AU753475C

AU753475C - Thermostable phytases in feed preparation and plant expression

Info

Publication number: AU753475C
Application number: AU33267/99A
Authority: AU
Inventors: Svend Petersen
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 1998-03-23
Filing date: 1999-03-22
Publication date: 2003-04-17
Anticipated expiration: 2019-03-22
Also published as: CN1293542A; AU3326799A; JP2002508942A; WO1999048380A1; BR9909006A; KR20010042063A; CA2325440A1; EP1065941A1; AU753475B2

Description

1

Thermostable phytases in feed preparation and plant expression

Technical Field This application relates to thermostable phytases, viz. their use in processes for the production of animal feed, and their expression in plants.

Background art WO 91/14782 describes transgenic tobacco and rapeseed plants expressing a phytase derived from Aspergillus ficuum NRRL 3135. The transgenic tobacco seeds are fed to broilers.

US 5,824,779 describes in standard fashion how to produce transgenic alfalfa expressing the same A. ficuum phytase, and the preparation of a phytase-containing concentrate which can be used per se as an animal feed supplement.

EP 0 556 883 Bl describes a method for preparing feed pellets based on an extrusion technique. The addition of temperature sensitive agents, one example of which is phytase, takes place after extrusion of the feed pellets, and the sensitive agents are loaded onto the pellets under reduced pressure.

As acknowledged in EP 0 556 883 Bl the loss of activity of heat-sensitive substances during feed preparation processes is a well-known problem. The above EP-patent proposes to solve this problem by adding these substances under reduced pressure subsequent to the extrusion process. This solution, however, requires a liquid form of the sensitive substance, as well as the installation of additional expensive process equipment. The present invention provides an improved process for preparing animal feed, as well as improved phytase-expressing transgenic plants.

Summary of the Invention

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.

The transgenic plan"c or part thereof, e.g. seeds or leaves, 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.

Brief description of the Figures

In the detailed description of the invention below, reference is made to the drawings, of which

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 α-mutant; and Fig. 5 shows the design of the consensus-phytase-1 a ino acid sequence; 3

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 α-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,

Y305H, A314T, S364G, M365I, A397S, S398A, G404A, and A405S.

Detailed description of the invention In the present context a "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.

The concept of "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 (or for short myo-inositol hexakisphosphate) 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.

This leads to a need of supplementing food and feed preparations with e.g. inorganic phosphate. 5 The non-metabolizable phytic acid phosphorous 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.

10 Phytic acid is degradable by phytases. In the present context 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)

15 inorganic phosphate.

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.

20 There are several descriptions of phytase producing filamentous fungi belonging to the fungal phylum of Ascomycota

(ascomycetes) . In particular, there are several references to phytase producing ascomycetes of the Aspergillus genus such as

Aspergillus terreus (Yamada et al., 1986, Agric. Biol. Chem.

25 322:1275-1282). Also, the cloning and expression of the phytase gene from Aspergillus niger var. awamori has been described (Piddington et al., 1993, Gene 133:55-62). EP 0420358 describes the cloning and expression of a phytase of Aspergillus ficuum (niger) . EP 0684313 describes the cloning and expression of

30 phytases of the ascomycetes Aspergillus niger, Myceliophthora thermophila, Aspergillus terreus. Still further, some partial 6 sequences of phytases of Aspergillus nidulans, Talaromyces thermophilus, Aspergillus fumigatus and another strain of Aspergillus terreus are given.

The cloning and expression of a phytase of Thermomyces lanuginosus is described in WO 97/35017.

WO 98/28409 describes the cloning and expression αf several basidiomycete phytases, e.g. from Peniophora lycii, Agrocybe pediades, Paxillus involutus and Trametes pubescens.

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

Many assays of phytase activity are known, and any of these can be used for the purpose of the present invention. Preferred phytase assays are included in the examples.

The concept of "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.

"Under the influence of heat" means that the temperatur-ε is at least 65°C, as measured on the product at the outlet of the agglomeration unit. More preferred temperatures are at least

70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or even at least 130°C.

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. Preferably, by properly adjusting 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.

