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WO1999048380A1 - Phytases thermostables dans la preparation de nourriture pour animaux et leurs expression chez les plantes - Google Patents

Phytases thermostables dans la preparation de nourriture pour animaux et leurs expression chez les plantes Download PDF

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WO1999048380A1
WO1999048380A1 PCT/DK1999/000154 DK9900154W WO9948380A1 WO 1999048380 A1 WO1999048380 A1 WO 1999048380A1 DK 9900154 W DK9900154 W DK 9900154W WO 9948380 A1 WO9948380 A1 WO 9948380A1
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phytase
plant
consensus
thermostable
phytases
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Svend Petersen
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Novo Nordisk AS
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Novo Nordisk AS
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Priority to KR1020007010410A priority Critical patent/KR20010042063A/ko
Priority to JP2000537445A priority patent/JP2002508942A/ja
Priority to EP99914443A priority patent/EP1065941A1/fr
Priority to BR9909006-6A priority patent/BR9909006A/pt
Priority to AU33267/99A priority patent/AU753475C/en
Priority to CA002325440A priority patent/CA2325440A1/fr
Publication of WO1999048380A1 publication Critical patent/WO1999048380A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • 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
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • 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

  • thermostable phytases viz. their use in processes for the production of animal feed, and their expression in plants.
  • EP 0 556 883 Bl describes a method for preparing feed pellets based on an extrusion technique.
  • the present invention provides a process of preparing an animal feed, which process comprises an agglomeration of feed ingredients, wherein a thermostable phytase is added before or during the agglomeration. Also provided is a transgenic plant or part thereof which comprises a DNA-construct encoding a thermostable phytase.
  • transgenic plan e.g. seeds or leaves
  • the transgenic plan may be used in the feed preparation process of the invention, to thereby provide - in a preferred embodiment - at the same time a nutrient (feed ingredient) and the feed additive phytase.
  • Fig. 1 is a differential scanning calorimetry (DSC) chart of consensus phytase-1 and consensus phytase-10;
  • Fig. 2 a DSC of consensus phytase-10-thermo-Q50T and consensus phytase-10-thermo-Q50T-K91A;
  • Fig. 3 a DSC of consensus phytase-1-thermo [8] -Q50T and consensus phytase-1-thermo [8 ] -Q50T-K91A;
  • Fig. 4 a DSC of the phytase from A. fumigatus ATCC 13073 and of its ⁇ -mutant; and
  • Fig. 5 shows the design of the consensus-phytase-1 a ino acid sequence;
  • Fig. 6 an alignment and the basidiomycete consensus sequence of five Basidiomycete phytases
  • Fig. 7 the design of the consensus-phytase-10 amino acid sequence
  • Fig. 8 an alignment for the design of consensus-phytase-11 (all Basidiomycete phytases were used as independent sequences using an assigned vote weight of 0.2 for each Basidiomycete sequence; still further the amino acid sequence of A. niger T213 was used) ;
  • Fig . 9 the DNA and amino acid sequence of consensus- phytase-1-thermo (8 ) -Q50T-K91A;
  • Fig . 10 the DNA and amino acid sequence of Consensus- phytase-10-thermo (3) -Q50T-K91A;
  • Fig . 11 the DNA and amino acid sequence of A. fumigatus ATCC 13073 ⁇ -mutant; and Fig . 12 the DNA and amino acid sequence of Consensus- phytase-7 which comprises the following mutations as compared to Consensus-phytase-1 : S89D, S92G, A94K, D164S, P201S, G203A, G205S, H212P, G224A, D226T, E255T, D256E, V258T, P265S, Q292H, G300K,
  • feed or an “animal feed” means any natural or artificial diet, meal or the like intended or suitable for being eaten, taken in, digested, by an animal.
  • Food for human beings is included in the above definition of feed.
  • Animals include all animals, be it polygastric animals (ruminants) ; or monogastric animals such as human beings, 4 poultry, swine and fish. Preferred animals are the mono-gastric animals, in particular pigs and broilers.
  • feed ingredients includes the raw materials from which a feed is to be, or is, produced; or the intended, or actual, component parts of a feed.
  • Feed ingredients for non-human animals are usually, and preferably, selected from amongst the following non-exclusive list: plant derived products such as seeds, grains, leaves, roots, tubers, flowers, pods, husks - and they may take the form of flakes, cakes, grits, flour, and the like; animal derived products such as fish meal, milk powder, bone extract, meat extract, blood extract and the like; additives such as minerals, vitamins, aroma compounds, and feed enhancing enzymes.
