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WO1998030681A1 - Combinaisons de phytase - Google Patents

Combinaisons de phytase Download PDF

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Publication number
WO1998030681A1
WO1998030681A1 PCT/DK1997/000586 DK9700586W WO9830681A1 WO 1998030681 A1 WO1998030681 A1 WO 1998030681A1 DK 9700586 W DK9700586 W DK 9700586W WO 9830681 A1 WO9830681 A1 WO 9830681A1
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WO
WIPO (PCT)
Prior art keywords
phytase
phytases
phosphate
lns
inositol
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PCT/DK1997/000586
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English (en)
Inventor
Anders Ohmann
Inge Helmer Knap
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Novo Nordisk A/S
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Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU54778/98A priority Critical patent/AU5477898A/en
Publication of WO1998030681A1 publication Critical patent/WO1998030681A1/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • 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

Definitions

  • the present invention relates generally to the use of at least two phytases of different position specificity.
  • the invention relates to animal feed and animal feed additives comprising such phytases.
  • 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. 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, including digestive enzymes, by electrostatic interaction.
  • 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 absorbabie from the gastro intestinal system, i.e. neither the phosphorous thereof, nor the chelated metal ions, nor the bound proteins are nutritionally available. Accordingly, since phosphorus is an essential element for the growth of all organisms, food and feed preparations need to be supplemented with inorganic phosphate. Quite often also the nutritionally essential ions such as iron and calcium, must be supplemented. And, besides, the nutritional value of a given diet decreases, because of the binding of proteins by phytic acid. Accordingly, phytic acid is often termed an anti-nutritional factor.
  • 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.
  • 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 of e.g. wheat-bran and maize root have been described (Thomlinson et al, Biochemistry, 1 (1962), 166-171 ; and H ⁇ bel, F. & E. Beck; Plant Physiology (1996), 112: 1429-1436, respectively).
  • An alkaline phytase from lilly pollen has been described by Barrientos et al, Plant. Physiol., 106 (1994), 1489-1495.
  • These plant phytases are generally 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.
  • Phytase producing yeasts are also described, such as Saccharomyces cerevisiae (Nayini et al, 1984, Strukturtician und Technologie 17:24-26. However, this enzyme is probably a myo-inositol monophosphatase (Wodzinski et al, Adv. Appl. Microbiol., 42, 263-303).
  • AU-A-24840/95 describes the cloning and expression of a phytase of the yeast Schwanniomyces occidentalis.
  • 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, IP1 , IP2, IP3, IP4, IP5 and P, respectively. This means that by action of a phytase, IP6 is degraded into P + one or more of the components IP5, IP4, IP3, IP2, IP1 and I.
  • lns(p,q,r,..)Pn myo-inositol carrying in total n phosphate groups attached to positions p, q, r,..
  • lns(p,q,r,..)Pn myo-inositol carrying in total n phosphate groups attached to positions p, q, r,..
  • PA lns(1 ) 2,3,4,5,6)P6 (phytic acid)
  • Inositolphosphate phosphate esters of myo-inositol
  • 1D- (or 1 L-) -lns(r,s,t,u,w,x)Pn n indicating the number 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 (NCIUB) cited above (and the references herein), and 1 D or 1 L is used so as to make a substituent have the lowest possible locant or number ("lowest-locant rule").
  • Phytase specificity As said above, phytases are divided according to their specificity in the initial hydrolysis step, viz. according to which phosphate-ester group is hydrolyzed first.
  • 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 feed consumed has a long residence time in the acid environment in the stomach which has a negative influence on the perfomance of any added enzyme. Consequently a rapid and more efficient removal of phosphorus groups from the phytate molecule is of similar importance in pigs.
  • the invention provides compositions, which comprise at least two phytases of different position specificity.
  • compositions are food and animal feed as well as additives for use in food and feed.
  • the invention provides processes for preparing these compositions, and the use of these compositions for various purposes.