The addition of water vapour or steam is often included in agglomeration, but not as an absolute requirement.

Agglomeration includes, but is not limited to, the well- known processes called extrusion, expansion (or pressure conditioning) and pelleting (or pellet pressing) .

Extrusion is i.a. described at pp. 149-153 of a handbook which is available on request from the Danish Company Sprout- Matador, Glentevej 5-7, DK-6705 Esbjerg 0 or Niels Finsensvej 4, DK-7100 Vejle ("Handbog i Pilleteringsteknik 1996") . However, in the agglomeration process of the invention, the following 8 process steps mentioned in the above handbook are entirely optional:

(i) pre-treating the feed ingredients in a cascade mixer; (ii) cutting the product leaving the nozzle-section into pieces (iii) of a desired size;

(iv) acclimatizing or conditioning it; -

(v) coating it; (vi) drying it; (vii) cooling it. The process of expansion (pressure conditioning) is i.a. described in the same handbook at pp. 61-66. Also for expansion, the above process steps (i)-(vi), in particular steps (i) and (vi) , are entirely optional steps.

This is so also for the following process steps: (ii' ) comminuting the product (using e.g. a blade granulator as shown at p. 65) ; (vii) pelleting the product (using e.g. a pellet press as shown at p. 62) ;

The process of pelleting is i.a. described in the same handbook at pp. 71-107. Also here, 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.

In a preferred agglomeration process of the invention, one or more of the above mentioned further process steps (i)-(vii) are included.

A particularly preferred further step is step (i) .

In a most preferred embodiment, the feed-ingredients are pre-heated in a first step (a) to a temperature of at least

45°C, preferably at least 50, 55, 60, 65, 70, 75, 80 °C; and then heated in a second step (b) to a temperature of at least 9

65°C, preferably 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or even at least 130°C.

The addition of 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

(melting temperature) as measured on purified phytase protein by

Differential Scanning Calorimetry (DSC) of at least 65°C, preferably using for the DSC a constant heating rate, more preferably of 10°C/min. In preferred embodiments, the Tm is at least 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75°C. Preferably, 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. Accordingly, 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.

Particularly preferred ranges for Tm are 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. In Example 3 below, the measurement of Tm by DSC is described, and the T ' s of a number of phytases are shown.

The optimum temperatures are also indicated, since - in the alternative - a thermostable phytase can be defined as a phytase having a temperature-optimum of at least 60 °C. Preferably, 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.

In preferred embodiments, the optimum temperature is at least 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70°C. Preferably, 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. Accordingly, 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-

10 130°C; - (etc.) - 60-100°C, 61-100°C, - (etc.) - 70-100°C.

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

15 sequence of Consensus-phytase-10-thermo-Q50T-K91A - of at least 48%, preferably at least 50, 52, 55, 60, 62, 65, 67, 70, 73, 75, 77, 80, 82, 85, 88, 90, 92, 95, 98 or 99%.

The degree of similarity or homology, alternatively identity, can be determined using any alignment programme known

20 in the art. 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

25 Molecular Biology, 48, 443-453) . Using GAP with the following settings for polypeptide sequence comparison: GAP weight of 3.000 and GAP lengthweight of 0.100.

A multiple sequence alignment can be made using the program PileUp (Program Manual for the Wisconsin Package,

30 Version 8, August 1994, Genetics Computer Group, 575 Science 11

Drive, Madison, Wisconsin, USA 53711) , with a GapWeight of 3.000 and a GapLengthWeight of 0.100.

Using the program GAP, some selected phytases exhibit the following percentage similarity (identity in brackets) to the Consensus-phytase-10-thermo (3) -Q50T-K91A amino acid sequence:

A. fumigatus ATCC-13073 α-mutant 86.7% (81.8%)

Basidiomycet consensus 64.1% (49.0%)

Consensus-phytase-1 98.7% (97.9%) Consensus-phytase-10 96.6% (94.4%)

Consensus-phytase-l-thermo(8)-Q50T-K91A 97.4% (95.5%) Consensus-phytase-11 96.5% (94.2%)

Consensus-phytase-12 92.5% (89.9%)

Consensus-phytase-7 95.5% (93.4%)

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.

