.

[0551] Results and discussion:

[0552]
FIG. 21 (pH 5.5) shows, that at this pH the Peniophora phytase liberates P from corn at significantly improved rate as compared to the Aspergillus phytase.

[0553] From FIG. 22 (pH 3.5) it is clearly apparent that at this pH the Peniophora phytase is much faster in the liberation of phosphorous from ground corn as compared to the Aspergillus phytase (0-120 minutes).

[0554] The passage time of the digestive system of for instance chickens/broilers is normally is of the order of magnitude of 30 minutes to 2 hours, so the observed difference is for sure important, whatever the pH. Nevertheless the pH value of 3.5 is more relevant in this respect than the pH 5.5 value.

[0555] This implies that the Peniophora enzyme is surprisingly more efficient than the known Aspergillus phytase as a P-liberator in the digestive system of e.g. broilers.

Example 13

Fermentation, Purification and Characterization of the Phytase of Agrocybe Pediades Expressed in Yeast

[0556] A seed culture is prepared by incubation of the yeast strain in 100 ml medium A at 250 rpm over night at 30° C. 100 ml medium B is inoculated with 2 ml seed culture and the strains incubate for 7 to 12 days at 30° C. 250 rpm.

[0557]

Agrocybe pediades
phytase was expressed in yeast as described in Example 2. The yeast clone comprises a cloned sequence encoding a phytase of the invention having the amino acid sequence shown in SEQ ID NO: 22.

[0558] Filter aid was added to the culture supernatant which was filtered through a filtration cloth. This solution was further filtered through a Seitz depth filter plate resulting in a clear solution. The filtrate was concentrated by ultrafiltration on 3kDa cut-off polyethersulphone membranes and refiltered on a germ filter plate. The pH of the filtrate was adjusted to pH 7.5 and the conductivity was adjusted to 2 mS/cm by dilution with distilled water.

[0559] The phytase was applied to a Q-sepharose FF column equilibrated with 20 mM Tris/CH3COOH, pH 7.5 and the enzyme was eluted with an increasing linear NaCl gradient (0→0.5 M). The phytase containing fractions from the Q-sepharose column were pooled and (NH4)2SO4 was added to 1.3 M final concentration. A Phenyl Toyopearl 650S column was equilibrated with 1.3 M (NH4)2SO4, 10 mM succinic acid/NaOH, pH 6.0 and the phytase was applied to this column and eluted with a decreasing linear (NH4)2SO4 gradient (1.3→0 M). Phytase containing fractions were pooled and buffer was exchanged with 20 mM Tris/CH3COOH, pH 7.5 on a Sephadex G25 column. Phytase was further purified on a SOURCE Q column equilibrated with 20 mM Tris/CH3COOH, pH 7.5, and eluted with a linear NaCl gradient (0→0.5 M). Finally, phytase containing fractions from the SOURCE Q column were pooled, concentrated on a 10 kDa cut-off regenerated cellulose membrane, and applied to a Superdex 200 column equilibrated in 25 mM CH3COOH/NaOH, 100 mM NaCl, pH 5.0.

[0560] Fractions from the Superdex 200 column were analyzed by SDS-PAGE. The phytase migrates in the gel as a very broad and diffuse band with approx. Mr=150 kDa indicating that the enzyme was highly glycosylated.

[0561] N-terminal amino acid sequencing of the 150 kDa component was carried out following SDS-PAGE and elctroblotting onto a PVDF membrane.

[0562] Two N-terminal sequences could be deduced in the relative amounts of approximately 4:1 (upper sequence:lower sequence):

[0563] Val-Gln-Pro-Phe-Phe-Pro-Pro-Gln-Ile-Gln-Asp-Ser-Trp-Ala-Ala-Tyr-Thr-Pro-Tyr-Tyr-Pro-Val-Gln- and

[0564] Thr-Phe-Val-Gln-Pro-Phe-Phe-Pro-Pro-Gln-Ile-Gln-Asp-Ser-Trp-Ala-Ala-Tyr-Thr-Pro-Tyr-Tyr-Pro-

[0565] The two N-terminal amino acids “Val” and “Thr” are found in position 27 and 25, respectively, in SEQ ID NO: 22. This indicates that the mature phytase enzyme of the invention, when expressed in yeast, starts at position 27 or 25 in SEQ ID NO: 22.

[0566] Accordingly the mature amino acid sequence of the phytase when expressed in yeast is supposed to be no. 27-453 or 25-453 of SEQ ID NO: 22.

EXAMPLE 14

Purification and characterization of the phytase from Agrocybe pediades expressed in Aspergillus oryzae

[0567] The Agrocybe pediades phytase was expressed in and excreted from Aspergillus oryzae IFO 4177.

[0568] Filter aid was added to the culture broth which was filtered through a filtration cloth. This solution was further filtered through a Seitz depth filter plate resulting in a clear solution. The filtrate was concentrated by ultrafiltration on 3kDa cut-off polyethersulphone membranes followed by diafiltration with distilled water to reduce the conductivity. The pH of the concentrated enzyme was adjusted to pH 7.5.