  • the invention provides a method of enzymatic release of phosphorous from a phytase substrate, in particular a phytate containing substrate, which method comprises subjecting the substrate to the simultaneous action of a 3-phytase (EC 3.1.3.8) and a 6-phytase (EC 3.1.3.26).
  • the invention relates to the use of at least two phytases of different position specificity.
  • the at least two phytases are distinct or different enzymes.
  • the phytases are selected from amongst the group consisting of 2-phytases, 3- phytases, 5-phytases and 6-phytases (as defined below to include 1 -phytase in the definition of 3-phytase and 4-phytase in the definition of 6-phytase).
  • any sub-group selected from this group which sub-group consists of any set of two, any set of three or all four members of the group, is within the scope of this invention.
  • Preferred examples of sub-groups of two phytases are, using in this paragraph the notation "N" for "a N-phytase:" 3+6 (viz. a 3-phytase and a 6-phytase); 2+6; 5+6; 3+2; 3+5; 2+5.
  • N-phytase 2+5+6 (viz. a 2-phytase and a 5-phytase and a 6- phytase); 2+5+3; 3+6+2; 3+6+5; 6+2+3; 6+2+5.
  • the phytase activity can be determined using any assay in which a phytase substrate is used.
  • a phytase substrate examples (non-exhaustive list) of phytase substrates are the various myo- inositol phosphates.
  • any stereoisomer of the mono-, di-, tri-, tetra- or penta-phosphates of myo-inositol might serve as a phytase substrate.
  • the phytase activity is determined in the unit of FYT, one FYT being the amount of enzyme that liberates 1 ⁇ moi inorganic ortho-phosphate per min. under the following conditions: pH 5.5; temperature 37°C; substrate: sodium phytate (C 6 H 6 O 24 P 6 Na 12 ) in a concentration of 0.0050 mol/l.
  • a suitable phytase assay is described below.
  • the phytase specificity can be examined in several ways, e.g by HPLC or by NMR spectroscopy, as further outlined in the experimental part. 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- lns(1 , 2,3,4, 5)P5 and L-lns(1 , 2,3,4, 5)P5, are enantiomers (mirror images), and therefore have identical NMR spectres.
  • N 2, 3, 5 or 6
  • 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 a D/L-6-phytase.
  • a 3-phytase means either of a D-3-, L-3- or D/L-3-phytase.
  • a 5-phytase and a 2-phytase comprise only one type of enzymes each, viz. those in which the penta phosphate ester resulting from the first step of the hydrolysis of phytic acid is unambiguously defined.
  • a 6-phytase preferably more than 50% of the hydrolysis product of the first step is lns(1 , 2,3,4, 5)P5 and/or lns(1 , 2,3,5, 6)P5.
  • 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.
  • N 2, 3 or 5
  • more than 50, 60, 70, 80, 90, or mostly preferred more than 95% of the hydrolysis product of the first step is ins(1 ,3,4,5,6)P5; (lns(2,3,4,5,6)P5 and/or lns(1 ,2,4,5,6)P5); or lns(1 ,2,3,4,6)P5, respectively.
  • Corresponding preferred embodiments (more than 50, 60, 70, 80, 90, 95%) apply with respect to a D-6-; a L-6-; a D/L-6-; a D-3-; a L-3-; and a D/L-3-phytase.
  • sub-groups of three phytases are (using the same notation as in the paragraph above): D6+L6+D3; D6+L6+L3; D3+L3+D6; D3+L3+L6 D6+D3+2; D6+D3+5; D6+L3+2; L6+L3+2; L6+D3+2; D6+L3+5; L6+L3+5; L6+D3+5 D/L6+L3+L6; D/L6+L3+D6; D/L6+L3+5; D/L6+D3+5; D/L6+D3+5; D/L6+D3+5; D/L6+D3+2 D/L6+L3+2 etc.
  • the 3-phytase is of microbial origin. In a more preferred embodiment, the 3-phytase is of fungal origin. In a most preferred embodiment, the 3- phytase is derived from a strain of Aspergillus, in particular from a strain of Aspergillus awamori, a strain of Aspergillus ficuum, or a strain of Aspergillus niger.