  • Phytic acid or myo-inositol 1, 2, 3, 4, 5, 6-hexakis dihydrogen phosphate is the primary source of inositol and the primary storage form of phosphate in plant seeds and grains. In the seeds of legumes it accounts for about 70% of the phosphate content. Seeds, cereal grains and legumes are important feed ingredients.
  • Phytic acid, or its salts phytates - said terms being, unless otherwise indicated, in the present context used synonymously or at random - is an anti-nutritional factor. This is partly due to its binding of nutritionally essential ions such as calcium, trace minerals such as mangane, and also proteins (by electrostatic interaction) . And partly due to the fact that the phosphorous thereof is not nutritionally available 5 either, since phytic acid and its salts, phytates, are often not metabolized.
  • Phytic acid is degradable by phytases.
  • a "phytase” is an polypeptide or enzyme which exhibits phytase activity, viz. 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)
  • 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.
  • EP 0420358 describes the cloning and expression of a phytase of Aspergillus ficuum (niger) .
  • EP 0684313 describes the cloning and expression of
  • WO 98/28409 describes the cloning and expression ⁇ f several basidiomycete phytases, e.g. from Peniophora lycii, Agrocybe pediades, Paxillus involutus and Trametes pubescens.
  • the 3-phytase hydrolyses first the ester bond at a 3- position, whereas the 6-phytase hydrolyzes first an ester bond at the 6-position of phytic acid. Both of these types of phytases are included in the above definition of phytase.
  • agglomeration is defined as a process in which various components are mixed under the influence of heat.
  • the resulting product is preferably an "agglomerate” or conglomerate in which the components adhere to each other while forming a product of a satisfactory physical stability.
  • the formation of dust from such agglomerate is an indication of its 7 physical stability - the less dust being formed, the better.
  • a suitable assay for dust formation from agglomerates is ASAE standard S 269-1.
  • a satisfactory agglomerate has below 20%, preferably below 15%, more preferably below 10%, even more preferably below 6% dust.
  • temperatur- ⁇ is at least 65°C, as measured on the product at the outlet of the agglomeration unit. More preferred temperatures are at least
  • a preferred agglomeration process is operated at an increased pressure.
  • the pressure is typically due to a compacting of the ingredients, optionally in combination with a reduction of the cross-sectional or throughput area.
  • process parameters such as temperature and pressure
  • the resulting shear forces and shear velocities are of such magnitude, that the starch- and protein-containing feed ingredients become fluid.
  • Increased pressure means increased as compared to normal atmospheric pressure, and the maximum pressure as measured within the agglomeration unit.
  • Agglomeration includes, but is not limited to, the well- known processes called extrusion, expansion (or pressure conditioning) and pelleting (or pellet pressing) .
  • steps (i)-(vii) above are entirely optional steps. These steps are i.a. described in more detail at pp. 29-70 of the above handbook.
  • a particularly preferred further step is step (i) .
  • the feed-ingredients are pre-heated in a first step (a) to a temperature of at least
  • 65°C preferably 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or even at least 130°C.
  • thermostable phytase takes place before or during step (a) and/or before or during step (b) .
  • Water is preferably added in step (a) . More preferably, heated steam is added during the mixing of the ingredients (steps (a) and/or (b) ) .
  • Process step (a) is preferably performed in a cascade mixer (see the above cited handbook p. 44) .
  • a "thermostable" phytase is a phytase which has a Tm
  • the Tm is at least 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75°C.
  • the Tm is equal to or lower than 150°C, more preferably equal to or lower than 145, 140, 135, 130, 125, 120, 115 or 110°C.
  • preferred intervals of Tm are: 65-150°C, 66-150°C, - (etc.) - 75-150°C; 65-145°C, 66-145°C, - (etc.) - 75-145°C; 65-140°C, - (etc.) - 75-140°C; - (etc.) - 65-110°C, 66-110°C, - (etc.) - 75-110°C.
  • Tm is the following: between 65 and 110°C; between 70 and 110°C; between 70 and 100°C; between 75 and 95°C, or between 80 and 90°C.
  • Example 3 the measurement of Tm by DSC is described, and the T ' s of a number of phytases are shown.
  • thermostable phytase can be defined as a phytase having a temperature-optimum of at least 60 °C.
  • the optimum temperature is determined on the substrate phytate at pH 5.5, or on the substrate phytic acid at 10 pH 5.0.
  • Preferred units are FYT, FTU or the units of Example 3.