[0569] The phytase was applied to a Q-sepharose FF column equilibrated in 20 mM Tris/CH3COOH, pH 7.5 and the enzyme was eluted with an increasing linear NaCl gradient (0→0.5 M). The phytase activity eluted as a single peak. This peak was pooled and (NH4)2SO4 was added to 1.3 M final concentration. A Phenyl Toyopearl 650S column was equilibrated in 1.3 M (NH4)2SO4, 10 mM succinic acid/NaOH, pH 6.0 and the phytase was applied to this column and eluted with a decreasing linear (NH4)2SO4 gradient (1.3→0 M). Phytase containing fractions were pooled and the buffer was exchanged for 20 mM Tris/CH3COOH, pH 7.5 by dialysis. The phytase was applied to a SOURCE 30Q column equilibrated in 20 mM Tris/CH3COOH, pH 7.5 and the enzyme was eluted with an increasing linear NaCl gradient (0→0.25 M). The phytase activity was pooled and (NH4) 2SO4 was added to 1.6 M final concentration. A SOURCE Phenyl column was equilibrated in 1.6 M (NH4)2SO4, 25 mM succinic acid/NaOH, pH 6.0 and the phytase was applied to this column and eluted with a decreasing linear (NH4)2SO4 gradient (1.6→0 M). Fractions from the SOURCE Phenyl column were analyzed by SDS-PAGE and pure phytase fractions were pooled. The phytase pool was dialysed against 20 mM Tris/CH3COOH, pH 7.5, applied to a HighTrap Q column equilibrated in the same buffer, and stepeluted with 20 mM Tris/CH3COOH, 0.5 M NaCl, pH 7.5.

[0570] The Agrocybe phytase migrates on SDS-PAGE as a band with Mr=60 kDa.

[0571] N-terminal amino acid sequencing of the 60 kDa component was carried out following SDS-PAGE and electroblotting onto a PVDF-membrane. Two N-terminal amino acid sequences could be deduced in relative amounts of approximately 2:1 (upper sequence:lower sequence).

[0572] Phe-Pro-Pro-Gln-Ile-Gln-Asp-Ser-Trp-Ala-Ala-Tyr-Thr-Pro-Tyr-Tyr-Pro-Val -Gln- and Gln-Pro-Phe-Phe-Pro-Pro-Gln-Ile-Gln-Asp-Ser-Trp-Ala-Ala-Tyr-Thr-Pro-Tyr-Tyr-

[0573] The upper sequence corresponds to amino acid residues 31-49 in the cDNA derived amino acid sequence while the lower sequence corresponds to amino acid residues 28-46.

[0574] Accordingly the mature amino acid sequence of the phytase when expressed in Aspergillus is supposed to be no. 31-453 or 28-453 of SEQ ID NO: 22.

[0575] Accordingly, summing up the results of example 13 and the present example, in SEQ ID NO: 21, the following sequences are phytase encoding sub-sequences: position 79 to 1362, 73-1362, 91-1362 or 82-1362 (i.e. corresponding to amino acid positions 27-453, 25-453, 31-453 or 28-453, respectively, in SEQ ID NO: 22).

[0576] Accordingly, there is a slight variability in the N-terminal sequence of the mature phytase enzyme. This variability is observed as well when the enzyme is expressed in a single strain, as when expressed in different strains. In yeast, the mature phytase enzyme starts at amino acid no. 27 or 25 (relative abundance about 80%:20%, respectively); in Aspergillus the mature phytase enzyme starts at amino acid no. 31 or 28 (relative abundance: about 65%:35%, respectively).

Example 15

Characterization of the purified phytase of Agrocybe pediades

[0577] The phytase of Agrocybe pediades was expressed in Aspergillus and purified as described in Example 14.

[0578] The phytase activity is measured using the following assay: 10 μl diluted enzyme samples (diluted in 0.1 M sodium acetate, 0.01% Tween20, pH 5.5) were added into 250 μl 5 mM sodium phytate (Sigma) in 0.1 M sodium acetate, 0.01 % Tween20, pH 5.5 (pH adjusted after dissolving the sodium phytate; the substrate was preheated) and incubated for 30 minutes at 37° C. The reaction was stopped by adding 250 μl 10 % TCA and free phosphate was measured by adding 500 μl 7.3 g FeSO4 in 100 ml molybdate reagent (2.5 g (NH4)6Mo7024.4H20 in 8 ml H2SO4 diluted to 250 ml). The absorbance at 750 nm was measured on 200 μl samples in 96 well microtiter plates. Substrate and enzyme blanks were included. A phosphate standard curve was also included (0-2 mM phosphate). 1 FYT equals the amount of enzyme that releases 1 μmol phosphate/min at the given conditions.

[0579] Temperature profiles were obtained by running the assay at various temperatures (preheating the substrate).