  • a microbial 3-phytase, Phytase NovoTM, derived from Aspergillus niger, is commercially available from Novo Nordisk A/S, Denmark.
  • the 6-phytase is of plant origin.
  • the 6-phytase is derived from a wheat, rye or maize plant. Wheat phytase is commercially available, e.g. from Sigma (P-1259).
  • the 6-phytase is of bacterial origin, preferably derived from a strain of Escherichia coli, in particular the strain Escherichia coli K12, ATCC 33965.
  • a 6-phytase from the strain Escherichia coli K12, ATCC 33965 has been purified and characterized by Greiner et al. [Greiner R, Konietzny U and Jany Kl-D; Archives of Biochemistry and Biophysics, 1993, 303 (1 ), 107-113].
  • the 6-phytase is of fungal origin.
  • a most preferred 6-phytase is derived from Peniophora lycii, in particular Peniophora lycii CBS 686.96, or a mutant or a variant thereof. The isolation and purification of this phytase is described in Examples 1 and 2 below.
  • a preferred 5-phytase is derived from I Illy pollen.
  • the phytases are preferably added in the form of mono-component preparations, i.e. enzyme preparations in which for instance essentially all of the 3-phytase activity or the 6-phytase activity (the phytase activity detectable) is owing to single phytase components.
  • the invention relates to compositions comprising at least two phytases of different position specificity and processes for preparing such compositions.
  • a composition comprising only a mixture of the at least two phytases (and nothing more) is also included in this definition, the composition usually also contains other components. Examples of such compositions are food and feed, as well as phytase preparations, in particular food and feed additives.
  • a feed and a food means any natural or artificial diet, meal or the like or components of such meals intended or suitable for being eaten, taken in, digested, by an animal and a human being, respectively.
  • Such product is prepared by mixing feed or food components, to usually provide a desired amount of protein, carbohydrates, lipids, vitamins and minerals.
  • Phytase preparations formulated enzyme compositions or preparations
  • Liquid preparations need not contain anything more than the phytase enzymes, preferably in a highly purified form. Usually, however, a stabilizer such as glycerol, sorbitol or mono propylen glycol is also added.
  • the liquid preparation may also comprise other additives, such as salts, sugars, preservatives, pH-adjusting agents, proteins, phytate (a phytase substrate). Typical liquid preparations are aqueous or oil- based slurries. The liquid preparations can be added to a food or feed after an optional pelleting thereof.
  • Dry preparations may be spray dried preparations, in which case the preparation need not contain anything more than the enzymes in a dry form.
  • dry preparations 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 disodium 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.
  • Such mixture comprises coating agents, preferably hydrophobic coating agents, such as hydrogenated palm oil and beef tallow, and if desired other additives, such as calcium carbonate or kaolin.
  • phytase preparations may contain other substituents such as coloring 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 composed as indicated for phytase preparations above and intended for or suitable for being added to food or feed.
  • such additive is prepared by mixing the additive components, examples of which are mentioned above and below.
  • 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 preparations or additives of the invention additionally comprise 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- ⁇ -4-glucanases and endo- ⁇ -1 ,3(4)-glucanases, cellulases, xylosidases, galactanases, in particular arabinogalactan endo-1 ,4- ⁇ - galactosidases and arabinogalactan endo-1 ,3- ⁇ -galactosidases, endoglucanases, in particular endo-1 ,2- ⁇ -glucanase, endo-1 ,3- ⁇ -glucanase, and endo-1 ,3- ⁇ -glucanase, pectin degrad
  • the animal feed additive of the invention is supplemented to the animal before or simultaneously with the diet.
  • the animal feed additive of the invention is supplemented to a 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 overall 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.
  • the phytase may exert its effect in vitro or in vivo, i.e. before intake or in the stomach of the individual, respectively. Also a combined action is possible.
  • the invention relates to the use of at least two phytases of different position specificity for liberating phosphorous from a phytase substrate.
  • the two or more different types of phytase enzymes referred to above are active simultaneously.
  • they act sequentially, i.e. one type is acting first, the second type subsequently etc.