  • the phytase assay of Example 3 is most preferred.
  • the optimum temperature is at least 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70°C.
  • 5 the optimum temperature is equal to or lower than 140 °C, more preferably equal to or lower than 135, 130, 125, 120, 115, 110,-. 105 or 100°C.
  • preferred intervals of optimum temperature are: 60-140°C, 61-140°C, - (etc.) - 70-140°C; 60- 135°C, 61-135°C, - (etc.) - 70-135°C; 60-130°C, - (etc.) - 70-
  • Preferred phytases of the present invention exhibit a degree of similarity or homology, preferably identity, to the complete amino acid sequence of either -of the phytases mentioned below under (iii) - preferably to the complete amino acid
  • the degree of similarity or homology, alternatively identity can be determined using any alignment programme known
  • a preferred alignment programme is GAP provided in the GCG version 8 program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (see also Needleman, S.B. and Wunsch, CD., (1970), Journal of
  • a multiple sequence alignment can be made using the program PileUp (Program Manual for the Wisconsin Package,
  • Consensus-phytase-1 98.7% (97.9%)
  • Consensus-phytase-10 96.6% (94.4%)
  • a "purified" phytase 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.
  • thermostable phytases are the so-called consensus phytases of EP 98113176.6 (EP 0897985), viz. (i) any thermostable phytase which is obtainable by the processes described therein;
  • a phytase comprising the amino acid sequence shown in Fig. 2 thereof or any variant or mutein thereof, preferred muteins being those comprising the substitutions Q50L; Q50T; Q50G; Q50T-Y51N or Q50L-Y51N.
  • thermostable phytases are 12
  • thermostable phytase which comprises at least one of the following amino acid sequence (some of which are shown in Figs. 5-12 herein), preferably the following phytases: Consensus-phytase-1 (or simply Consensus phytase) ; Consensus-phytase-1-thermo (3) ; Consensus-phytase-l-Q50T; basidiomycete-consensus (or simply Basidio) ; Consensus-.. phytase-10 (or Fcp 10) ; Consensus-phytase-11 (or Consensus Seq.
  • Consensus-phytase-1-thermo (8) -Q50T-K91A; Consensus-phytase-1-thermo (8) -Q50T; Consensus-phytase-1- thermo(8); Consensus-phytase-10-thermo (3) -Q50T-K91A; Consensus-phytase-10-thermo (3) -Q50T (sometimes, "(3)" is deleted from this expression) ; Aspergillus fumigatus ATCC 13073 phytase ⁇ -mutant; Aspergillus fumigatus ATCC 13073 phytase ⁇ -mutant plus the mutations E59A, S126N, R329H, S364T, G404A; Aspergillus fumigatus
  • thermostable variants and muteins of the phytases of (iv) and (v) in particular those comprising one or more of the following substitutions: Q50L,T,G; Q50L-Y51N; Q50T-Y51N.
  • plant is intended to include not only whole plants as such, but also plant parts or organs, such as leaves, seeds or grains, stem, root, tubers, flowers, callus, fruits etc.; tissues, cells, protoplats etc.; as well as any combinations or sub-combinations thereof. Plant tissue cultures and plant cell lines as well as plant protoplasts are specifically included herein. 13
  • transgenic plant is a plant as defined above, which has been genetically modified, as well as its progeny and propagating material thereof having retained the genetical modification.
  • the transgenic plant comprises at least one specific gene introduced into an ancestral plant by recombinant gene technology.
  • the term is not confined to a. single plant variety.
  • the invention relates to a transgenic plant which comprises a DNA-construct encoding a thermostable phytase.
  • the transgenic plant is a plant grouping which is characterized in that it comprises a DNA- construct encoding a thermostable phytase.
  • the members of this plant grouping may very well possess individuality, but are clearly distinguishable from other varieties by their common characteristic feature of the the thermostable phytase DNA- construct.
  • the present teaching is applicable to more than one plant variety. No naturally occuring plant varieties are included amongst the plants of the invention.
  • the invention relates to a transgenic plant variety or a variant thereof; a transgenic plant species, a transgenic plant genus, a transgenic plant family, and/or a transgenic plant order. More preferably, plant varieties as such are disclaimed.
  • Any thermostable phytase may be used in the present invention, e.g. any wild-type phytases, genetically engineered phytases, consensus phytases, phytase muteins, and/or phytase variants.