[0580] Temperature stability was investigated by preincubating the phytases in 0.1 M sodium phosphate, pH 5.5 at various temperatures before measuring the residual activity.

[0581] The pH-stability was measured by incubating the enzyme at pH 3 (25 mM glycine-HCl), pH 4-5 (25 mM sodium acetate), pH 6 (25 mM MES), pH 7-9 (25 mM Tris-HCl) for 1 hour at 40° C., before measuring the residual activity.

[0582] The pH-profiles were obtained by running the assay at the various pH using the same buffer-systems (50 mM, pH was re-adjusted when dissolving the substrate).

[0583] The results of the above pH-profile, pH-stability, temperature-profile and temperature stability studies are shown in FIGS. 23, 24, 25 and 26, respectively.

[0584] From FIG. 23 it appears that the phytase of Agrocybe pediades has a reasonable activity at pH 3-6 (i.e. more than 50% of the maximum activity). At pH 4-6 more than 70% of the maximum activity is found, at pH 5-6 more than 90%. Optimum pH seems to be in the area of pH 5.5-6.

[0585] It is apparent from FIG. 24 that the phytase of Agrocybe pediades is very stable (i.e. more than 80% of the maximum activity retained) for 1 hour at 40° C. in the whole range of pH 3-9.

[0586] As regards the temperature profile, it is apparent from FIG. 25, that the Agrocybe pediades phytase has a reasonable activity at temperatures of 35-55° C. (i.e. more than 60% of the maximum activity), whereas at temperatures of 40-52° C. the activity is more than 70% of the maximum activity, and the optimum temperature is close to 50° C.

[0587] And finally, as regards the temperature stability results shown at FIG. 26, the phytase is very stable at temperatures of 0 to about 55° C. (i.e. more than 60% residual activity). A sharp decline in residual activity is seen after preincubation at 60° C. Anyhow, at 60° C. at least 20%, preferably 25% and more preferably 30% of the residual activity still remains. Also at pre-incubation temperature above 60° C., e.g. at 70° C. and 80° C., a surprisingly high residual activity remains, viz. more than 20%, preferably more than 30%, especially more than 40% remains.

[0588] This fact is contemplated to be due to the enzyme being surprisingly capable of refolding following its thermal denaturation. The degree of refolding will depend on the exact conditions (pH, enzyme concentration).

[0589]
FIG. 27 shows the result of differential scanning calorimetry (DSC) measurements on the Agrocybe phytase.

[0590] In DSC the heat consumed to keep a constant temperature increase in the sample-cell is measured relative to a reference cell. A constant heating rate is kept (e.g. 90° C./hour). An endothermal process (heat consuming process—e.g. the unfolding of an enzyme/protein) is observed as an increase in the heat transferred to the cell in order to keep the constant temperature increase.

[0591] DSC was performed using the MC2-apparatus from MicroCal. Cells were equilibrated 20 minutes at 20° C. before scanning to 90° C. at a scan rate of 90°/h. Samples of around 2.5 mg/ml Agrocybe phytase in 0.1 M sodium acetate, pH 5.5 were loaded.

[0592] The temperature stability studies were confirmed by DSC, since from FIG. 5 it is apparent that the Agrocybe phytase has a denaturation or “melting” temperature of about 58° C. at pH 5.5. The re-scan of the Agrocybe phytase shows a minor peak at 58° C., and this is also indicative of the fact that a fraction of the enzyme is actually refolded folding the thermal inactivation in the first scan.

Example 16

Time-resolved Product-profiling of Phytase-catalyzed Hydrolysis of Phytic Acid by 1H NMR Spectroscopy

[0593] The hydrolysis of phytic acid (PA) catalyzed by the Agrocybe phytase and by a commercial Aspergillus niger phytase (Phytase Novo®) was investigated (27 mM phytate, 1 FYT/ml, pH 5.5, and 27° C.) by 1H NMR profiling the product mixture in the course of 24 hours.

[0594] In the following (Ins(p,q,r, . . . )Pn denotes myo-inositol carrying in total n phosphate groups attached to positions p, q, r, . . . For convenience Ins(1,2,3,4,5,6)P6 (phytic acid) is abbreviated PA. Please refer, however, to the section “Nomenclature and position specificity of phytases” in the general part of this application.

[0595] The technique provides specific information about initial points of attack by the enzyme on the PA molecule, as well as information about the identity of the end product. On the other side the evolving patterns of peaks reflecting the composition of the intermediate product mixtures, provide a qualitative measure, a finger print, suitable for identification of similarities and differences between individual enzymes.

[0596] NMR, like most other analytical methods, can distinguish between stereo-isomers which are not mirror images (diastereomers), but not between a set of isomers, which are mirror-images (enantiomers), since they exhibit identical NMR spectra.

[0597] Thus, Ins(1,2,4,5,6)P5 (3-phosphate removed) exhibits a NMR spectrum different from Ins(1,2,3,4,5)P5 (6-phosphate removed) because the isomers are diastereomers.