  • the first type may be active or non-active, when the second type excerts its effect etc., viz. a simultaneous and sequential, and a pure sequential action, respectively.
  • a synergistic effect is observed on the release of phosphorous from the phytase substrate, in particular inorganic phosphate from phytate.
  • the enzymes are active simultaneously.
  • the effect can be exerted in vitro, viz. e.g. by pre-treating animal feed, or in vivo, i.e. in the digestive system of the animal.
  • the phytases are preferably active in vivo.
  • compositions which comprises at least one phytase substrate; i.e. at least one substrate towards which the phytases show an activity.
  • This composition may be an essentially pure mono component substrate or - which is usually the case - based on or originating from a complex biological material, such as cereals and legumes, in particular seeds and cereal grains, e.g. seeds of legumes.
  • a complex biological material such as cereals and legumes, in particular seeds and cereal grains, e.g. seeds of legumes.
  • phytase substrates examples are various myo-inositol phosphates.
  • any stereoisomer of the mono-, di-, tri-, tetra- or penta-phosphates of myo-inositol might serve as a phytase substrate.
  • the phytase substrate is present in amounts significant for being a source of phosphorous. More preferably, the phytase substrate comprises phytate (as broadly defined previously to include phytic acid and phytin).
  • a preferred composition is an animal feed composition
  • an animal feed composition comprising soy bean, field beans, peas, lupines, linseed, sunflower, rape, or cereals, in particular barley, wheat, oat, corn, sorghum, etc.
  • these animal feed compositions are intended for mono-gastric animals.
  • mono-gastric animals include i.a. fish, poultry, in particular broiler chicks, layers and turkeys, pigs, in particular piglets, and young calves.
  • phosphorous When using the phytases, "phosphorous" is released.
  • the term “phosphorous” is to be interpreted broadly, e.g. it is intended to include i.a. inorganic and organic bound phosphorous, whatever the form, e.g. inorganic ortho phosphate, and any P- comprising substituents.
  • the use of the invention may be carried out at conditions usually employed for release of phosphorous from a phytase substrate, e.g. phytate.
  • a phytase substrate e.g. phytate.
  • the conditions are chosen balancing on one hand the optimum conditions of the two enzymes in question, and on the other hand the optimum conditions for the particular application. Some examples of such conditions which could be relevant in practice are described below.
  • the pH may be in the range of from about pH 2 to about pH 8, preferably of from about 3 to about 7, in particular of from about 2 to about 6, still more preferably of from about 4 to about 6.
  • the temperature may be in the range of from about 20°C to about 80°C, preferably of from about 20°C to about 60°C, still more preferably 30-60°C, alternatively around 37-58°C.
  • the reaction time may be of from about 15-30 minutes to about 24 hours, preferably of from about 30 minutes to 6 hours, still more preferably 30 minutes to 4 hours; 30 minutes to 3hours, 30 minutes to 2 hours, even more preferably 30 minutes to 1 hour. Also a reaction time of from about 2 to about 8 hours is highly relevant in feed for pigs.
  • An effective overall 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.
  • animal feed has a dry matter content of 10-20%.
  • the animal feed additive comprises phytase enzymes in total amounts corresponding to of from about 200 to about 50,000 Phytase Units (FYT) per gram of the composition, preferably of from about 500 to about 10,000 FYT per gram of the composition, more preferably of from about 2000 to about 6000 FYT per gram of the composition, most preferably of from about 2500 to about 5000 FYT/g.
  • FYT Phytase Units
  • Nitrilothacetic acid 1.50 g
  • YPD 30 10 g yeast extract, 20 g peptone, H 2 O to 900 ml. Autoclaved, 100 ml 20% glucose (sterile filtered) added. YPM:
  • the expression plasmid (shuttle vector) pYES 2.0 comprising the full length cDNA sequence encoding this phytase has been transformed into a strain of the Escherichia coli which was deposited according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH., Masheroder Weg 1 b, D-38124 Braunschweig, Germany, (DSM).
  • 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; prd ::HIS3; prbl :: LEU2; cir+).