  • Genetically engineered phytases include, but are not limited to, phytases prepared by site-directed mutagenesis, gene shuffling, random mutagenesis, etc. 14
  • the nucleotide sequence encoding a wild-type thermostable phytase may be of any origin, including mammalian, plant and microbial origin and may be isolated from these sources by conventional methods.
  • the nucleotide sequence is derived from a microorganism, such as a fungus, e.g. a yeast or a filamentous fungus, or a bacterium.
  • the DNA sequence encoding- a thermostable phytase may be isolated from the cell producing it, using various methods well known in the art (see e.g. WO 98/28409 and EP 0897985) .
  • thermostable genetically engineered or consensus phytase including muteins and variants thereof, may be prepared in any way, e.g. as described in Example 3 hereof and in EP 0897985.
  • the nucleotide sequence encoding the phytase is inserted into an expression construct containing regulatory elements or sequences capable of directing the expression of the nucleotide sequence and, if necessary or desired, to direct secretion of the gene product or targetting of the gene product to the seeds of the plant.
  • the nucleotide sequence encoding the thermostable phytase is operably linked to a suitable promoter capable of mediating transcription in the plant in question.
  • the promoter may be an inducible promoter or a constitutive promoter.
  • an inducible promoter mediates transcription in a tissue-specific or growth-stage specific manner, whereas a constitutive promoter provides for sustained transcription in all cell tissues.
  • An example of a suitable constitutive promoter useful for the present invention is the cauliflower mosaic virus 35 S promoter. Transcription initiation sequences from the tumor-inducing plasmid (Ti) of 15
  • Agrobacterium such as the octopine synthase, nopaline synthase, or mannopine synthase initiator, are further examples of preferred constitutive promoters.
  • suitable inducible promoters include a seed- specific promoter such as the promoter expressing alpha-amylase in wheat seeds (see Stefanov et al, Acta Biologica Hungarica. Vol. 42, No. 4 pp. 323-330 (1991), a promoter of the gene encoding a rice seed storage protein such as glutelin, prolamin, globulin or albumin (Wu et al., Plant and Cell Physiology Vol. 39, No. 8 pp. 885-889 (1998)), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba described by Conrad U. et al, Journal of Plant Physiology Vol. 152, No. 6 pp. 708-711 (1998), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, eg as described in WO 91/14772.
  • a seed- specific promoter such as the promoter expressing al
  • the promoter enhancer may be an intron which is placed between the promoter and the amylase gene.
  • the intron may be one derived from a monocot or a dicot.
  • the intron may be the first intron from the rice Waxy (Wx) gene (Li et al., Plant Science Vol. 108, No. 2, pp. 181-190 (1995)), the first intron from the maize Ubil (Ubiquitin) gene (Vain et al., Plant Cell Reports Vol. 15, No. 7 pp.
  • a seed specific enhancer may be used for increasing the expression of the thermostable phytase in seeds.
  • An example of a seed specific enhancer is the one derived from the beta- phaseolin gene encoding the major seed storage protein of bean 16
  • the expression construct preferably contains a terminator sequence to signal transcription termination of the thermostable phytase gene such as the rbcS2' and the nos3' terminators .
  • the expression construct should also preferably include one or more selectable markers, e.g. an antibiotic resistance selection marker or a selection marker providing resistance to a herbicide.
  • selectable markers e.g. an antibiotic resistance selection marker or a selection marker providing resistance to a herbicide.
  • One widely used selection marker is the neomycin phosphotransferase gene (NPTII) which provides kanamycin resistance.
  • NPTII neomycin phosphotransferase gene
  • suitable markers include a marker providing a measurable enzyme activity, e.g. dihydrofolate reductase, luciferase, and b-glucoronidase (GUS) .
  • Phosphinothricin acetyl transferase may be used as a selection marker in combination with the herbicide basta or bialaphos.
  • the transgenic plant of the invention may be prepared by methods known in the art.
  • the transformation method used will depend on the plant species to be transformed and can be selected from any of the transformation methods known in the art such as Agrobacterium mediated transformation (Zambryski et al., EMBO Journal 2, pp 2143-2150, 1993), particle bombardment, electroporation (From et al. 1986, Nature 319, pp 791-793), and virus mediated transformation.
  • Agrobacterium mediated transformation Zeambryski et al., EMBO Journal 2, pp 2143-2150, 1993
  • particle bombardment electroporation
  • electroporation From et al. 1986, Nature 319, pp 791-793
  • virus mediated transformation for transformation of monocots particle bombardment (ie biolistic transformation) of embryogenic cell lines or cultured embryos are preferred.
