Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
  • C12Y301/03026—4-Phytase (3.1.3.26), i.e. 6-phytase
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/189—Enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
  • A23L29/06—Enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
  • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
  • A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

• TITLE Phytase polypeptides
• 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.
• the invention relates to phytases derived from the phyllum Basidiomycota, phytases of certain consensus sequences and fungal ⁇ -phytases.
• Phytic acid or myo-inositol 1, 2, 3, 4, 5, ⁇ -hexakis dihydrogen phosphate 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.
• the phosphate moieties of phytic acid chelates divalent and trivalent cations such as metal ions, i.a. the nutritionally essential ions of calcium, iron, zinc and magnesium as well as the trace minerals mangane, copper and molybdenum.
• the phytic acid also to a certain extent binds proteins by electrostatic interaction.
• pi the isoelectric point, of the protein, the positively charged protein binds directly with phytate.
• the negatively charged protein binds via metal ions to phytate.
• Phytic acid and its salts, phytates are often not metabolized, since they are not absorbable from the gastro intestinal system, i.e. neither the phosphorous thereof, nor the chelated metal ions, nor the bound proteins are nutritionally available.
• phosphorus is an essential element for the growth of all organisms
• food and feed preparations need to be supplemented with inorganic phosphate.
• the nutritionally essential ions such as iron and calcium, must be supplemented.
• 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.
• 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.
• Phytic acid or phytates are degradable by phytases.
• 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.
• 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.
• 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.
• the endogenous phytase might contribute to a greater extent to the degradation of phytate.
• phytases 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.
• phytases have been described which are derived from Bacillus subtllis (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. coll has been purified and characterized by Greiner et al, Arch. Biochem. Biophys., 303, 107-113, 1993). However, this enzyme is probably an acid phosphatase.
• Phytase producing yeasts are also described, such as Saccharomyces cerevlslae (Nayini et al, 1984, Lebensml ttel Wlssenschaft und Technologle 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 Schwannlomyces occldentalls .
• 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.
• 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.
• phytate myo-inositol hexakisphosphate
• IP6 I, IPl, 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, IPl and I.
• myo-inositol carrying in total n phosphate groups attached to positions p, q, r, .. is denoted Ins (p, q, r, .. ) P n .
• Ins (1, 2, 3, , 5, 6) P 6 (phytic acid) is abbreviated PA.
• Inositolphosphate, phosphate esters of .myo- inositol are generally designated ID- (or 1L-) -Ins (r, s, t ,u, w,x) P n , 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 ID or 1L is used so as to make a substituent have the lowest possible locant or number ( "lowest-locant rule").
• lL-tnyo- inositol - 1 - phosphate (lL-Ins(l)P, an intermediary product in the biosynthesis of inositol) and lD-_uyo-inositol -1 -phosphate (ID- Ins (1)P, a component of phospholipids), are numbered as it is apparent from Fig. 38.
• phytases are divided according to their specificity in the initial hydrolysis step, viz. according to which phosphate-ester group is hydrolyzed first.
• plant phytases are generally said to be 6-phytases.
• the lilly pollen phytase is said to be a 5 -phytase.
• the microorganism derived phytases are mainly said to be 3 -phytases.
• 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)).
• 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.
• 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) P 5 and L- Ins (1, 2, 3, 4, 5) P 5 , are enantiomers (mirror images), and therefore have identical NMR spectres.
• 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.
• the present inventors have surprisingly found a whole subfamily of fungal phytases of interesting properties.
• This subfamily 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.
• phytase consensus sequences of the present invention are common to all basidiomycete phytases.
• 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") .
• Ascomycetes phyllum Ascomycota
• NASH Data Base (Entrez)
• 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 .
• the invention relates to an isolated polypeptide having phytase activity and being derived from the phyllum Basidiomycota.
• the invention in a second aspect, relates to an isolated polypeptide having phytase activity and comprising at least one of several consensus sequences.
• 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.
• the invention relates to an isolated polypeptide having 6-phytase activity and being derived from a fungus .
• the invention relates to isolated polypeptides having phytase activity and being homologous to five specific sequences.
• the invention provides cloned DNA sequences encoding the above polypeptides, as well as vectors and host cells comprising these cloned DNA sequences.
• 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.
• 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.
• 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 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.
• polypeptide or enzyme exhibiting phytase activity is intended to cover any enzyme capable of effecting the liberation of inorganic phosphate or phosphorous from various myo-inositol phosphates.
• myo-inositol phosphates are phytic acid and any salt thereof, e.g. sodium phytate or potassium phytate or mixed salts.
• any stereoisomer of the mono-, di-, tri-, tetra- or penta-phosphates of myo-inositol might serve as a phytase substrate.
• the phytase activity can be determined using any assay in which one of these substrates is used.
• the phytase activity is determined in the unit of FYT, one FYT being the amount of enzyme that liberates 1 ⁇ mol inorganic ortho-phosphate per min. under the following conditions: pH 5.5; temperature 37 °C; substrate: sodium phytate (C 6 H 6 0 24 P 6 Na 12 ) in a concentration of 0.0050 mol/1.
• 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, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (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.
• 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) .
• more than 50% of the hydrolysis product of the first step is Ins (1, 2, 3, 4 , 5) P 5 and/or Ins (1, 2, 3, 5, 6) P 5 .
• 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.
• 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 .
• 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.
• 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.
• the signal sequence guides the secretion of the polypeptide and the pro-peptide guides the folding of the polypeptide.
• 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.
• any fragment of such sequence encoding a polypeptide fragment, which still retains some phytase activity, is to be included in this definition.
• 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 5 incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated.
• the nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
• the 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
• 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
• the filter is then washed twice for 30 minutes in 2 x 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).