[0598] However, the NMR spectra of Ins(1,2,4,5,6)P5 and Ins(2,3,4,5,6)P5 (1-phosphate removed) are identical because the isomers are enantiomers. The same holds for the pair Ins(1,2,3,4,5)P5 and Ins(1,2,3,5,6)P5 (4-phosphate removed).

[0599] Thus, by NMR it is not possible to distinguish between a 3- and a 1-phytase, and it is not possible to distinguish between a 6- and a 4-phytase (or a L-6- and a D-6-phytase using the lowest-locant rule).

[0600] Biased by the description of 3- and 6-phytases in the literature, we have used the terms 3- and 6-phytases for our enzymes, but, though unlikely, we do not actually know if we have a 1- and a 4-phytase instead.

[0601] Experimental

[0602] NMR spectra were recorded at 300 K (27° C.) on a Bruker DRX400 instrument equipped with a 5 mm selective inverse probe head. 16 scans preceded by 4 dummy scans were accumulated using a sweep width of 2003 Hz (5 ppm) covered by 8 K data points. Attenuation of the residual HOD resonance was achieved by a 3 seconds presaturation period. The spectra were referenced to the HOD signal (δ 4.70).

[0603] PA samples for NMR analysis were prepared as follows: PA (100 mg, Phytic acid dipotassium salt, Sigma P-5681) was dissolved in deionized water (4.0 ml) and pH adjusted to 5.5 by addition of aqueous NaOH (4 N). Deionized water was added (ad 5 ml) and 1 ml portions, each corresponding to 20 mg of phytic acid, were transferred to screw-cap vials and the solvent evaporated (vacuum centrifuge). The dry samples were dissolved in deuterium oxide (2 ml, Merck 99.5% D) and again evaporated to dryness (stored at −18° C. until use).

[0604] For NMR analysis one 20 mg phytic acid sample was dissolved in deuterium oxide (1.0 ml, Merck 99.95% D). The solution was transferred to a NMR tube and the 1H NMR spectrum recorded. Enzyme solution (1 FTU, dissolved in/diluted, as appropriate, with deuterium oxide) was added followed by thorough mixing (1 minute). 1H NMR spectra were recorded immediately after addition of enzyme (t=0), then after 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105, 120, 135 150, 165, 180, 195, 210 minutes (=3.5 hours), 4.5, 5.5 6.5, 7.5, 8.5, 9.5, 11.5, 13.5, 15.5, 17.5, 19.5, 21.5, and 23.5 hours. The pH in the NMR tube was measured. Additional spectra were acquired after 48 and 120 hours (5 days), where a portion of substrate (PA, 6 mg) was added to probe if the enzyme retained its catalytic activity.

[0605] By means of 2D NMR analysis of inositol phosphate mixtures obtained by partial digestion of PA, in conjunction with published NMR data (Scholz, P.; Bergmann, G., and Mayr, G. W.: Methods in Inositide Research (Ed. Irvine, R. F.), pp. 65-82, Raven Press, Ltd., New York (1990)), characteristic 1H NMR signals attributable to Ins(1,2,3,4,5,6)P6 (PA), Ins(1,2,4,5,6)P5, Ins(1,2,3,4,5)P5, Ins(1,2,5,6) P4, Ins(1,2,6)P3, Ins(1,2)P2, and Ins(2)P, were identified and permitted relative quantification of these species during the course of the reaction.

[0606] Stacked plots of product profiles for the Aspergillus phytase and the Agrocybe phytase covering 24 hours of reaction time is presented in FIG. 28 and FIG. 29, respectively.

[0607] The signal at δ 3.25(t) represents H-5 in Ins(1,2)P2 whereas the signal at δ 3.18(t) represents H-5 in Ins(2)P. Ins(1,2)P2 starts accumulating after about 4 hours of reaction time with the Aspergillus phytase and after about 2 hours of reaction time with the Agrocybe phytase. Ins(2)P is observed after about 10 hours of reaction with the Aspergillus phytase and after about 5 hours of reaction with the Agrocybe phytase. After 24 hours of reaction the amount or level of Ins(1,2)P2 is very low for both phytases, whereas the amount of Ins(2)P is maximum for both phytases after 24 hours.

[0608] Accordingly, the profiles observed after 24 hours of reaction time demonstrate that both phytases degrade PA to Ins(2)P. The fully dephosphorylated species, inositol (Ins), was not observed at all.

[0609] For both enzymes the reaction mixture at 24 h comprised in addition to Ins(2)P minor amounts of Ins(1,2)P2. Prolonged reaction times (several days) resulted in disappearance of the residual Ins(1,2)P2, but the fully dephosphorylated species, inositol (Ins), was not observed at all. The observation is not explained by irreversible inhibition/denaturation of the enzyme, since the enzymes retained their catalytic activities for prolonged periods, as demonstrated by their ability to digest fresh portions of PA added to the NMR tubes after keeping them 5 days at room temperature.