  • references are listed, which disclose various methods for transforming various plants: Rice (Cristou et al. 1991, Bio/Technology 9, pp.
  • Agrobacterium mediated transformatiosi is conveniently achieved as follows:
  • a vector system carrying the thermostable phytase is constructed.
  • the vector system may comprise of one vector, but it can comprise of two vectors.
  • the vector system is referred to as a binary vector system (Gynheung An et al.(1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19) .
  • An Agrobacterium based plant transformation vector consists of replication origin (s) for both E.coli and Agrobacterium and a bacterial selection marker.
  • a right and preferably also a left border from the Ti plasmid from Agrobacterium tumefaciens or from the Ri plasmid from Agrobacterium rhizogenes is nessesary for the transformation of the plant. Between the borders the expression construct is placed which contains the thermostable phytase gene and appropriate regulatory sequences such as promotor and terminator sequences.
  • a selection gene e.g.
  • NPTII neomycin phosphotransferase type II
  • GUS betha-glucuronidase
  • the invention also relates to a method of preparing a transgenic plant capable of expressing a thermostable phytase, said method comprising the steps of (i) isolating a nucleotide sequence encoding a thermostable phytase; (ii) inserting the nucleotide sequence of (i) in an expression construct capable of mediating the expression of the nucleotide sequence in a selected host plant; and (iii) transforming the selected host plant with the expression construct.
  • thermostable phytase when used in relation to the thermostable phytase, is also within this invention.
  • This method is an essentially non-biological method.
  • Any plant may be a selected host plant. More specifically, the plant can be dicotyledonous or monocotyledonous, for short a dicot or a monocot. Of primary interest are such plants which are potential food or feed components. These plants may comprise phytic acid. 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) .
  • grasses such as meadow grass (blue grass, Poa)
  • forage grass such as festuca, lolium
  • temperate grass such as Agrostis
  • 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.
  • legumes such as lupins, pea, bean and soybean
  • cruciferous (family Brassicaceae) such as cauliflower, oil seed rape and the closely related model organism Arabidopsis thaliana.
  • crops or cereal plants such as wheat (Triticum, e.g. aestivum) , barley (Hardeum, e.g. vulgare) , oats, rye, rice, sorghum and corn (Zea, e.g. mays).
  • crops or cereal plants such as wheat (Triticum, e.g. aestivum) , barley (Hardeum, e.g. vulgare) , oats, rye, rice, sorghum and corn (Zea, e.g. mays).
  • dicotyledonous plants such as those mentioned above.
  • the ancestral plant or host plant is per se a desired feed ingredient. 19
  • the phytase activity can be measured using the following assay: 10 ⁇ l diluted enzyme samples (diluted in 0.1 M sodium acetate, 0.01 % Tween20, pH 5.5) are added into 250 ⁇ l 5 mM sodium phytate (Sigma) in 0.1 M sodium acetate, 0.01 % Tween20, pH 5.5 (pH adjusted after dissolving the sodium phytate; the substrate is preheated) and incubated for 30 minutes at 37 °C.
  • the reaction is stopped by adding 250 ⁇ l 10 % TCA and free phosphate is measured by adding 500 ⁇ l 7.3 g FeS04 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 is measured on 200 ⁇ l samples in 96 well microtiter plates. Substrate and enzyme blanks are included. A phosphate standard curve is also included (0-2 mM phosphate) .
  • 1 FYT equals the amount of enzyme that releases 1 ⁇ mol phosphate/min at the given conditions. This assay is preferred for phytase enzyme preparations (when not in admixture with other feed ingredients) .
  • FTU assay - for analyzing phytase in admixture with feed ingredients
  • One FTU is defined as the amount of enzym, which at stan- dard conditions (37°C, pH 5,5; reaction time 60 minutes and start concentration of phytic acid 5 mM) releases phosphate equivalent to 1 ⁇ mol phosphate per minute .
  • the FTU assay is preferred for phytase activity measure- ments on animal feed premixes and the like complex compositions.
  • This buffer is also used for preparation of P0 4 -standards and further dilution of premix samples. 0.22 M acetate buffer with Tween 20 pH 5,5
  • the sodium acetate is dissolved in demineralised water. Tween 20 is added, and pH adjusted to 5,50 ⁇ 0,05 with acetic acid.
  • the ingredients are dissolved in demineralised water, and pH is adjusted to 5,50 ⁇ 0,05 with acetic acid.
  • This buffer is used for production of phytic acid substrate) .