• Molecules to which the oligonucleotide probe hybridizes under 5 these conditions are detected using an x-ray film.
• 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 15 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) .
• Tm(hetero) 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 .
• Tm(homo) melting temperature between two fully complementary DNA strands (homoduplex formation)
• Tm denotes the molar cation concentration in wash buffer, 35 “%GC” % Guanine (G) and Cytosine (C) of total number of bases in the DNA sequence, form” % formamid in the wash buffer, and X" the length of the DNA sequence.
• 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).
• 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 ) .
• vector is intended to include such terms/objects as "nucleic acid constructs,” “DNA constructs,” expression vectors” or “recombinant vectors.”
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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, NY) .
• 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. 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 .
• 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.
• 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.
• 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.
• the host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote.
• 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.
• 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, Alnsworth and Blsby' 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 ) .
• 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 .
• 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.
• the transgenic plant can be dicotyledonous or monocotyledonous, for short a dicot or a monocot.
• 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) .
• 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.
• 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.
• the plant or plant part e.g. the seeds
• the plant or plant part e.g. the seeds
• the use e.g. intake, thereof, with a view to adapting the speed of the enzymatic degradation to the actual use.
• the plant produced enzyme can also be recovered from the plant.
• the recovery from the plant is to be preferred with a view to securing a heat stable formulation in a potential subsequent pelleting process.
• plant parts are stem, callus, leaves, root, fruits, seeds, tubers etc. But also any plant tissue is included in this definition.
• 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) .
• the transformants are screened using any method known to the skilled man, following which they are regenerated into whole plants.
• 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).
• 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.
• 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) .
• 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- 378 ) .
• 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.
• Preferred host cells are a strain of. Fusarlum, Trlchoderma or Asperglllus, in particular a strain of Fusarlum gramlnearum, Fusarlum venena tum, Fusarlum cerealls , Fusarlum sp . having the identifying characteristic of Fusarlum ATCC 20334, as further described in PCT/US/95/07743, Trlchoderma harzlanum or Trlchoderma reesel , Asperglllus nlger or Asperglllus oryzae .
• fig. 1 is 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) ;
• fig. 2 is the nucleotide sequence of a phytase from Agrocybe pediades, as in fig. 1;
• fig. 3 is the nucleotide sequence of a first phytase, PHYA1, from Paxillus involtus, as in fig. 1, except for the box referring to the SignalP VI .1 prediction of the signal peptide;
• fig . 4 is the nucleotide sequence of a second phytase, PHYA2, from Paxillus involtus, as in fig. 3;
• fig. 5 is the nucleotide sequence of a phytase from Trametes pubescens, as in fig. 3;
• fig. 6 is an alignment of the deduced amino acid sequences of the encoded phytases of figs. 1-5, identical residues in at least three of the sequences being indicated by a grey box;
• fig. 7 is 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;
• fig. 8 is a pH-activity curve of the Peniophora phytase
• fig. 12 a Differential Scanning Calorimetry (DSC) curve thereof;
• NMR spectra NMR spectra, stacked plots (up to 24h) , showing the product profiling of an Aspergillus niger and the Peniophora phytase, respectively;
• fig. 23 is a pH-activity curve of the Agrocybe phytase
• fig. 27 a Differential Scanning Calorimetry (DSC) curve thereof;
• 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 Escherlchia coll which were deposited according to the
• the phytases of the invention derived from basidiomycetes are preferably derived from the classes Gasteromycetes, Hymenomycetes, Urediniomycetes, Ustilaginomycetes or from unclassified Basidiomycota , more preferably from the class Hymenomycetes .
• the phytases derived from the class Hymenomycetes are preferably derived from strains of the orders Agaricales, Aphyllophorales, Auriculariales, Boletales, Cantharellales, Ceratobasidiales, Dacrymyce tales , Echinodontiaceae, Hericiales, Stereales, Thelephorales , Tremellales, Tulasnellales or from the class of mitosporic Hymenomycetes .
• Preferred families of the order Aphyllophorales are Polyporaceae (e.g. genus Trametes, Bj erkandera , Irpex, Oxyporus, Trichaptum, Daedalea, Fomes) , Coniophoraceae (e.g. genus Coniophora) , Corticiaceae (e.g. genus Hyphoderma, Trechispora, Steccherinum, Merulius, Penlophora) ,
• Polyporaceae e.g. genus Trametes, Bj erkandera , Irpex, Oxyporus, Trichaptum, Daedalea, Fomes
• Coniophoraceae e.g. genus Coniophora
• Corticiaceae e.g. genus Hyphoderma, Trechispora, Steccherinum, Merulius, Penlophora
• Schizophyllaceae e.g. genus Schizophyllum
• Bolbi tiaceae e.g. genus Agrocybe, Conocybe, Bolbi tius
• Coprinaceae e.g. genus Coprinus, Panaeolus
• Pluteaceae e.g. genus
• Volvariella Volvariella
• Podaxaceae e.g. genus Podaxis
• Tricholomataceae e.g. genus Marasmiellus, Strobilurus, Lyophyllum, Cystoderma, Merismodes
• Strophariaceae e.g. genus Stropharia, Hypholoma
• a preferred family of the order Auriculariales is Exidiaceae (e.g. genus Exidia) .
• a preferred family of the order Dacrymetales is Dacrymycetaceae (e.g. genus Ferns jonia) .
• Hyphoderma taceae e.g. genus Hyphodontia
• Stereaceae e.g. genus Amylostereum and Stereum
• a preferred family of the order Boletales is Paxillaceae (e.g. genus Paxillus and Hygrophoropsis) .
• a preferred family of the order Thelophorales is Thelephoraceae (e.g. genus Typhula ) .