[0610] Turning now to FIGS. 30 and 31, these depict in more detail the profiles evolving during the initial 4.5 hours. It is inferred from FIG. 32 that H-3 in Ins(1,2,4,5,6)P5 (designated A) shows a signal at δ 3.66(dd), H-6 in Ins(1,2,3,4,5)P5 (B) a signal at 6 3.87(t) and H-3 in Ins(1,2,5,6)P4 (C) a signal at 6 3.56(dd). Now, compound A corresponds to phosphate in position 3 having been hydrolyzed, B position 6 and C position 3 and 4.

[0611] It is apparent from FIG. 30 that compound A appears as the major primary product (t=5 min) using the Aspergillus phytase, whereas compound B does not appear. Compound C appears after 20-25 minutes.

[0612] From FIG. 31 (the Agrocybe phytase) one infers that compound A as well as compound B appear very early, i.e. within the first 15 minutes, probably more of the compound B than A.

[0613] The signals at δ 4.82(dt, H-2), 4.38 (q, H-4/H-6), 4.13(q, H-5) and 4.11(dt,H1/H3) are attributable to the substrate, phytic acid, PA. Comparing FIGS. 30 and 31 it is apparent, that these peaks diminish much faster (i.e. within an hour) with the Agrocybe phytase than with the Aspergillus phytase.

[0614] These differences are highlighted in FIG. 32, which present the profiles observed after 20 min with the above indicated diagnostic signals (A,B,C) labelled.

[0615]
FIG. 33 shows the final result (under these conditions) of the hydrolysis of phytic acid (i.e. corresponding to the upper line of FIGS. 28 and 29). All signals labelled at the upper Agrocybe embodiment represent the compound Ins(2)P, viz. the protons thereof, from the right to the left: H-5, H1 and H3, H4 and H6 and finally H-2. Relative intensity: 1:2:2:1. The corresponding signals are found in the bottom embodiment of Aspergillus. This means that the end product is in both embodiments Ins(2)P. However, a minor amount of Ins(1,2)P2 is also detected in both embodiments, the corresponding peaks being indicated at the Aspergillus embodiment only.

[0616] Marked differences are observed:

[0617] Aspergillus: The initial major product was identified as Ins(1,2,4,5,6)P5 (A) followed by appearance of Ins(1,2,5,6)P4(C), and Ins(1,2,6)P3 (D) (H-3 at δ 3.49(dd) after 1½ hours) corresponding to consecutive removal of the phosphate groups in the 3-, 4- and 5-positions. The concentration of Ins(1,2)P2 (E) builds up slowly starting at 2 hours and decreases very steeply between 12 and 14 hours with a concomitant rapid increase of the Ins(2)P (F) level. This is visualized in FIG. 34 and 35, representing the time dependent concentration of Ins(1,2)P2 and Ins(2)P, respectively, constructed from slices along the time-dimension in FIG. 28-29 at the chemical shift values (δ) of H-5 in Ins(1,2)P2 and Ins(2)P, respectively (note that the time scale is only linear in segments).

[0618] Agrocybe: Both the 3- and 6-positions are initially attacked, with some preference for the 6-position. A characteristic feature is that PA is digested at a faster rate compared to the Aspergillus phytase. Additional characteristic features are that the end product, Ins(2)P (F) appear very early (5 hours) and builds up slowly, in contrast to the very steep increase in the Ins(2)P-level towards the end of the reaction observed for the Aspergillus phytase.

[0619] The data generated permit i.a. the following conclusions:

[0620] The Aspergillus phytase attacks with a high degree of selectivity PA in the 3-position, whereas the Agrocybe phytase appear less specific.

[0621] The Agrocybe phytase digests PA at a faster rate compared to the Aspergillus phytase.

[0622] The end-product is in both cases, under the conditions applied, Ins (2)P (F).

[0623] The overall reaction rates (PA→Ins(2)P) were comparable, approximately 20 hours (FIG. 35).

[0624] Accordingly, the Aspergillus phytase prove to be an essentially clean 3-phytase, whereas the Agrocybe phytase appear to be less specific, however, with some preference for the 6-position.

[0625] By application of 2D-homo- and heteronuclear (1H, 13C) correlation techniques, the latter circumventing problems with severely overlapping 1H-resonances by taking advantage of the larger chemical shift dispersion of the 13C-nuclei, in combination with suitable computer software, it would in principle be possible to identify and quantify intermediates present at any given time and thereby completely map out the reaction sequence. In other words, curves like those shown in FIG. 34 and 35 representing concentration as a function of time, could in theory be constructed for other intermediate inositol phosphates.

Example 17

Comparative Assay, Aspergillus and Agrocybe Phytase Release of Inorganic Phosphate From Corn

[0626] The present example gives a simple assay for the phytase 2by catalyzed liberation of phosphorous from corn at pH 3.5 and 5.5. Two parameters have been focused on—velocity and level of P-liberation

[0627] Materials and methods

[0628] Corn was obtained from North Carolina State University (sample No. R27), and ground at a mill (Bühler Universal) at point 6.8.