Preferred 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;

(ii) 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.

Other preferred thermostable phytases are 12

(iii) a 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. 11); 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 α-mutant; Aspergillus fumigatus ATCC 13073 phytase α-mutant plus the mutations E59A, S126N, R329H, S364T, G404A; Aspergillus fumigatus

ATCC 13073 phytase α-mutant plus the mutations E59A, K68A,

S126N, R329H, S364T, G404A; Consensus-phytase-7 ;

Consensus-phytase-12.

(iv) as well as 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.

The term "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

The term "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. Preferably, 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. In a preferred embodiment 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.

Accordingly, the present teaching is applicable to more than one plant variety. No naturally occuring plant varieties are included amongst the plants of the invention. In another preferred embodiment 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. Preferably, 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) . The nucleotide sequence encoding a 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.

In order to accomplish expression of the thermostable phytase in a plant of the invention 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.

In order for transcription to occur 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. Typically, 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.

Examples of 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.

In order to increase the expression of the thermostable phytase it is desirable that a promoter enhancer element is used. For instance, 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. For instance, 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. 489-494 (1996)) or the first intron from the Actl (actin) gene. As an example of a dicot intron the chsA intron (Vain et al . op cit.) is mentioned. Also, 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

(Phaseolus vulgaris) disclosed by Vandergeest and Hall, Plant Molecular Biology Vol. 32, No. 4, pp. 579-588 (1996).

Also, 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 . -

To facilitate selection of successfully transformed plants, 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. One widely used selection marker is the neomycin phosphotransferase gene (NPTII) which provides kanamycin resistance. Examples of other 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. For transformation of monocots particle bombardment (ie biolistic transformation) of embryogenic cell lines or cultured embryos are preferred. Below, references are listed, which disclose various methods for transforming various plants: Rice (Cristou et al. 1991, Bio/Technology 9, pp. 957-962), Maize (Gordon-Kamm et al. 1990, Plant Cell 2, pp. 603-618), Oat (Somers et al. 1992, 17

Bio/Technology 10, pp 1589-1594), Wheat (Vasil et al. 1991, Bio/Technology 10, pp. 667-674, Weeks et al. 1993, Plant Physiology 102, pp. 1077-1084) and Barley (Wan and Lemaux 1994, Plant Physiology 102, pp. 37-48, review Vasil 1994, Plant Mol. Biol. 25, pp 925-937) .

More specifically, 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. In the case 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. Additionally, a selection gene e.g. the neomycin phosphotransferase type II (NPTII) gene from transposon Tn5 and a reporter gene such as the GUS (betha-glucuronidase) gene is cloned between the borders. A disarmed Agrobacterium strain harboring a helper plasmid containing the virulens genes is transformed with the above vector. The transformed Agrobacterium strain is then used for plant transformation. 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.

The above method in which "at least one" replaces "a," 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) .

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

Of particular interest are monocotyledonous plants, in particular 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).

Of further particular interest are dicotyledonous plants, such as those mentioned above.

In a preferred embodiment, the ancestral plant or host plant is per se a desired feed ingredient. 19

Examples

Example 1

FYT-assay - for analyzing phytase enzyme preparations

The phytase activity can be measured using the following assay: 10 μl diluted enzyme samples (diluted in 0.1 M sodium acetate, 0.01 % Tween20, pH 5.5) are added into 250 μl 5 mM sodium phytate (Sigma) in 0.1 M sodium acetate, 0.01 % Tween20, pH 5.5 (pH adjusted after dissolving the sodium phytate; the substrate is preheated) and incubated for 30 minutes at 37 °C. The reaction is stopped by adding 250 μl 10 % TCA and free phosphate is measured by adding 500 μl 7.3 g FeS04 in 100 ml molybdate reagent (2.5 g (NH₄) ₆Mo₇0₂₄.4H₂0 in 8 ml H₂S0₄ diluted to 250 ml). The absorbance at 750 nm is measured on 200 μl 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 μmol phosphate/min at the given conditions. This assay is preferred for phytase enzyme preparations (when not in admixture with other feed ingredients) .