• Stropharia cubensis (in particular ATCC 13966) , Agrocybe pediades (in particular CBS 900.96), Bjerkandera adusta (in particular CBS 580.95), Trametes zonatella , Trametes pubescens
• Penlophora quercina Hyphoderma argillaceum, Scizophyllum sp .
• 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.
• 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 2 2 . Likewise, if there are "N" such multiple choices in a given formula, the number of sequences covered by this formula is 2 N .
• 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) .
• 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.
• 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) .
• 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 Penlophora lycii , Agrocybe pedlades, Paxillus involtus and Trametes pubescens.
• 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 X SSC and a temperature of 65°C
• 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.
• 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.
• a target sequence e.g. a nucleotide sequence
• genomic DNA or cDNA can be e.g. genomic DNA or cDNA.
• 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.
• the PCR reaction is performed with the wild-type gene of any PE variant thereof.
• at least one PCR band is obtained using at least one primer set on the wild type gene .
• 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.
• 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.”
• the phytase is a D6-phytase.
• the phytase is a L6-phytase.
• 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.
• 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.
• 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 5 "Detailed description of the invention.”
• the (3+6) -phytase has a slight preference for one of these positions, in particular the 6- position.
• the phytases of claims 11, 14, 17, 19 and 21 are all more than 50% homologous to the isolated phytases of Agrocybe pedlades, Peniophora lycii , Paxillus involtus (phyAl and phyA2) and Trametes pubescens, respectively, corresponding to SEQ ID Nos:
• 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.
• all amino acid homologies of the present application are at least 55%, or at least 60%, or at least 65%, especially at least 70%.
• the degree of 30 homology is at least 80%, more preferably at least 90%, still more preferably at least 95%, especially at least 97%.
• Claim 12 relates to certain fragments of the amino acid sequence of SEQ ID NO 22 derived from Agrocybe, these 35 fragments, however, still exhibiting phytase activity.
• SEQ ID NO 22 amino acid sequence of SEQ ID NO 22 derived from Agrocybe
• Claim 12 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) .
• claim 15 relates to a fragment of the amino acid sequence of SEQ ID NO 24 derived from Penlophora , this fragment, however, still exhibiting phytase activity.
• the Peniophora phytase when expressed in Aspergillus, 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.
• Claims 13, 16, 18, 20 and 22 are drawn to phytases homologous to the isolated phytases of Agrocybe pedlades, Peniophora lycii , Paxillus invol tus (phyAl and phyA2) and Trametes pubescens, respectively.
• phytases are here defined as being encoded by a phytase encoding part of i) the DNA sequences listed in the sequence listing as SEQ
• 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, NY).
• Claims 23-26 relate to nucleotide sequences encoding the phytases of the invention, in particular to DNA molecules.
• 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 .
• the DNA molecule of claim 25 is defined by encoding a phytase, and being selected from: (a) the specific nucleotide sequences of the Agrocybe, Peniophora, Paxillus and Trametes phytases (phyAl and phyA2 of Paxillus) , e.g. DNA as shown in the sequence listings having the SEQ ID nos indicated (or their complementary strands) ; (b) the same sequences as under (a) but expressed via the deposited plasmid clones;
• hybridization For a definition of "hybridization,” please refer to the section headed “General definitions,” which also lists some preferred hybridization conditions.
• 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) .
• 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%.
• 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.
• 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 X 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) .
• a target nucleotide sequence e.g. DNA, for instance genomic DNA or cDNA (or mRNA) ; wild-type gene of any PE variant thereof.
• DNA sequences of the invention can also be cloned by any general method involving
• 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) .
• a method of identifying further phytase producing cells, in particular microorganisms, is disclosed in claim 31.
• 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.
• 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.
• the amplified PCR fragment derived from the template is specific.
• Suitable verification procedures are: a) running an electrophoresis in agarose gels revealing the existence of a PCR band corresponding to the amplified PCR fragment; and, if desired, also b) controlling that the size of the amplified PCR-fragment is as expected; and, if desired, also 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.
• 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.
• 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.
• 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.
• Ad c) Preferably the degree of homology is more than 50, 60, 70, 75, 80, 85, 90, 95% homology (determined as described above) .
• 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.
• Claim 34 relates to a process for preparing the phytase polypeptide, said process logically following from this screening method of claim 31.
• Steps a)+d) of claim 34 relate to the preparation of the phytase from the wild-type cell, in particular microorganism strain.
• 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).
• 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) .
• DNA molecules may be isolated from any source (target sequence, template) which comprises polynucleotides, such as genomic DNA, cDNA, mRNA.
• Hybridization experiments and in particular hybridization conditions, including preferred conditions, and isolation procedures are as generally hereinbefore described.
• DNA molecule is transferred to a host cell, as is also generally hereinbefore described.
• 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.
• 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 (i) reducing the phytate level of manure,
• the phytases of the invention can also be used in chicken food to improve egg shell quality (reduction of 5 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. 10 439-444 (1995 ); Poultry Science Vol. 74 , No. 5 pp. 784-787 (1995) and Poultry Science Vol. 73 , No. 10 pp. 1590-1596 (1994) .
• a "feed” and a “food,” respectively, means any natural or 15 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.
• the phytase may exert its effect in vi tro or in vivo, i.e. 20 before intake or in the stomach of the individual, respectively. Also a combined action is possible.
• a phytase composition according to the invention always comprises at least one phytase of the invention.
• phytase compositions are liquid or dry.
• Liquid compositions need not contain anything more than the phytase enzyme, preferably in a highly purified form. Usually,
• 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
• liquid compositions can be added to a food or feed after an optional pelleting thereof.
• Dry compositions may be spraydried compositions, in which case the composition need not contain anything more than the enzyme in a dry form.
• 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.
• Agglomeration granulates are prepared using agglomeration technique in a high shear mixer (e.g. L ⁇ dige) 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.