  • E. coli strain DH10B (Life Technologies) Plasmids:
  • the Aspergillus expression vector pHD414 is a derivative of the plasmid p775
  • pHD414 (described in EP 238 023).
  • the construction of pHD414 is further described in WO
  • 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/l, malto dextrin 15 g/l, bacto peptone 5 g/l, pluronic 0.2 g/l). The culture was incubated stationary at 26°C for 15 days. The resulting culture broth was filtered through miracloth and the mycelium was frozen down in liquid nitrogen.
  • medium B soya 30 g/l, malto dextrin 15 g/l, bacto peptone 5 g/l, pluronic 0.2 g/l
  • 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.
  • First-strand cDNA was synthesized by incubating the reaction mixture at 45°C for 1 hour. After synthesis, the mRNA:cDNA hybrid mixture was gelfiltrated through a MicroSpin S-400 HR (Pharmacia) spin column according to the manufacturer's instructions.
  • the hybrids were diluted in 250 ⁇ l second strand buffer (20 mM Tris-CI, pH 7.4, 90 mM KCI, 4.6 mM MgCI 2 , 10 mM (NH 4 ) 2 SO 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-CI, pH 7.4, 90 mM KCI, 4.6 mM MgCI 2 , 10 mM (NH 4 ) 2 SO 4 , 0.16 mM bNA
  • 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
  • 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
  • the cDNAs were recovered by centrifugation, washed in 70% EtOH and dried.
  • the cDNA pellet was resuspended in 25 ⁇ l ligation buffer (30 mM Tris-CI, pH 7.8, 10 mM MgCI 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 10x 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 1x 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. Construction of libraries:
  • the directional, size-selected cDNA was recovered by centrifugation, washed in 70% EtOH, dried and resuspended in 30 ⁇ l 10 mM Tris-CI, 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 containing 0.2M CaCI2 is poured over the plates and after 1-4 days the phytase positive colonies are identified as colonies surrounded by a clearing zone. Cells from enzyme-positive colonies were spread for single colony isolation on agar, and an enzyme-producing single colony was selected for each of the phytase- producing colonies identified.
  • 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 0.1 M Sodiumacetate pH 4.5 and 0.1 % Inositol hexaphosphoric acid. The plates are left over night at 37°C. A buffer consisting of 0.1 M CaCI2 and 0.2M Sodium acetate pH 4.5 is poured over the plates and the plates are left at room temperature for 1 h. Phytase activity is then identified as a clear zone.
  • 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.
  • the phytase encoding part of the DNA sequence 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. 9x10 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.
  • 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. In order to express the phytase in Aspergillus, the DNA was digested with appropriate restriction enzymes, size fractionated on gel, and a fragment corresponding to the phytase gene was purified. The gene was subsequently ligated to pHD414, digested with appropriate restriction enzymes, resulting in the plasmid pA2phy2.
  • the plasmid was transformed into Aspergillus oryzae as described above.
  • 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.
  • Peniophora lycii phytase Purification of the phytase from Peniophora lycii expressed in Aspergillus oryzae
  • 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 polyethersul- phone 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 NaCI gradient (0 -> 0.5M). The phytase activity eluted as a single peak. This peak was pooled and (NH 4 ) 2 SO 4 was added to 1.5M final concentration.
  • Phenyl Toyopearl 650S column was equilibrated in 1.5M (NH 4 ) 2 SO 4 , 10mM succinic acid/NaOH, pH 6.0 and the phytase was applied to this column and eluted with a decreasing linear (NH 4 ) 2 SO 4 gradient (1.5 -> 0M).
  • Phytase containing fractions were pooled and the buffer was exchanged for 20mM Tris/CH 3 COOH, pH 7.5 on a Sephadex G25 column.
  • the G25 filtrate was applied to a Q-sepharose FF column equilibrated in 20mM Tris/CH 3 COOH, pH 7.5.
  • the phytase activity is measured using the following assay:
  • n denotes myo-inositol carrying in total n phosphate groups attached to positions p, q, r,..