Example 2

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 μmol phosphate per minute .

1 FTU = 1 FYT

The FTU assay is preferred for phytase activity measure- ments on animal feed premixes and the like complex compositions. 20

Reagents /substrates

Extraction buffer for feed etc.

This buffer is also used for preparation of P0₄-standards and further dilution of premix samples. 0.22 M acetate buffer with Tween 20 pH 5,5

30 g sodium acetate trihydrate (MW = 136,08 g/mol) e.g. Merck Art 46267 per liter and 0,1 g Tween 20 e.g. Merck Art 22184 pr. liter are weighed out.

The sodium acetate is dissolved in demineralised water. Tween 20 is added, and pH adjusted to 5,50 ± 0,05 with acetic acid.

Add demineralised water to total volume.

Extraction buffer for premix

0,22 M acetate buffer with Tween 20, EDTA, P0₄ ^3~ og BSA. 30 g sodium acetate trihydrate e.g. Merck Art 6267 per liter.

0,1 g Tween 20 e.g. Merck Art 22184 per liter.

30 g EDTA f .eks. Merck Art 8418 pr. liter.

20 g Na₂HP0₄, 2H₂0 e.g. Merck Art 6580 per liter. 0,5 g BSA (Bovine Serum Albumine, e.g. Sigma Art A-9647 per liter.

The ingredients are dissolved in demineralised water, and pH is adjusted to 5,50 ± 0,05 with acetic acid.

Add demineralised water to total volume. BSA is not stable, and must therefore be added the same day the buffer is used. 21 50 mM PQ₄ ^3~stock solution

0,681 g KH2P04 (MW = 136,09 g/mol) e.g. Merck Art 4873 is weighed out and dissolved in 100 ml 0,22 M sodium acetat with Tween, pH 5,5. Storage stability: 1 week in refrigerator.

0.22 M acetate buffer pH 5 , 5 without Tween

This buffer is used for production of phytic acid substrate) .

150 g sodium acetate trihydrate (MW = 136,08) e.g. Merck Art 6267 is weighed out and dissolved in demineralised water, and pH is adjusted with acetic acid to 5,50 ± 0,05.

Add demineralised water to 5000 ml.

Storage stability: 1 week at room temperature.

Phytic acid substrate: 5 mM phytic acid The volume of phytic acid is calculated with allowance for the water content of the used batch.

If the water content is e.g. 8,4 % the following is obtained:

0,005 moU /x 923,8 g / mol 7 ^ = 5,04g / l

(l÷ 0,084)

Phytic acid (Na-salt) (MW = 923,8 g/mol) e.g. Sigma P-8810 is weighed out and dissolved in 0,22 M acetate buffer (without tween) . Addition of (diluted) acetic acid increases the dissolution speed. pH is adjusted to 5,50 ± 0,05 with acetic acid.

Add 0,22 M acetate buffer to total volume. 21.7 % nitric acid solution 22

For stop solution.

1 part concentrated (65%) nitric acid is mixed into 2 parts demineralised water. Molybdate reagent For stop solution.

100 g ammonium heptamolybdate tetrahydrate (NH₄) 6Mo₇0₂₄, H₂0 e.g. Merck Art 1182 is dissolved in demineralised water. 10 ml 25 % NH₃ is added.

Add demineralised water to 1 liter. 0.24 % Ammonium vanadate

Bought from fra Bie & Berntsen.

Molybdat/vanadat stop solution

1 part vanadate solution (0,24 % ammonium vanadate) + 1 part molybdate solution are mixed. 2 parts 21,7 % nitric acid solution are added.

The solution is prepared not more than 2 hours before use, and the bottle is wrapped in tinfoil.

Samples

Frozen samples are defrosted in a refrigerator overnight. Sample size for feed samples: At least 70 g, preferably 100 g.

Feed samples

Choose a solution volume which allows addition of buffer corresponding to 10 times the sample weight, e.g. 100 g is dis- solved in 1000 ml 0,22 M acetate buffer with Tween, see enclosure 1. Round up to nearest solution volume.