• Typical filler materials are salts such as disodiu sulphate.
• Other fillers are kaolin, talc, magnesium aluminium silicate and cellulose fibres.
• binders such as dextrins are also included in agglomeration granulates.
• Typical carrier materials are starch, e.g. in the form of cassava, corn, potato, rice and wheat. Salts may also be used.
• the granulates are coated with a coating mixture.
• a coating 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.
• 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 .
• 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.
• 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 ⁇ -galactosidases, ⁇ -galactosidases, in particular lactases, other phytases, ⁇ - glucanases, in particular endo- ⁇ -1, 4-glucanases and endo- ⁇ - 1, 3 ( ) -glucanases, cellulases, xylosidases, galactanases, in particular arabinogalactan endo-1, - ⁇ -galactosidases and arabinogalactan endo-1, 3- ⁇ -galactosidases, endoglucanases, in particular endo-1, 2- ⁇ -glucanase, endo-1, 3- ⁇ -glucanase, and endo-1, 3- ⁇ -glucanase, pectin degrading enzymes, in particular pectin degrading
• the animal feed additive of the invention is supplemented to the mono-gastric animal before or simultaneously with the diet.
• the animal feed additive of the invention is supplemented to the mono-gastric animal simultaneously with the diet.
• the animal feed additive is added to the diet in the form of a granulate or a stabilized liquid.
• 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.
• Examples of other specific uses of the phytase of the invention is in soy processing and in the manufacture of inositol or derivatives thereof.
• 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.
• a feed comprising an effective amount of the phytase of the invention.
• 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.
• 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.
• Nitrilotriacetic acid 1.50 g MgS0 4 ,7 H 2 0 3.00 g
• YPD 15 10 g yeast extract, 20 g peptone, H 2 0 to 900 ml. Autoclaved, 100 ml 20% glucose (sterile filtered) added.
• SC-variant agar 35 20 g agar, 20 ml 10 x Basal salt, H 2 0 ad 900 ml, autoclaved Phytase activity assay
• the phytase activity can be measured using the following assay:
• the absorbance at 750 nm was measured on 200 ⁇ l 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 ⁇ mol phosphate/min at the given conditions.
• Peniophora lycii CBS No. 686.96 comprises a phytase encoding
• 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.
• 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+) .
• E. coli strain DH10B (Life Technologies)
• 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.
• Peniophora lycii CBS 686.96 Fermentation procedure of Peniophora lycii CBS No. 686.96 for mRNA isolation: 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/1, malto dextrin 15 g/1, bacto peptone 5 g/1, pluronic 0.2 g/1). 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.
• 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) .
• 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 ⁇ g in 5 ⁇ l of DEPC-treated water) was heated at 70°C for 8 min.
• the hybrids were diluted in 250 ⁇ l second strand buffer (20 mM Tris-Cl, pH 7.4, 90 mM KCl, 4.6 mM MgCl 2 , 10 mM (NH 4 ) 2 S0 4 , 0.16 mM bNAD+) containing 200 ⁇ l 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.
• second strand buffer 20 mM Tris-Cl, pH 7.4, 90 mM KCl, 4.6 mM MgCl 2 , 10 mM (NH 4 ) 2 S0 4
• Mung bean nuclease treatment The double-stranded cDNA was precipitated at -20 °C for 12 hours by addition of 2 vols 96% EtOH, 0.2 vol 10 M NH 4 Ac, recovered by centrifugation, washed in 70% EtOH, dried and resuspended in 30 ⁇ l Mung bean nuclease buffer (30 mM NaAc, pH 4.6, 300 mM NaCl, 1 mM ZnS0 4 , 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 ⁇ l 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.
• 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. 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.
• T4 DNA polymerase buffer 20 mM Tris- acetate, pH 7.9, 10 mM MgAc, 50 mM KAc, 1 mM DTT
• T4 DNA polymerase New England Biolabs
• Adaptor ligation, Not I digestion and size selection After the fill-in reaction the cDNAs were recovered by centrifugation, washed in 70% EtOH and dried. The cDNA pellet was resuspended in 25 ⁇ l ligation buffer (30 M Tris-Cl, pH 7.8, 10 mM MgCl 2 , 10 mM DTT, 0.5 mM ATP) containing 2.5 ⁇ g 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 ⁇ l water, 5 ⁇ l lOx 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 lx TBE to separate unligated adaptors and small cDNAs.
• FMC SeaPlaque GTG low melting temperature agarose gel
• 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.
• the directional, size-selected cDNA was recovered by centrifugation, washed in 70% EtOH, dried and resuspended in 30 ⁇ l 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.
• 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, NY). 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
• 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 0.2M CaC12 is poured over the plates and after 1-4 days the phytase positive colonies are identified as colonies surrounded by a clearing zone.
• 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.
• 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.
• 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.
• the cDNA inset of Qiagen purified plasmid DNA of pA2phy2 was sequenced with the Taq deoxy terminal cycle sequencing kit (Perkin Elmer, USA) and synthetic oligonucleotide primers using an Applied Biosystems ABI PRISMTM 377 DNA Sequencer according to the manufacturers instructions.
• Transformation of Aspergillus oryzae or Aspergillus niger Protoplasts are prepared as described in WO 95/02043, p. 16, line 21 - page 17, line 12.
• 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.
• 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.
• the phytase activity is identified by applying 20 ⁇ l supernatant to 4 mm diameter holes punched out in 1% LSB- agarose plates containing 0. IM Sodiumacetate pH 4.5 and 0.1% Inositol hexaphosphoric acid. The plates are left over night at 37°C A buffer consisting of 0. IM CaC12 and 0.2M Sodium acetate pH 4.5 is poured over the plates and the plates are left at room temperature for lh. Phytase activity is then identified as a clear zone.