  • PA lns(1 ,2,3,4,5,6)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.
  • 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.
  • lns(1 ,2,4,5,6)P 5 (3-phosphate removed) exhibits a NMR spectrum different from lns(1 , 2,3,4, 5)P 5 (6-phosphate removed) because the isomers are diastereo- mers.
  • 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 di- potassium 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 Aspergillus phytase prove to be an essentially clean 3-phytase, whereas the Peniophora phytase at pH 5.5 appear to be an essentially clean 6- phytase.
  • the 3-phytase is Phytase NovoTM which is commercially available from Novo Nordisk A/S, Denmark
  • the 6-phytase is a phytase originating from Peniophora lycii CBS 686.96 (obtained and purified as described in Examples 1 and 2).
  • Male broiler chickens are housed on deep litter under conventional conditions from 1 to 14 days of age and fed a commercial starter diet. On day 14 the birds are weighed individually and birds with low or high body weights are discarded. Groups of 4 birds are then assigned randomly to 35 digestibility cages and for each treatment 5 replicates are used.
  • the Ca level is 0.65 % and the P-level in the negative control 0.41 %.
  • the P-level in the normal diet is 0.41 % + 0.18 % mineral P giving a total of 0.59 % P.
  • a Both diets are supplemented with monensin (100 mg/kg) and avoparcin (10 mg/kg).
  • the in vivo balance trial is conducted according to the European reference method which consists of a 7-day period of adaptation and a 4-day balance period. During the main balance period the birds are fed the respective diets at a level of 90 % of ad libitum intake and their excreta is collected quantitatively each day.
  • Dietary samples approximately 500 g, are analyzed for in-feed enzymatic activity.
  • Inositol phosphate spectrum by Anion exchange HPLC and post-column Ferri / Sulfosalicylic acid reaction Phytate is the fully phosphorylated inositol with phosphates in position 1 to 6, which is de-phosphated by phytase.
  • Acetate buffer 100 mM. pH 5.0 5.80 ml of Acetic acid in 500 mL of water
  • Phytase is diluted with Acetate buffer, pH 5.0 until 0.36 FYT/mL Experiments with phytase (0.18 FYT/mL incubation mixture) and reaction time from 0 to 48 h are being performed.
  • Enzyme mixtures of 3- and 6-specific phytase are prepared as: 25, 50 and 75% of one of the specific phytases while the other is added up to 100%, 0.36 FYT/ml. Thus all incubation mixtures have a total phytase activity of 0.18 FYT/ml.
  • Incubation Test tube with stirring bar, cross type, is placed in water bath at 37°C.
  • reaction times are: 0 min - 5 min - 10 min - 30 min - 1 - 3 - 24 and 48 hours. Stop of incubation
  • the reaction is stopped by adding 70 mL 4 M HCI/tube to the incubation mixture, mixing and placing at room temperature.
  • peaks Five peaks are identified in each assay. They are inositol di-phosphate (IP 2 ), inositol tri-phosphate (IP 3 ), inositol tetra-phosphate (IP 4 ), inositol penta-phosphate (IP 5 ) and Phytic acid (inositol hexa-phosphate ,IP 6 ).
  • IP 2 inositol di-phosphate
  • IP 3 inositol tri-phosphate
  • IP 4 inositol tetra-phosphate
  • IP 5 inositol penta-phosphate
  • IP 6 Phytic acid

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Abstract

L'invention concerne de manière générale l'utilisation d'au moins deux phytases présentant une spécificité de position différente, notamment n'importe quelle combinaison de 1-, 2-, 3-, 4-, 5-, et 6-phytases. En combinant des phytases présentant une spécificité de position différente, on obtient un effet synergique. L'invention concerne en outre des compositions telles que des aliments pour humains et animaux et des additifs alimentaires pour humains et animaux contenant ces phytases combinées.