  • Phytic acid substrate 5 mM phytic acid The volume of phytic acid is calculated with allowance for the water content of the used batch.
  • the solution is prepared not more than 2 hours before use, and the bottle is wrapped in tinfoil.
  • Sample size for feed samples At least 70 g, preferably 100 g.
  • sample size is approx. 100 g all the sample is ground in a coffee grinder and subsequently placed in tared 23 beakers. The sample weight is noted. It is not necessary to grind not-pelleted samples. If a sample is too big to handle, it is sample split into parts of approx. 100 g.
  • Magnets are placed in the beakers and 0,22 M acetate buffer with Tween is added.
  • the samples are extracted for 90 minutes.
  • the samples are centrifuged for 10 minutes at 4000 rpm.
  • Magnets are placed in the beakers and 0,22 M acetate buffer with Tween, EDTA og P0 4 3 ⁇ is added.
  • the samples are extracted for 60 minutes.
  • the samples are centrifuged for 10 minutes at 4000 rpm.
  • Extracts of feed samples are analysed directly.
  • Extracts of premix are diluted to approx. 1,5 FTU/g (A 415 (main sample) ⁇ 1,0 ) .
  • the samples are taken off the water bath and 2 , 0 ml stop solution is added (exactly 60 minutes after addition of substrate) .
  • the samples are stirred for 1 minute or longer.
  • Feed samples are centrifuged for 10 minutes at 4000 rpm (It is not necessary to centrifuge premix samples) .
  • 100 ml of the supernatant from the extracted and centri- fuged samples are placed in marked glass test tubes, and a magnet is placed in each tube.
  • the samples are incubated for 60 minutes at room temperature.
  • the feed samples are centrifuged for 10 minutes at 4000 rpm (it is not necessary to centrifuge premix samples) .
  • 2 x 100 ml are taken from each of the 8 standards and also 4 x 100 ml 0,22 M acetate buffer (reagent blind) .
  • a 415 is measured on all samples.
  • 100 ⁇ l is taken from the extracted and centrifuged sample.
  • FTU /g sample ⁇ (x - b) x 0,0001 x 1000 x 10000 ⁇ / ⁇ a x 60 x C ⁇
  • thermostability of various phytases has been determined, viz. the melting temperature, Tm, and/or the optimum temperature.
  • the phytase of Aspergillus niger NRRL 3135 was prepared as described in EP 0420358 and van Hartingsveldt et al (Gene, 127, 87-94, 1993).
  • Consensus-phytase-1 (Fig. 5) and Consensus-phytase-l-Q50T are shown in and were prepared as described in EP 0897985. 27
  • Consensus-phytase-10 was derived and prepared according to the teachings of EP-0897985 (Examples 1-2 and 3-7, respectively), however adding to the alignment at Fig. 1 thereof the phytase sequence of Thermomyces lanuginosa (Berka et al, Appl. Environ. Microbiol. 64, 4423-4427, 1998) and a basidiomycete consensus sequence (derivation described below) ⁇ omitting the sequence of A. niger T213, and assigning a vote weight of 0.5 for the remaining A. niger phytase sequences. The derivation of the sequence of Consensus-phytase-10 is shown in Fig. 7.
  • the basidiomycete consensus sequence was also derived according to the principles of EP-0897985, viz. from the five basidiomycete phytases of WO 98/28409, starting with the first amino acid residue of the mature phytases (excluding signal peptide). A vote weight of 0.5 was assigned to the two Paxillus phytases, all other genes were used with a vote weight of 1.0 - see Fig. 6.
  • Consensus-phytase-10-thermo Consensus-phytase-10-thermo-Q50T-K91A (Fig. 10) and Consensus-phytase-10- thermo-Q50T were prepared from consensus-phytase-10, in analogy to Examples 5-8 of EP-0897985, by introducing the three back- mutations K94A, V158I and A396S ("thermo(3)” or "thermo") and, where applicable, also the mutations Q50T or Q50T-K91A.
  • Consensus-phytase-1-thermo (8) was prepared from consensus-phytase-1, in analogy to Example 8 of EP-0897985, by introducing the eight mutations E58A, D197N, E267D, R291I, R329H, S364T, A379K and G404A ("thermo (8) ") and, where applicable, also the mutations Q50T or Q50T-K91A. 28
  • Consensus-phytase-1-thermo (3) was prepared from consensus- phytase-1 by introduction of the three mutations K94A, V158I and A396S.