• 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.
• 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, NY).
• Cloning and expression was done by using the Expression cloning in yeast technique as described above.
• mRNA was isolated from Peniophora lycii , CBS No. 686.96, grown as described above.
• 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. 9xl0 5 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.
• 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 ⁇ l water. The DNA was transformed into E. coli by standard procedures. Plasmid DNA was isolated from E.
• 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 PRISMTM 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.
• SEQ ID No. 23 DNA nucleotides from No 1 to No. 1320 define a phytase encoding region.
• 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.
• the cDNA is obtainable from the plasmid in DSM 11312.
• Total DNA was isolated from a yeast colony and plasmid DNA was rescued by transformation of E. coli as described above.
• 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.
• 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 .
• Agrocybe pediades CBS No. 900.96 comprises a phytase encoding
• 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.
• 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
• mRNA was isolated from Agrocybe pediades, CBS No. 900.96, grown as described above with agitation to ensure sufficient aeration.
• 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. 9xl0 5 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.
• 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.
• SEQ ID No. 21 DNA nucleotides from No 1 to No. 1362 define the phytase encoding region.
• the part of the DNA sequence in SEQ ID NO 21, which is encoding the mature part of the phytase is position 79 to
• the cDNA is obtainable from the plasmid in DSM 11313.
• Total DNA was isolated from a yeast colony and plasmid DNA was rescued by transformation of E. coli as described above.
• 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.
• Example 3 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 phytase in Aspergillus oryzae .
• Example 3
• Paxillus invol tus CBS No. 100231 comprises two phytase encoding DNA sequences of the invention and Trametes pubescens CBS 100232 comprises a phytase of the invention.
• 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. PhyAl, PhyA2 of Paxillus invol tus and the phytase of Trametes pubescens, respectively.
• 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, NY).
• mRNA was isolated from the respective microorganisms, grown under phytase producing conditions, e.g. as described above with agitation to ensure sufficient aeration.
• Mycelia were harvested after 3-5 days' growth, immediately frozen in liquid nitrogen and stored at -80°C.
• Libraries consisting of approx. 9xl0 5 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 . 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.
• the cDNA is obtainable from the plasmids in DSM 11842, 11843 and 11844.
• Total DNA was isolated from a yeast colony and plasmid DNA was rescued by transformation of E. coli as described above.
• 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.
• the two phytases of Paxillus invol tus CBS 100231 (PhyAlP.i. and PhyA2P.i.) and the phytase of Trametes pubescens CBS 100232 (PhyAT.p.) have the following characteristics:
• Directional cDNA libraries are constructed as described in the previous examples from phytase induced mycelia from the four basidiomycetes A. pediades, P. lycii, P. invol tus and T. pubescens, in the yeast expression vector pYES2.0.
• the cDNA libraries are screened for phytase activity, resulting in isolation of five different phytase cDNAs, phyA P. lycii , phyA A. pediades, phyAl P. invol tus, phyA2 P. invol tus, and phyA T. pubescens .
• the five new phytases are transformed and overexpressed in A. oryzae in order to facilitate the purification and characterization of the recombinant enzymes.
• 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 (pClphy2) , phyA A. pediades (pClphy3), phyAl P. invol tus (pClphy5) , phyA2 P. invol tus (pClphy7), and phyA T. pubescens (pClphy ⁇ ) .
• the primary structure of the phyA cDNA encoding PhyA of Peniophora lycii is shown in Fig. 1.
• the 1593 bp cDNA from pClphy2 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.
• the phyA cDNA sequence and the deduced sequence of PHYA from 5 A. pediades are presented in Fig. 2.
• the 1501 bp cDNA from pClphy3 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 ⁇ o acid sequences are included in the sequence listing as (SEQ ID NO: 21) and (SEQ ID NO: 22), respectively.
• nucleotide sequences of the phyAl and the phyA2 cDNA cloned from Paxilus invol tus, and the deduced sequences of 15 PHY1 and PHY2, are shown in Fig. 3. and Fig. 4. respectively.
• the 1522 bp insert in pClphy5 contains a 1329 bp ORF coding for a 442 amino acid polypeptide. According to the SignalP VI .1 prediction (Henrik Nielsen, Jacob Engelbrecht,
• the plasmid pClphy7 contains a 1642 bp insert with a 30 1329 bp ORF coding for a 442 residue polypeptide.
• the SignalP VI.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 35 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. In Fig. 5. the phyA cDNA sequence and the deduced sequence of PHYA from T. pubescens are shown.
• the 1536 bp insert in pClphy ⁇ contains a 1332 bp ORF coding for a 443 residue polypeptide.
• 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.
• the first five phytases in the leftmost column are basidiomycete phytases, whereas the rest are ascomycete phytases.
• the complete cDNA sequences were compared.
• 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.
• the DNA homology of the basidiomycet phytases versus the ascomycet phytases 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.
• Table 2 shows the results when comparing cDNA sequences of ORF and peptide sequences of the mature protein (signal peptide cleaved off) .
• Consensus Sequences I, II and III are the so-called Consensus Sequences I, II and III below, the corresponding alignments of which are shown in Tables 3 and 4 below.
• 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.
• Consensus Sequence I I-Q-R-H-G-A-R- [F/W] -P-T-S-G-A-X-X-R (SEQ ID NO: 3)
• thermophila phyA. P. involtus phyA2 P. involtus phyA T. pubescens phyA A. pediades phyA P. lycii phyA A. fumigatus phyA A. niger phyA A. terreus phyA T. lanuginosa phyA M. thermophila
• thermophila AA pos. P. lycii phyA 1 P. involtus phyA2 P.involtus phyA T. pubescens phyA A. pediades phyA P. lycii phyA A. fumigatus phyA A. niger phyA A. terreus phyA T. lanuginosa phyA . thermophila
• thermophila Consensus Sequence I (SEQ ID NO: 3), residue position 68 to 83 with the numbering for PHYA P. lycii in Fig.