[0629] A corn-suspension (16.7% w/w) was prepared by weighing 20 g of ground corn into a 250 ml blue cap bottle and adding 100 ml of buffer.

[0630] The following buffers were used:

[0631] pH 5.5: 0.22 M acetate-buffer

[0632] pH 3.5: 0.05 M citrate-buffer.

[0633] Enzymes tested: Two phytases was tested: A commercial phytase of Aspergillus niger (Phytase Novo®) and an Agrocybe phytase of the invention, purified as described in example 3 and 4.

[0634] Dosage: All enzymes were applied at 25 FYT/20 g corn (correspond to 1250 FYT/kg).

[0635] The bottles with the corn suspension were closed by caps, and immediately placed in a water bath at 37° C. and subjected to constant stirring. pH was measured at this stage and again after 24 hours. After 30 min of stirring a sample of 5 ml was collected.

[0636] Then the phytase enzymes were added at a dosage of 25 FYT/20 g of corn.

[0637] Samples were then collected 10, 20, 30, 40, 50, 60, 120 min, and approx. 20 hours after the addition of the phytases, and the content of release P determined as follows:

[0638] Phytase containing samples were diluted 1+4 in buffer. Then the samples were centrifuged at 3000 rpm for 5 min, and 1.0 ml of the supernatant was collected. 2.0 ml buffer and 2.0 ml MoV stop solution (cfr. the FYT assay of Example 15) was added. The samples were placed in a refrigerator at 3-5° C. until all samples could be measured at the spectrophotometer at 415 nm.

[0639] pH was measured at time 0 and 20 hours.

[0640] For the determinations a phosphate standard or stock solution of 50 mM was used prepared. 0.5, 1.0, 1.5 and 2.0 ml stock solution is diluted to a total volume of 50 ml using buffer. 3.0 ml of each solution is added 2.0 ml MoV stop solution.

[0641] Two experiments were conducted: at pH 5.5 and at pH 3.5. The analysis results are shown at FIGS. 36 and 37 (pH 5.5 and 3.5, respectively). At these figures, symbol “u” represents the control experiment, “s” the Agrocybe phytase and “g” the Aspergillus phytase.

[0642] Results and Discussion

[0643]
FIGS. 36 and 37 clearly show that the Agrocybe phytase initially liberates phosphorous from ground corn faster than the Aspergillus phytase at pH 5.5 and at pH 3.5.

[0644] However, after 40 min. at pH 5.5 (FIG. 36) and after 120 min. at pH 3.5 (FIG. 37), the Aspergillus phytase is approximately at the same level of released phosphate as is the Agrocybe phytase.

[0645] But considering the passage time of the digestive system of for instance chickens/broilers, which normally is of the order of magnitude of 30 minutes to 2 hours, this difference is for sure important. Besides, it should be mentioned, that the pH value of 3.5 is more relevant in this respect than the pH 5.5 value.

[0646] This implies that the Agrocybe enzyme is surprisingly more efficient than the known Aspergillus phytase as a P-liberator in the digestive system of e.g. broilers.

Example 18

Purification and Characterization of the Phytases From Paxillus Involtus and Trametes Pubescens

[0647] The PhyA1 Phytase from Paxillus Involtus

[0648] The Paxillus involtus PhyA1 phytase was expressed in and excreted from Aspergillus oryzae IFO 4177 as described in Examples 3 and 1.

[0649] Filter aid was added to the culture broth which was filtered through a filtration cloth. This solution was further filtered through a Seitz depth filter plate resulting in a clear solution. The filtrate was concentrated by ultrafiltration on 3kDa cut-off polyethersulphone membranes and the phytase was transferred to 10 mM succinic acid/NaOH, pH 6.0 on a Sephadex G25 column. The pH of the G25 filtrate was adjusted to pH 7.0.

[0650] The phytase was applied to a Q-sepharose FF column equilibrated in 20 mM HEPES/NaOH, pH 7.0. The enzyme turned out to be in the run-through from the column. (NH4)2SO4 was added to the run-through to 2.0 M final concentration. A Butyl Toyopearl 650S column was equilibrated in 2.0 M (NH4)2SO4, 10 mM CH3COOH/NaOH, pH 5.5 and the phytase was applied to this column and eluted with a decreasing linear (NH4)2SO4 gradient (2.0→0 M). Phytase containing fractions were pooled and the buffer was exchanged for 20 mM HEPES/NaOH, pH 7.5 on a Sephadex G25 column. The G25 filtrate was applied to a Q-sepharose FF column equilibrated in 20 mM HEPES/NaOH, pH 7.5. After washing the column extensively with the equilibration buffer, the phytase was eluted with an increasing linear NaCl gradient (0→0.5 M). The phytase activity was pooled and the buffer was exchanged for 20 mM Tris/CH3COOH, pH 8.0 on a Sephadex G25 column. The G25 filtrate was applied to a SOURCE 30Q column equilibrated in 20 mM Tris/CH3COOH, pH 8.0. After washing the column thoroughly with the equilibration buffer en phytase was eluted with an increasing linear NaCl gradient (0→0.3 M). Phytase containing fractions were pooled and (NH4)2SO4 was added to 2.0 M final concentration. A Phenyl Toyopearl 650S column was equilibrated in 2.0 M (NH4)2SO4, 10 mM CH3COOH/NaOH, pH 5.5 and the phytase was applied to this column and eluted with a decreasing linear (NH4)2SO4 gradient (2.0→0 M). Phytase containing fractions were pooled and reapplied to the same Phenyl Toyopearl column after adding (NH4)2SO4 to 2.0 M final concentration. Fractions from the second Phenyl Toyopearl column were analyzed by SDS-PAGE and pure phytase fractions were pooled.