  • Tm unfolding temperature or melting temperature
  • differential scanning calorimetry was applied as previously published by Brugger et al (1997) : “Thermal denaturation of phytases and pH 2.5 acid phosphatase studied by differential scanning calorimetry," in The Biochemistry of phytate and phytase (eds. Rasmussen, S.K; Raboy, V.; Dalb ⁇ ge, H. and Loewus, F.; Kluwer Academic Publishers).
  • phytase activity was determined basically as described by Mitchell et al (Microbiology 143, 245-252, 1997) : The activity was measured in an assay mixture containing 0.5% phytic acid ( ⁇ 5 mM) in 200 mM 29 sodium acetate, pH 5.0. After 15 min of incubation at 37 °C, the reaction was stopped by addition of an equal volume of 15% trichloroacetic acid.
  • the liberated phosphate was quantified by mixing 100 ⁇ l of the assay mixture with 900 ⁇ l H 2 0 and 1 ml of 0.6 M H 2 S0 4 , 2% ascorbic acid and 0.5% ammonium molybdate. Standard solutions of potassium phosphate were used as- reference.
  • One unit of enzyme activity was defined as the amount of enzyme that releases 1 ⁇ mol phosphate per minute at 37 °C.
  • the protein concentration was determined using the enzyme extinction coefficient at 280 nm calculated according to Pace et al (Prot.Sci. 4, 2411-2423, 1995): Consensus phytase, 1.101; consensus phytase 7, 1.068; consensus phytase 10, 1.039.
  • enzyme (lOO ⁇ l) and substrate solution (lOO ⁇ l) were pre-incubated for 5 in at the given temperature.
  • the reaction was started by addition of the substrate solution to the enzyme. After 15 min incubation, the reaction was stopped with trichloroacetic acid and the amount of phosphate released was determined.
  • Phytase- activity-versus-temperature is plotted, and the temperature optimum is determined as that temperature at which the acitivity reaches its maximum value.

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Abstract

L'invention concerne des phytases thermostables dans la préparation de nourriture pour animaux et l'expression de ces phytases chez les plantes. Pour la préparation de nourriture pour animaux, une phytase thermostable est ajoutée avant ou pendant l'étape d'agglomération. Les étapes sont, de préférence, la granulation, l'extrusion et l'expansion. Une plante transgénique exprimant une phytase thermostable peut être utilisée directement dans la préparation de nourriture pour animaux.
PCT/DK1999/000154 1998-03-23 1999-03-22 Phytases thermostables dans la preparation de nourriture pour animaux et leurs expression chez les plantes Ceased WO1999048380A1 (fr)

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KR1020007010410A KR20010042063A (ko) 1998-03-23 1999-03-22 사료 제제 및 식물 발현에 있어서의 열안정성 피타제
JP2000537445A JP2002508942A (ja) 1998-03-23 1999-03-22 飼料調製物及び植物発現における熱安定性フィターゼ
EP99914443A EP1065941A1 (fr) 1998-03-23 1999-03-22 Phytases thermostables dans la preparation de nourriture pour animaux et leurs expression chez les plantes
BR9909006-6A BR9909006A (pt) 1998-03-23 1999-03-22 Processo de preparar uma alimentação animal, planta transgênica, construção de expressão, vetor, e, processo de preparar uma planta transgênica
AU33267/99A AU753475C (en) 1998-03-23 1999-03-22 Thermostable phytases in feed preparation and plant expression
CA002325440A CA2325440A1 (fr) 1998-03-23 1999-03-22 Phytases thermostables dans la preparation de nourriture pour animaux et leurs expression chez les plantes

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000043503A1 (fr) * 1999-01-22 2000-07-27 Novozymes A/S Phytases ameliorees
WO2001025411A1 (fr) 1999-10-01 2001-04-12 Novozymes A/S Produit enzymatique seche par atomisation
WO2002038774A3 (fr) * 2000-11-10 2003-01-09 Aventis Animal Nutrition Sa Phytases bacteriennes et procede de production de ces phytases