• Consensus Sequence III (SEQ ID NO: 9), AA pos. 415-433 in P. lycii , 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).
• the five phyA cDNAs from A. pediades, P. lycii , P. invol tus, and T. pubescens were subcloned into pHD 14, 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.
• PCR-primers specific for molecular screening of related phytases can be designed (Example 5) .
• N A, C, G or T
• R A or G
• Y C or T
• M A or C
• W A or T.
• the design of the primers is based on the alignment in Fig. 7.
• the 522/538 primerset is tested on genomic DNA from selected ascomycetes and basidiomycetes shown in Table 6 below.
• the genomic DNA is isolated according to the following procedure : Procedure for isolation of fungal genomic DNA.
• Lysis buffer 25 100 mM EDTA
• PE buffer please refer to the QIAprepTM Plasmid Handbook from
• PCR buffer 10 mM Tris-HCl, pH 8.3, 50 mM KCl
• PCR buffer 10 mM Tris-HCl, pH 8.3, 50 mM KCl
• the total volume is 50 ⁇ l.
• the PCR reaction is caried out in a Perkin - Elmer GeneAmp PCR System 2400.
• the PCR reaction is performed using a cycle profile of:
• Table 6 shows the results of the test of this primerset, viz. whether a specific PCR band was detected or not.
• Primersets 522/525, 539/540, 539/538, 539/525 and 537/525 are tested as described in Example 5 above, using 100 pmol of each
• the PCR fragments can be purified and sequenced using the Jet sorb Gel extraction Kit (Genomed GmbH, Germany) according to
• 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
• the doublestranded cDNA was synthesized as described in Example 1.
• Peniophora lycii phytase was expressed in and excreted from Aspergillus oryzae IFO 4177.
• 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.
• the phytase was applied to a Q-sepharose FF column equilibrated in 20mM Tris/CH 3 COOH, pH 7.5 and the enzyme was eluted with an increasing linear NaCl gradient (0 - 0.5M).
• the phytase activity eluted as a single peak. This peak was pooled and (NH 4 ) 2 S0 4 was added to 1.5M final concentration.
• Phenyl Toyopearl 650S column was equilibrated in 1.5M
• the dialysed phytase was applied to a SOURCE 30Q column equilibrated in 20mM Tris/CH 3 COOH, pH 7.5. After washing the column thoroughly with the equilibration buffer a phytase was eluted with an
• 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:
• the sequence corresponds to amino acid residues 31-37 in the cDNA derived amino acid sequence.
• the phytase of Peniophora lycii was expressed in Aspergillus and purified as described in Example 8.
• the phytase activity is measured using the following assay:
• the absorbance at 750 nm was measured on 200 ⁇ l 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 ⁇ mol phosphate/min at the given conditions.
• Temperature profiles were obtained by running the assay at various temperatures (preheating the substrate) .
• 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.
• 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.
• the pH-profiles were obtained by running the assay at the various pH using the same buffer-systems (50 mM, pH was readjusted when dissolving the substrate) .
• Fig. 12 shows the result of differential scanning calorimetry (DSC) measurements on the Peniophora phytase.
• DSC differential scanning calorimetry
• 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
• 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 Peniophora phytase in 0.1 M sodium acetate, pH 5.5 were loaded.
• 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) .
• the protein concentration in the phytase sample was determined by amino acid analysis as follows: An aliquot of the phytase sample was hydrolyzed in 6N 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.
• 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.
• the specific activity is calculated to 987 FYT/mg enzyme protein.
• P n denotes myo-inositol carrying in total n phosphate groups attached to positions p, q, r, ..
• P 6 phytic acid
• 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.
• 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.
• Ins (1, 2, , 5, 6) P 5 (3-phosphate removed) exhibits a NMR spectrum different from Ins (1, 2, 3, 4, 5) P 5 (6-phosphate removed) because the isomers are diastereomers.
• the NMR spectra of Ins (1, 2, 4 , 5, 6) P 5 and Ins (2, 3, 4, 5, 6) P 5 (1-phosphate removed) are identical because the isomers are enantiomers. The same holds for the pair Ins (1,2, 3,4, 5) P 5 and Ins (1, 2 , 3, 5, 6) P 5 (4-phosphate removed).
• 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 ( ⁇ 4.70) .
• 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) .
• the signal at ⁇ 3.25 (t) represents H-5 in Ins(l,2)P
• the signal at ⁇ 3.18(t) represents H-5 in Ins (2) P.
• Ins(l,2)P 2 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) P 2 is very low for both phytases, whereas the amount of Ins(2)P is maximum for both phytases after 24 hours.
• the reaction mixture at 24 h comprised in addition to Ins(2)P minor amounts of Ins (1,2) P 2 .
• Prolonged reaction times resulted in disappearance of the residual Ins(l,2)P 2 , 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.
• 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, HI 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(l,2)P 2 is also detected in both embodiments, the corresponding peaks being indicated at the Aspergillus embodiment only.
• Aspergillus The initial major product was identified as Ins (1, 2, 4, 5, 6) P 5 (A) followed by appearance of
• 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.
• 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.
• the Peniophora phytase digests PA at a faster rate compared to the Aspergillus phytase.
• the end-product is, at pH 3.5 as well as 5.5, under the conditions applied, Ins(2)P (F) .
• the Aspergillus phytase prove to be an essentially clean 3-phytase
• 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.
• 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 .
• Other ways of determining the exact specificity is by determining optical rotation or using a chiral HPLC column.