PCT/DK1997/000586 1997-01-09 1997-12-19 Combinaisons de phytase WO1998030681A1 (fr)

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GB2340727A (en) * 1998-08-19 2000-03-01 Univ Saskatchewan Process for converting phytate into inorganic phosphate
WO2001012792A1 (fr) * 1999-08-13 2001-02-22 The Victoria University Of Manchester Enzymes phytase, acides nucleiques codant pour ces enzymes phytase, et vecteurs et cellules hotes incorporant lesdites enzymes phytase
US6284502B1 (en) 1998-08-21 2001-09-04 University Of Saskatchewan Process for converting phytate into inorganic phosphate
US6511699B1 (en) 1999-03-31 2003-01-28 Cornell Research Foundation, Inc. Enzymes with improved phytase activity
WO2002054881A3 (fr) * 2001-01-10 2003-10-30 Dsm Ip Assets Bv Utilisation d'aliments et de boissons en tant que systeme de distribution de phytase chez les etres humains
WO2002098442A3 (fr) * 2001-06-01 2003-12-04 Krueger Gmbh & Co Kg Composition renfermant une phytase
US7026150B2 (en) 1998-06-25 2006-04-11 Cornell Research Foundation, Inc. Overexpression of phytase genes in yeast systems
FR2888249A1 (fr) * 2005-07-08 2007-01-12 Adisseo France Sas Soc Par Act Effet synergique de l'association de phytases sur l'hydrolyse de l'acide phytique
US7300781B2 (en) 1999-11-18 2007-11-27 Cornell Research Foundation, Inc. Site-directed mutagenesis of Escherichia coli phytase
US7309505B2 (en) 2002-09-13 2007-12-18 Cornell Research Foundation, Inc. Using mutations to improve Aspergillus phytases
US7320876B2 (en) 2001-10-31 2008-01-22 Phytex, Llc Phytase-containing animal food and method
US7323200B2 (en) 2003-08-18 2008-01-29 Abbott Laboratories Calcium fortified, soy based, infant nutritional formulas
WO2010135588A2 (fr) 2009-05-21 2010-11-25 Verenium Corporation Phytases, acides nucléiques codant pour elles et procédés de fabrication et d'utilisation associés
WO2011005577A1 (fr) 2009-06-24 2011-01-13 Soparkar Charles N S Supplémentation en zinc pour augmenter la faculté de réponse à une thérapie par métalloprotéase
US7919297B2 (en) 2006-02-21 2011-04-05 Cornell Research Foundation, Inc. Mutants of Aspergillus niger PhyA phytase and Aspergillus fumigatus phytase
EP2397486A1 (fr) 2006-09-21 2011-12-21 Verenium Corporation Phytases, acides nucléiques encodant celles-ci et méthodes pour leur fabrication et leur utilisation
US8540984B2 (en) 2006-08-03 2013-09-24 Cornell Research Foundation, Inc. Phytases with improved thermal stability
EP3854221A1 (fr) * 2020-01-21 2021-07-28 Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen Procédé de fourniture du phosphate à partir d'une biomasse contenant du phytate, biomasse réduite en phytate et phosphate, et utilisations correspondantes

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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
WO1995028850A1 (fr) * 1994-04-22 1995-11-02 Novo Nordisk A/S Procede destine a ameliorer la solubilite de proteines vegetales

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
EP0619369A1 (fr) * 1993-04-05 1994-10-12 Aveve N.V. Hydrolyse de la phytate et composition enzymatique pour hydrolyser la phytate
WO1995028850A1 (fr) * 1994-04-22 1995-11-02 Novo Nordisk A/S Procede destine a ameliorer la solubilite de proteines vegetales

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US7026150B2 (en) 1998-06-25 2006-04-11 Cornell Research Foundation, Inc. Overexpression of phytase genes in yeast systems
US7312063B2 (en) 1998-06-25 2007-12-25 Cornell Research Foundation, Inc. Overexpression of phytase genes in yeast systems
GB2340727B (en) * 1998-08-19 2002-05-22 Univ Saskatchewan Process for converting phytate into inorganic phosphate