If the 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.

After extraction the samples rest for 30 minuts to allow for the feed to sediment. A 5 ml sample is withdrawn with a pipette. The sample is taken 2 - 5 cm under the surface of the so- lution and placed in a centrifuge glass, which is covered by a lid.

The samples are centrifuged for 10 minutes at 4000 rpm.

Premix samples

Choose a solution volume which allows addition of buffer corresponding to 10 times the sample weight. Round up to nearest solution volume.

If the samples have been weighed (50 - 100 g) all of the sample is placed in tared beakers. The sample weight is noted.

If a sample is too big to handle, it is split into parts of ap- prox. 100 g.

Magnets are placed in the beakers and 0,22 M acetate buffer with Tween, EDTA og P0₄ ^3~ is added.

The samples are extracted for 60 minutes.

After extraction the samples rest for 30 minutes to allow for the premix to sediment. A 5 ml sample is withdrawn with a pipette. The sample is taken 2 - 5 cm under the surface of the solution and placed in a centrifuge glass, which is covered by a lid. 24

The samples are centrifuged for 10 minutes at 4000 rpm.

Analysis

Extracts of feed samples are analysed directly.

Extracts of premix are diluted to approx. 1,5 FTU/g (A₄₁₅ (main sample) < 1,0 ) .

0,22 M acetate buffer with Tween 20 is used for the dilution.

Main Samples

2 x 100 ml of the supernatant from the extracted and cen- trifuged samples are placed in marked glass test tubes and a magnet is placed in each tube.

When all samples are ready they are placed on a water bath with stirring. Temperature: 37 °C.

3 , 0 ml substrate is added. Incubation for exactly 60 minutes after addition of substrate .

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) .

Blind 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.

2,0 ml stop solution is added to the samples. 25 3 , 0 ml substrate is added to the samples.

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) .

Standards

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) .

A₄₁₅ is measured on all samples.

CALCULATION

FTU/g = μmol P0₄ ^{3 "} / (min * g ( sample) )

C g sample is weighed out (after grinding) .

100 μl is taken from the extracted and centrifuged sample.

P0₄ ^3~ standard curve is linear.

From the regression curve for the P0₄ ^3" standard the actual con- centration of the sample is found (concentration in mM) :

[P0₄ ^3~] = (x - b) / a x = A₄₁₅ a = slope b = in tercept wi th y-axis

μmol P0₄ ³7min = { [P0₄ ^3~] (mM) x Vol (liter) x 1000 μmol/mmol } /t t = incubation time in minutes. Vol = sample volume in liter = 0,0001 liter 1000 = conversion factor from mmol to μmol 26

FTU /g_prβve = { (x - b) x Vol x 1000 x F_p} / { a x t x C)

C - gram sample weighed out F_p = Relation between the sample taken out and the total sample (after extraction). Example: 0,100 ml taken from 1000 ml -» F_p = 1000/0,100 = 10000.

Reduced expression with insertion of the following values: t = 60

Vol = 0,0001 1

F_p = 10000

FTU /g_sample = { (x - b) x 0,0001 x 1000 x 10000} / { a x 60 x C}

Example 3

Determination of optimum temperature and melting point Tm of various phytases

The 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).

The phytases of Aspergillus fumigatus ATCC 13073, Aspergillus terreus 9A-1, Aspergillus terreus CBS 116.46,

Aspergillus nidulans, Myceliophthora thermophila, and

Talaromyces thermophilus were prepared as described in

EP-0897985 and the references therein.

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)_τ 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.

The muteins 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.

The muteins Consensus-phytase-1-thermo (8) , Consensus- phytase-1-thermo (8) -Q50T-K91A (Fig. 9) and Consensus-phytase-1- thermo (8) -Q50T, were 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.

An Aspergillus fumigatus so-called α-mutant (with the mutations Q51(27)T, F55Y, V100I, F114Y, A243L, S265P, N294D) and the further muteins thereof shown in Table 1 were prepared as* generally described above. The position numbering refers to Fig. 11 hereof, except for the number in parentheses which refers to the numbering used in EP 0897010. DNA constructs encoding the above thermostable phytases can be prepared e.g. according to the teachings of EP 0897985. For expression thereof in plants, reference is made to the present description.