• Corn was obtained from North Carolina State University (sample No. R27), and ground at a mill (B ⁇ hler Universal) at point 6.8.
• 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.
• the pH value of 3.5 was adjusted by 8N HCl/NaOH.
• 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.
• 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 415nm.
• pH was measured at time 0 and 20 hours.
• a phosphate standard or stock solution of 50mM 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.
• Fig. 21 shows, that at this pH the Peniophora phytase liberates P from corn at significantly improved rate as compared to the Aspergillus phytase.
• Peniophora phytase is much faster in the liberation of phosphorous from ground corn as compared to the Aspergillus phytase (0-120 minutes) .
• 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.
• Peniophora enzyme is surprisingly more efficient than the known Aspergillus phytase as a P-liberator in the digestive system of e.g. broilers.
• 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.
• 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.
• 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 2mS/cm by dilution with distilled water.
• the phytase was applied to a Q-sepharose FF column equilibrated with 20mM Tris/CH 3 C00H, pH 7.5 and the enzyme was eluted with an increasing linear NaCl gradient (0 - 0.5M) .
• the phytase containing fractions from the Q-sepharose column were pooled and (NH 4 ) 2 S0 4 was added to 1.3M final concentration.
• Phenyl Toyopearl 650S column was equilibrated with 1.3M (NH 4 ) 2 S0 4 , lOmM succinic acid/NaOH, pH 6.0 and the phytase was applied to this column and eluted with a decreasing linear (NH 4 ) 2 S0 4 gradient (1.3 - 0M) .
• Phytase containing fractions were pooled and buffer was exchanged with 20mM Tris/CH 3 COOH, pH 7.5 on a Sephadex G25 column.
• Phytase was further purified on a SOURCE Q column equilibrated with 20mM Tris/CH 3 COOH, pH 7.5, and eluted with a linear NaCl gradient (0
• 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.
• the phytase was applied to a SOURCE 30Q column equilibrated in 20mM Tris/CH 3 COOH, pH 7.5 and the enzyme was eluted with an increasing linear NaCl gradient (0 -> 0.25M). The phytase activity was pooled and (NH 4 ) 2 S0 4 was added to 1.6M final concentration. A SOURCE Phenyl column was equilibrated in 1.6M (NH 4 ) 2 S0 4 , 25mM succinic acid/NaOH, pH 6.0 and the phytase was applied to this column and eluted with a decreasing linear (NH 4 ) 2 S0 4 gradient (1.6 -> 0M) .
• 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).
• 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.
• 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.
• 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).
• the phytase of Agrocybe pediades was expressed in Aspergillus and purified as described in Example 14.
• the phytase activity is measured using the following assay:
• the reaction was stopped by adding 250 ⁇ l 10 % TCA and free phosphate was measured by adding 500 ⁇ l 7.3 g FeS0 4 in 100 ml molybdate reagent (2.5 g (NH 4 ) 6 Mo 7 0 24 .4H 2 0 in 8 ml H 2 S0 4 diluted to 250 ml) .
• the absorbance at 750 nm was measured on 200 ⁇ l 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 ⁇ mol phosphate/min at the given conditions.
• Temperature profiles were obtained by running the assay at various temperatures (preheating the substrate) .
• 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.
• 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.
• the pH-profiles were obtained by running the assay at the various pH using the same buffer-systems (50 mM, pH was readjusted when dissolving the substrate) .
• 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.
• 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.
• 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.
• Fig. 27 shows the result of differential scanning calorimetry (DSC) measurements on the Agrocybe phytase.
• 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. Samples of around 2.5 mg/ml Agrocybe phytase in 0.1 M sodium acetate, pH 5.5 were loaded.
• the temperature stability studies were confirmed by DSC, since 5 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 ⁇ o inactivation in the first scan.
• P n denotes yo-inositol carrying in total n phosphate groups attached to positions p, q, r, ..
• P 6 phytic acid
• the technique provide specific information about initial 30 points of attack by the enzyme on the PA molecule, as well as information about the identity of the end product.
• the evolving patterns of peaks reflecting the composition of the intermediate product mixtures provide a qualitative measure, a finger print, suitable for 35 identification of similarities and differences between individual enzymes .
• NMR 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.
• 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 ( ⁇ 4.70) .
• 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) . 5
• the signal at ⁇ 3.25 (t) represents H-5 in Ins (1,2) P 2 whereas the signal at ⁇ 3.18 (t) represents H-5 in Ins (2) P.
• Ins (1,2) P 2 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(l,2)P 2 is 5 very low for both phytases, whereas the amount of Ins(2)P is maximum for both phytases after 24 hours.
• reaction mixture at 24 h comprised in addition to Ins(2)P minor amounts of Ins (1,2) P 2 . Prolonged
• A) shows a signal at ⁇ 3.66 (dd) , U- 6 in Ins (1, 2 , 3 , 4 , 5) P 5 (B) a signal at ⁇ 3.87 (t) and H-3 in Ins (1, 2 , 5, 6) P 4 (C) a signal at ⁇ 3.56(dd).
• compound A corresponds to phosphate in position 30 3 having been hydrolyzed, B position 6 and C position 3 and 4.
• Fig. 32 present the profiles observed after 20 min with the above indicated diagnostic signals (A,B,C) labelled.
• 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, HI 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(l,2)P 2 is also detected in both embodiments, the corresponding peaks being indicated at the Aspergillus embodiment only.
• Aspergillus The initial major product was identified as Ins (1,2,4, 5, 6) P 5 (A) followed by appearance of
• Ins(l,2,5,6)P 4 (C) , and Ins(l,2,6)P 3 (D) H-3 at ⁇ 3.49(dd) after A hours) corresponding to consecutive removal of the phosphate groups in the 3-, 4- and 5-positions.