GB2340727A (en) * 1998-08-19 2000-03-01 Univ Saskatchewan Process for converting phytate into inorganic phosphate
US6284502B1 (en) 1998-08-21 2001-09-04 University Of Saskatchewan Process for converting phytate into inorganic phosphate
US6974690B2 (en) 1999-03-31 2005-12-13 Cornell Research Foundation, Inc. Phosphatases with improved phytase activity
US6511699B1 (en) 1999-03-31 2003-01-28 Cornell Research Foundation, Inc. Enzymes with improved phytase activity
US7022371B2 (en) 1999-08-13 2006-04-04 Genencor International, Inc. Phytase enzymes, nucleic acids encoding phytase enzymes and vectors and host cells incorporating same
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WO2001012792A1 (fr) * 1999-08-13 2001-02-22 The Victoria University Of Manchester Enzymes phytase, acides nucleiques codant pour ces enzymes phytase, et vecteurs et cellules hotes incorporant lesdites enzymes phytase
US7300781B2 (en) 1999-11-18 2007-11-27 Cornell Research Foundation, Inc. Site-directed mutagenesis of Escherichia coli phytase
WO2002054881A3 (fr) * 2001-01-10 2003-10-30 Dsm Ip Assets Bv Utilisation d'aliments et de boissons en tant que systeme de distribution de phytase chez les etres humains
AU2002250832B2 (en) * 2001-01-10 2006-08-10 Dsm N.V. The use of food and drink as a delivery system for phytase in humans
US7566466B2 (en) 2001-01-10 2009-07-28 Dsm Ip Assets B.V. Use of food and drink as a delivery system for phytase in humans
WO2002098442A3 (fr) * 2001-06-01 2003-12-04 Krueger Gmbh & Co Kg Composition renfermant une phytase
US7320876B2 (en) 2001-10-31 2008-01-22 Phytex, Llc Phytase-containing animal food and method
US7833743B2 (en) 2001-10-31 2010-11-16 Phytex, Llc Phytase-containing animal food and method
US7736680B2 (en) 2002-09-13 2010-06-15 Cornell Research Foundation, Inc. Using mutations to improve Aspergillus phytases
US7309505B2 (en) 2002-09-13 2007-12-18 Cornell Research Foundation, Inc. Using mutations to improve Aspergillus phytases
US7323200B2 (en) 2003-08-18 2008-01-29 Abbott Laboratories Calcium fortified, soy based, infant nutritional formulas
JP2009500028A (ja) * 2005-07-08 2009-01-08 アディッソ・フランス・エス．エー．エス． フィチン酸の加水分解におけるフィターゼの併用の相乗効果
WO2007006952A1 (fr) * 2005-07-08 2007-01-18 Adisseo France S.A.S. Effet synergique de l'association de phytases sur l'hydrolyse de l'acide phytique
NO339291B1 (no) * 2005-07-08 2016-11-21 Adisseo France Sas Preparat, som kombinerer minst to fytaser for hydrolysen av fytinsyre, anvendelse derav for fremstillingen av et ernæringstilskudd for dyr eller et dyrefor, sett eller sammensetning til foring av dyr samt en fremgangsmåte for hydrolyse av fytinsyre.
FR2888249A1 (fr) * 2005-07-08 2007-01-12 Adisseo France Sas Soc Par Act Effet synergique de l'association de phytases sur l'hydrolyse de l'acide phytique
TWI410496B (zh) * 2005-07-08 2013-10-01 Adisseo France Sas 植酸酶組合物用於植酸水解之協同效用
US8541036B2 (en) 2005-07-08 2013-09-24 Adisseo France S.A.S. Synergetic effect of the phytase combination on the hydrolysis of phytic acid
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US7919297B2 (en) 2006-02-21 2011-04-05 Cornell Research Foundation, Inc. Mutants of Aspergillus niger PhyA phytase and Aspergillus fumigatus phytase
US8540984B2 (en) 2006-08-03 2013-09-24 Cornell Research Foundation, Inc. Phytases with improved thermal stability
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US9463224B2 (en) 2009-06-24 2016-10-11 Cnss Ip Holdings, Inc. Compositions and methods for enhancing metal ion dependent drug therapies
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