In order to determine the unfolding temperature or melting temperature, Tm, of a phytase, 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.; Dalbøge, H. and Loewus, F.; Kluwer Academic Publishers).

Homogenous or purified phytase solutions of 50-60 mg/ml of protein are prepared, and extensively dialyzed against 10 mM sodium acetate, pH 5.0. A constant heating rate of 10°C/min is applied up to 90-95°C. The results of Tm determinations on the above phytases are shown in Table 1 below; for selected phytases also in Figs. 1-4.

In Table 1 below, the optimum temperature of various phytases is also indicated. For this determination, 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 μl of the assay mixture with 900μl H₂0 and 1 ml of 0.6 M H₂S0₄, 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 μmol 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.

For determination of the temperature optimum, enzyme (lOOμl) and substrate solution (lOOμl) 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.

Table 1

Temperature optimum and Tm for various phytases

Phytase Optimum temperature Tm (°C) (°C)

Aspergillus niger 55 63.3

NRRL 3135

Aspergillus 55 62.5 fumigatus ATCC 13073 30

Aspergillus terreus 49 57.5 9A-1

Aspergillus terreus 45 58.5 CBS 116.46

Aspergillus nidulans 45 55.7

Myceliophthora 55 thermophila

Talaromyces 45 thermophilus

Consensus-phytase- 82 89.3 10-thermo-Q50T-K91A

Consensus-phytase- 82 88.6 10-thermo-Q50T

Consensus-phytase-10 80 85.4

Consensus-phytase-1- 85.7 thermo ( 8 ) -Q50T-K91A

Consensus-phytase-1- 78 84.7 thermo(8)-Q50T

Consensus-phytase-1- 81 thermo (8)

Consensus-phytase-1- 78 84.7 thermo ( 8 ) -Q50T-K91A

Consensus-phytase-1- 75 thermo (3)

Consensus-phytase-1- 78.9 Q50T

Consensus-phytase-1 71 78.1

Aspergillus 63 fumigatus α-mutant, plus mutations E59A, 31

S126N, R329H, S364T, G404A

Aspergillus 63 fumigatus - as above, plus mutation

-

K68A

Aspergillus 60 67.0 fumigatus α-mutant

(Q51(27)T, F55Y,

V100I, F114Y, A243L,

S265P, N294D)

Claims

32CLAIMS

1. 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.

2. The process of claim 1, wherein the feed ingredients are heated to a temperature of at least 65┬░C.

3. The process of any of claims 1-2, wherein the thermostable phytase is a phytase with a Tm as measured by DSC of at least 65┬░C, using for the DSC a constant heating rate of 10┬░C/min.

4. The process of any of claims 1-3, when performed in a feed expander.

5. The process of any of claims 1-3, when performed in an extruder.

6. The process of any of claims 1-3, when performed in a pellet press.

7. The process of any of claims 1-6, wherein the thermostable phytase is present in a transgenic plant.

8. The process of any of claims 1-7, wherein the agglomeration includes the following steps:

(a) pre-heating the feed ingredients to a temperature of at least 45┬░C; and (b) heating the product of step (a) to a temperature of at least 65┬░C; 33 wherein the thermostable phytase is added prior to or during step (a) and/or (b) .

9. A transgenic plant which comprises a DNA-construct 5 encoding a thermostable phytase.

10. The transgenic plant of claim 9, wherein the DNA-construct encoding the thermostable phytase is operably linked to regulatory sequences capable of mediating expression of said

10 phytase encoding sequence in at least one part of the plant.

11. An expression construct which comprises a DNA construct encoding a thermostable phytase, operably linked to regulatory sequences capable of mediating expression of said phytase

15 encoding sequence in at least one part of a plant.

12. A vector which comprises the expression construct of claim 11.

20 13. 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; 25 (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.

30 34

14. The method of claim 13, which comprises the further step of extracting the phytase from the plant.