• the concentration of Ins(l,2)P 2 (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.
• Fig. 34 and 35 representing the time dependent concentration of Ins(l,2)P 2 and Ins(2)P, respectively, constructed from slices along the time-dimension in Fig. 28-29 at the chemical shift values ( ⁇ ) of H-5 in Ins(l,2)P 2 and Ins(2)P, respectively (note that the time scale is only linear in segments) .
• 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.
• the Agrocybe phytase digests PA at a faster rate compared to the Aspergillus phytase.
• the end-product is in both cases, under the conditions applied, Ins(2)P (F) .
• 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.
• 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.
• 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.
• pH 5.5 0.22 M acetate-buffer pH 3.5: 0.05 M citrate-buffer.
• 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.
• 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. Then the phytase enzymes were added at a dosage of 25 FYT/20 g of corn.
• 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 415nm.
• a phosphate standard or stock solution of 50mM 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.
• the Paxillus invol tus PhyAl phytase was expressed in and excreted from Aspergillus oryzae IFO 4177 as described in Examples 3 and 1.
• 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 lOmM succinic acid/NaOH, pH 6.0 on a Sephadex G25 column. The pH of the G25 filtrate was adjusted to pH 7.0.
• the phytase was applied to a Q-sepharose FF column equilibrated in 20mM HEPES/NaOH, pH 7.0.
• the enzyme turned out to be in the run-through from the column.
• (NH 4 ) 2 S0 4 was added to the run-through to 2.0M final concentration.
• a Butyl Toyopearl 650S column was equilibrated in 2.
• OM (NH 4 ) 2 S0 4 , lOmM CH 3 COOH/NaOH, pH 5.5 and the phytase was applied to this column and eluted with a decreasing linear (NH 4 ) 2 S0 4 gradient (2.0 - 0M) .
• Phytase containing fractions were pooled and the buffer was exchanged for 20mM HEPES/NaOH, pH 7.5 on a Sephadex G25 column.
• the G25 filtrate was applied to a Q-sepharose FF column equilibrated in 20mM 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
• Phenyl Toyopearl 650S column was equilibrated in 2.0M (NH 4 ) 2 S0 4 , lOmM CH 3 COOH/NaOH, pH 5.5 and the phytase was applied to this column and eluted with a decreasing linear (NH 4 ) 2 S0 4 gradient (2.0 - 0M) .
• Phytase containing fractions were pooled and reapplied to the same Phenyl Toyopearl column after adding (NH 4 ) 2 S0 4 to 2.0M final concentration. Fractions from the second Phenyl Toyopearl column were analyzed by SDS-PAGE and pure phytase fractions were pooled.
• 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.
• the sequence corresponds to amino acid residues 21-33 in the cDNA derived amino acid sequence.
• 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.
• the Paxillus involtus PhyA2 phytase was expressed in and excreted from Aspergillus oryzae IFO 4177 as described in Examples 3 and 1.
• 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 (NH 4 ) 2 S0 4 was added to 2. OM final concentration.
• the phytase was applied to a Phenyl sepharose FF column equilibrated in 2.
• OM (NH 4 ) 2 S0 4 20mM CH 3 COOH/NaOH, pH 5.5 and the enzyme was eluted with a decreasing linear (NH 4 ) 2 S0 4 gradient (2.0 - OM) .
• the phytase activity eluted as a single peak. This peak was pooled and (NH 4 ) 2 S0 4 was added to 2.
• OM final concentration NH 4 ) 2 S0 4
• a Butyl Toyopearl 650S column was equilibrated in 2.0M (NH 4 ) 2 S0 4 , lOmM CH 3 COOH/NaOH, pH 5.5 and the phytase was applied to this column and eluted with a decreasing linear (NH 4 ) 2 S0 4 gradient (2.0 - 0M) .
• Phytase containing fractions were pooled and the buffer was exchanged for 20mM HEPES/NaOH, pH 7.0 on a Sephadex G25 column.
• the G25 filtrate was applied to a Q-sepharose FF column equilibrated in 20mM HEPES/NaOH, pH 7.0.
• the phytase was eluted with an increasing linear NaCl gradient (0 -> 0.5M).
• the phytase activity was pooled and the buffer was exchanged for 20mM HEPES/NaOH, pH 7.0 by dialysis.
• the dialysed phytase was applied to a SOURCE 30Q column equilibrated in 20mM 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 -> 0.3M).
• the sequence corresponds to amino acid residues 25-30 in the cDNA derived amino acid sequence.
• 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 the sequence listing under the headings SEQ ID NOs: 27 and 28.
• 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 lOkDa 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 lOmM succinic acid/NaOH, pH 6.0 by dilution with deionised water.
• the phytase was applied to a Q-sepharose FF column equilibrated in lOmM succinic acid/NaOH, pH 6.0 and the enzyme was eluted with an increasing linear NaCl gradient (0 - 0.5M). The phytase activity eluted as a single peak. This peak was pooled and (NH 4 ) 2 S0 4 was added to 2.0M final concentration. A Butyl Toyopearl 650S column was equilibrated in 2.
• the Trametes pubescens phytase migrates on SDS-PAGE as a band with M r 57 kDa.
• the sequence corresponds to amino acid residues 31-41 in the cDNA derived amino acid sequence.
• 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.
• the molecular weights (kDa) of the three phytases of Trametes pubescens and PhyA2 and PhyAl of Paxillus invol tus are 65, 55 and 65 kDa, respectively.
• the termostability of the PhyAl phytase of Paxillus invol tus 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 PhyAl is around 60% and 40%, respectively.
• Td's of around 48°C, 59°C and 55°C are found for the phytases PhyA2 and PhyAl of Paxillus invol tus and for the phytase of Trametes pubescens, respectively.

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