[0651] The Paxillus involtus PhyA1 phytase migrates on SDS-PAGE as a band with Mr=65 kDa. N-terminal amino acid sequencing of the 65 kDa component was carried out following SDS-PAGE and electroblotting onto a PVDF-membrane. The following N-terminal amino acid sequence could be deduced. Ser-Val-Pro-Lys-Asn-Thr-Ala-Pro-Thr-Phe-Pro-Ile-Pro

[0652] The sequence corresponds to amino acid residues 21-33 in the cDNA derived amino acid sequence.

[0653] Accordingly a mature amino acid sequence of the phytase when expressed in Aspergillus is supposed to be no. 21-442 of SEQ ID NO: 26. Accordingly, the predicted signal peptide at FIG. 3 does not correspond with the actual signalpeptide when this phytase is expressed in Aspergillus. This is also so for the indications regarding mature peptide of the sequence listing under the headings SEQ ID NOS: 25 and 26.

[0654] The PhyA2 phytase from Paxillus involtus

[0655] The Paxillus involtus PhyA2 phytase was expressed in and excreted from Aspergillus oryzae IFO 4177 as described in Examples 3 and 1.

[0656] Filter aid was added to the culture broth which was filtered through a filtration cloth. This solution was further filtered through a Seitz depth filter plate resulting in a clear solution. The filtrate was concentrated by ultrafiltration on 3 kDa cut-off polyethersulphone membranes and (NH4)2SO4 was added to 2.0 M final concentration.

[0657] The phytase was applied to a Phenyl sepharose FF column equilibrated in 2.0 M (NH4)2SO4, 20 mM CH3COOH/NaOH, pH 5.5 and the enzyme was eluted with a decreasing linear (NH4)2SO4gradient (2.0→0 M). The phytase activity eluted as a single peak. This peak was pooled and (NH4)2SO4was added to 2.0 M final concentration. A Butyl Toyopearl 650S column was equilibrated in 2.0 M (NH4)2SO4, 10 mM CH3COOH/NaOH, pH 5.5 and the phytase was applied to this column and eluted with a decreasing linear (NH4)2SO4gradient (2.0 0 0 M). Phytase containing fractions were pooled and the buffer was exchanged for 20 mM HEPES/NaOH, pH 7.0 on a Sephadex G25 column. The G25 filtrate was applied to a Q-sepharose FF column equilibrated in 20 mM HEPES/NaOH, pH 7.0. After washing the column extensively with the equilibration buffer, the phytase was eluted with an increasing linear NaCl gradient (0 0.5 M). The phytase activity was pooled and the buffer was exchanged for 20 mM HEPES/NaOH, pH 7.0 by dialysis. The dialysed phytase was applied to a SOURCE 30Q column equilibrated in 20 mM HEPES/NaOH, pH 7.0. After washing the column thoroughly with the equilibration buffer en phytase was eluted with an increasing linear NaCl gradient (0→0.3 M). Fractions from the SOURCE 30Q column were analyzed by SDS-PAGE and pure phytase fractions were pooled.

[0658] The Paxillus involtus PhyA2 phytase migrates on SDS-PAGE as a band with Mr =52 kDa. N-terminal amino acid sequencing of the 52 kDa component was carried out following SDS-PAGE and electroblotting onto a PVDF-membrane. The following N-terminal amino acid sequence could be deduced. Asn-Ile-Ala-Pro-Lys-Phe-

[0659] The sequence corresponds to amino acid residues 25-30 in the cDNA derived amino acid sequence.

[0660] Accordingly a mature amino acid sequence of the phytase when expressed in Aspergillus is supposed to be no. 25-442 of SEQ ID NO: 28. Accordingly, the predicted signal peptide at FIG. 4 does not correspond with the actual signalpeptide when this phytase is expressed in Aspergillus. This is also so for the indications regarding mature peptide of SEQ ID NOS: 27 and 28.

[0661] The Phytase from Trametes Pubescens

[0662] The Trametes pubescens phytase was expressed in and excreted from Aspergillus oryzae IFO 4177.