WO2003066847A2 (fr) 2002-02-08 2003-08-14 Novozymes A/S Variants de phytase
US6720174B1 (en) 1999-01-28 2004-04-13 Novozymes A/S Phytases
US7186817B2 (en) 2000-05-04 2007-03-06 Forskningscenter Riso Polynucleotides encoding phytase polypeptides
US7238378B2 (en) 2002-02-08 2007-07-03 Novozymes A/S Phytase variants
WO2008017661A1 (fr) 2006-08-07 2008-02-14 Novozymes A/S Granules d'enzyme pour alimentation animale
EP2116136A1 (fr) 2008-05-08 2009-11-11 Nederlandse Organisatie voor toegepast- natuurwetenschappelijk onderzoek TNO Nouvelles phytases
EP2143339A1 (fr) 2004-09-27 2010-01-13 Novozymes A/S Granules d'enzyme
EP3072399A1 (fr) 2006-08-07 2016-09-28 Novozymes A/S Granules d'enzyme pour alimentation animale
WO2020115179A1 (fr) 2018-12-05 2020-06-11 Novozymes A/S Utilisation d'un granule enzymatique

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CN1294266C (zh) * 2002-08-05 2007-01-10 中国农业科学院饲料研究所 在转基因植物中表达耐热植酸酶
AU2013217568B2 (en) * 2012-02-07 2018-09-27 Danisco Us Inc. Glycosylation as a stabilizer for phytase
EP3257381A1 (fr) * 2016-06-16 2017-12-20 Clariant International Ltd Utilisation de bactéries 3-phytase pour aliments ou produits alimentaires
CN108614069B (zh) * 2018-07-26 2024-07-23 山东省科学院生物研究所 一种植酸酶发酵液中植酸酶活性的测定方法和装置

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EP0619369A1 (fr) * 1993-04-05 1994-10-12 Aveve N.V. Hydrolyse de la phytate et composition enzymatique pour hydrolyser la phytate
EP0682876A1 (fr) * 1994-05-20 1995-11-22 Soufflet Alimentaire Procédé de fabrication d'une purée de légumineuses déshydratées instantanée
WO1997016981A1 (fr) * 1995-11-07 1997-05-15 Gist-Brocades B.V. Compositions stables comprenant un materiau de plante transgenique
WO1997035016A1 (fr) * 1996-03-18 1997-09-25 Novo Nordisk Biotech Inc Polypeptides a activite phytase et acides nucleiques codant pour lesdites polypeptides

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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000043503A1 (fr) * 1999-01-22 2000-07-27 Novozymes A/S Phytases ameliorees
US6720174B1 (en) 1999-01-28 2004-04-13 Novozymes A/S Phytases
WO2001025411A1 (fr) 1999-10-01 2001-04-12 Novozymes A/S Produit enzymatique seche par atomisation
US7186817B2 (en) 2000-05-04 2007-03-06 Forskningscenter Riso Polynucleotides encoding phytase polypeptides
US7563878B2 (en) 2000-11-10 2009-07-21 Adisseo France S.A.S. Bacterial phytases and method for producing same
WO2002038774A3 (fr) * 2000-11-10 2003-01-09 Aventis Animal Nutrition Sa Phytases bacteriennes et procede de production de ces phytases
WO2003066847A3 (fr) * 2002-02-08 2003-12-18 Novozymes As Variants de phytase
WO2003066847A2 (fr) 2002-02-08 2003-08-14 Novozymes A/S Variants de phytase
US7238378B2 (en) 2002-02-08 2007-07-03 Novozymes A/S Phytase variants
CN100366736C (zh) * 2002-02-08 2008-02-06 诺维信公司 肌醇六磷酸酶变体
EP2295553A1 (fr) 2002-02-08 2011-03-16 Novozymes A/S Variants de phytase
EP2160950A1 (fr) 2004-09-27 2010-03-10 Novozymes A/S Granules d'enzyme
EP2143339A1 (fr) 2004-09-27 2010-01-13 Novozymes A/S Granules d'enzyme
EP2143338A1 (fr) 2004-09-27 2010-01-13 Novozymes A/S Granules d'enzyme
DE202005021810U1 (de) 2004-09-27 2010-04-22 Novozymes A/S Körnchen mit einem Kern und einer Beschichtung
EP2258209A1 (fr) 2004-09-27 2010-12-08 Novozymes A/S Granules d'enzyme
WO2008017661A1 (fr) 2006-08-07 2008-02-14 Novozymes A/S Granules d'enzyme pour alimentation animale
EP3072399A1 (fr) 2006-08-07 2016-09-28 Novozymes A/S Granules d'enzyme pour alimentation animale
EP2116136A1 (fr) 2008-05-08 2009-11-11 Nederlandse Organisatie voor toegepast- natuurwetenschappelijk onderzoek TNO Nouvelles phytases
WO2020115179A1 (fr) 2018-12-05 2020-06-11 Novozymes A/S Utilisation d'un granule enzymatique

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