[0663] Filter aid was added to the culture broth which was filtered through a filtration cloth. This solution was further filtered through a Seitz depth filter plate resulting in a clear solution. The filtrate was concentrated by ultrafiltration on 10 kDa cut-off polyethersulphone membranes followed by diafiltration with distilled water to reduce the conductivity. The pH of the concentrated enzyme was adjusted to pH 6.0 and the conductivity was adjusted to that of 10 mM succinic acid/NaOH, pH 6.0 by dilution with deionised water.

[0664] The phytase was applied to a Q-sepharose FF column equilibrated in 10 mM succinic acid/NaOH, pH 6.0 and the enzyme was eluted with an increasing linear NaCl gradient (0→0.5 M). The phytase activity eluted as a single peak. This peak was pooled and (NH4)2SO4was added to 2.0 M final concentration. A Butyl Toyopearl 650S column was equilibrated in 2.0 M (NH4)2SO4, 10 mM CH3COOH/NaOH, pH 5.5 and the phytase was applied to this column and eluted with a decreasing linear (NH4)2SO4gradient (2.0→0 M). Phytase containing fractions were pooled and the buffer was exchanged for 10 mM succinic acid/NaOH, pH 6.5 on a Sephadex G25 column. The G25 filtrate was applied to a Q-sepharose FF column equilibrated in 10 mM succinic acid/NaOH, pH 6.5. After washing the column extensively with the equilibration buffer, the phytase was eluted with an increasing linear NaCl gradient (0→0.3 M). The phytase activity was pooled and the buffer was exchanged for 10 mM succinic acid/NaOH, pH 7.0 on a Sephadex G25 column. The G25 filtrate was applied to a SOURCE 30Q column equilibrated in 10 mM succinic acid/NaOH, pH 7.0. After washing the column thoroughly with the equilibration buffer phytase was eluted with an increasing linear NaCl gradient (0→0.2 M). Fractions from the SOURCE 30Q column were analyzed by SDS-PAGE and pure phytase fractions were pooled.

[0665] The Trametes pubescens phytase migrates on SDS-PAGE as a band with Mr=57 kDa. N-terminal amino acid sequencing of the 57 kDa component was carried out following SDS-PAGE and electroblotting onto a PVDF-membrane. The following N-terminal amino acid sequence could be deduced.

[0666] Xxx-Ala-Cys-Leu-Asp-Val-Thr-Arg-Asp-(Ala/Val)-Gln-

[0667] The sequence corresponds to amino acid residues 31-41 in the cDNA derived amino acid sequence.

[0668] Accordingly a mature amino acid sequence of the phytase when expressed in Aspergillus is supposed to be no. 31-443 of SEQ ID NO: 30. Accordingly, the predicted signal peptide at FIG. 5 does not correspond with the actual signalpeptide when this phytase is expressed in Aspergillus. This is also so for the indications regarding mature peptide of the sequence listing under the headings SEQ ID NOS: 29 and 30.

[0669] The molecular weights (kDa) of the three phytases of Trametes pubescens and PhyA2 and PhyA1 of Paxillus involtus are 65, 55 and 65 kDa, respectively.

[0670] The pH profiles of these three phytases are similar to FIGS. 8 and 23 for the Peniophora and the Agrocybe phytases (optimum pH around 5-6; very little activity at pH above 7). As compared to the known phytase of Aspergillus ficuum (in this test having a temperature optimum of around 50° C.) PhyA1 of Paxillus involtus has a slightly higher temperature optimum around 60° C., while PhyA2 has a temperature optimum of around 40° C. The termostability of the PhyA1 phytase of Paxillus involtus is comparable to that of the Aspergillus ficuum phytase, and better than that of the PhyA2 phytase of Paxillus involtus and the phytase of Trametes pubescens. Following 60 minutes incubation at 70° C. and 80° C., respectively, the residual activity of PhyA1 is around 60% and 40%, respectively.

[0671] In DSC, Td's of around 48° C., 59° C. and 55° C. are found for the phytases PhyA2 and PhyA1 of Paxillus involtus and for the phytase of Trametes pubescens, respectively.

[0672] The specific activities are surprisingly high for all these phytases, viz. 1450, 1370 and 810 FYT/mg enzyme protein for the phytases of Trametes pubescens, Paxillus involtus PhyA2 and PhyA1, respectively (A280 of 3.58; 5.72 and 3.08; FYT/ml of 5184, 7808 and 2497; assumed extinction coefficient of 1 l/(g×cm), respectively).

Number	Date	Country
1480/96	Dec 1996	DK
1481/96	Dec 1996	DK
0301/97	Mar 1997	DK
0529/97	May 1997	DK
1388/97	Dec 1997	DK

Number	Date	Country
60046081	May 1997	US
60046082	May 1997	US
60067304	Dec 1997	US

	Number	Date	Country
Parent	09482558	Jan 2000	US
Child	09999214	Nov 2001	US
Parent	08993359	Dec 1997	US
Child	09482558	Jan 2000	US

Phytase polypeptides

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