US20210395719A1 - Method of Precipitating Phytase - Google Patents

Method of Precipitating Phytase Download PDF

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US20210395719A1
US20210395719A1 US17/288,676 US201917288676A US2021395719A1 US 20210395719 A1 US20210395719 A1 US 20210395719A1 US 201917288676 A US201917288676 A US 201917288676A US 2021395719 A1 US2021395719 A1 US 2021395719A1
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phytase
precipitation
polyanion
precipitated
sodium
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Katja Palmunen
Mirkka PERKKALAINEN
Leena LEHTIKARI
Imke KÜHN
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AB Enzymes GmbH
AB Enzymes Oy
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AB Enzymes Oy
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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
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

  • the present invention relates to the field of protein production, and in particular to concentrating phytase and to compositions comprising phytase.
  • Phytase enzymes (myo-inositol hexakisphosphate phosphohydrolases; EC 3.1.3.8 and 3.1.3.26) are a group of phosphatase enzymes that catalyze the hydrolysis of phytic acid (myo-inositol hexakisphosphate, also known as inositol polyphosphate, or phytate when in salt form) found in plants, especially in grains and oil seeds. The majority (50-70%) of dietary phosphate is bound to phytic acid with low availability for non-ruminant animals.
  • Phytases are able to degrade phytate (myo-inositol hexakisphosphate), which results into release of inorganic phosphorus. Simultaneously, compounds bound to the phytate are also released. Thus, phytases can be used to increase nutritional value of feed by releasing inorganic phosphate and other nutrients from phytate. In animal feeding supplementing feed with phytase also helps to decrease environmental impact of farming, because phytate bound phosphorus can be utilized by the animal, which reduces the amount of mineral phosphorus to be added and the phosphorus excretions to the manure.
  • HAPs histidine acid phosphatases
  • 6-phytases mostly bacterial phytases that initiate the dephosphorylation of phytic acid at position 6′
  • 3-phytases mostly fungal phytases that initiate the dephosphorylation of phytic acid at position 3′
  • hydrolase first usually referring to the D-configuration of the molecule
  • Protein precipitation is commonly used in downstream processing of biological products in order to concentrate and purify proteins from various impurities. Protein precipitation is a process where a protein is separated from a solution as a solid by altering the protein solubility with addition of a specific reagent. Repulsive electrostatic forces between proteins are manipulated by the reagent to favor generation of submicroscopic sized protein aggregates. The aggregates grow and stick to each other forming eventually microscopic amorphous precipitate particles lacking well-ordered structure or in a special case crystalline particles having well-ordered structure. If solids concentration is high enough these particles can be seen with bare eye as for example haziness, turbidity, flocs or sediment in the liquid.
  • the inventors have surprisingly found that phytase can be precipitated by a simple method, which is applicable in large scale production of phytase.
  • the method can be used e.g. to precipitate and concentrate phytase from spent fermentation broth, and to separate phytase from other components present in the spent fermentation broth. Further, the method can be applied to manufacture concentrated phytase compositions.
  • the phytase-polyanion complex formed by the present method is stable which makes it possible e.g. to wash the phytase-polyanion complex. After precipitation, it is also possible to formulate the phytase-polyanion complex into various formulations.
  • the precipitate can also be reconstituted to a liquid product in selected conditions.
  • the present method is very fast, inexpensive and effective, and it solves problems relating to previous methods that involve ultrafiltration or other purification steps to increase phytase concentration.
  • the present invention simplifies production of phytase compositions.
  • the invention is based on the discovery that phytase is capable of forming a complex with polyanions, resulting into controlled and effective precipitation of phytase.
  • the complex formation can be controlled by selecting conditions, such as concentration, the type of polyanion, ionic strength, and pH.
  • This present invention can be utilized in production of phytase-containing products e.g. by precipitating phytase from spent fermentation broth while leaving other components in a soluble form.
  • the precipitated phytase-polyanion complex is stable, allowing e.g. concentrating and washing to further increase purity without marked decrease in yield.
  • the phytase-polyanion complex precipitated with the present method can also be dissolved after precipitation with excellent yield. Further, the precipitate provides phytase in a very stable form and even allows dehydrating and reconstituting without significant changes in specific activity or yield.
  • a method of preparing a phytase composition comprising:
  • a phytase composition comprising phytase complexed with a polyanion.
  • a phytase composition comprising phytase and a polyanion.
  • a feed supplement comprising phytase composition of the third or fourth aspect and optionally at least one further enzyme.
  • FIG. 1 is a diagram showing the effect of sodium chloride and sodium alginate on the precipitation of E. coli phytase protein.
  • FIG. 2 is a diagram showing permeate formation during precipitated phytase harvesting by microfiltration.
  • FIG. 3 is a diagram showing permeate flow during precipitated phytase harvesting by microfiltration.
  • FIG. 4 is a diagram showing the relative height of precipitate surface level during settling experiment.
  • FIG. 5 is a diagram showing the residual phytase activities of E. coli phytase liquid formulations during storage at temperature 37° C.
  • FIG. 6 is a diagram showing the residual phytase activities of E. coli phytase powder formulations during storage at temperature 40° C.
  • FIG. 7 is a diagram showing recovery improvement in pelleting test when polyelectrolyte precipitated and spray dried phytase powder formulations are compared to the commercial dry phytase products without polyelectrolyte precipitation.
  • the enzyme activity analysed in feed pellets after heat treatment by conditioning at different temperatures before pelleting is compared to the enzyme activity in the mash feed without heat treatment and pelleting.
  • FIG. 8 is a diagram showing the effect of phytase:mg of polyanionic compound-ratio and different polyanionic compounds on soluble phytase activity.
  • the aqueous medium comprising phytase comprises fermentation broth, preferably clarified spent fermentation broth.
  • the aqueous medium comprising phytase is fermentation broth, preferably clarified spent fermentation broth.
  • the clarified spent fermentation broth is obtained by clarifying spent fermentation broth from recombinant production of the phytase.
  • the aqueous medium comprising phytase comprises clarified fermentation broth, spent fermentation broth, spent and clarified fermentation broth, or a combination thereof.
  • the dry matter content of the aqueous medium comprising phytase without solids is between 0.1-25% w/w before adding the polyanion.
  • the dry matter content of the aqueous medium comprising phytase without solids is selected from the range 0.1-25% w/w, such as 0.1-20, 0.1-15, 0.1-10, 1-20, 1-15, 1-10, 2-20, 2-15, 2-10, 3-20, 3-15, 3-10, 3-9, 3-8, 4-15, 4-10, 4-9 or 4-8% w/w.
  • the dry matter content refers to the dry matter present in the aqueous solution comprising phytase without calculating the effect of the added polyanion.
  • the aqueous medium comprising phytase does not contain CaCl 2 ), or the concentration of CaCl 2 ) is below 5 mM, 2 mM or 1 mM.
  • the pH is set to 3-5 for enhancing precipitation of phytase.
  • the pH is set to a value selected from the range 3-5.
  • the pH can be set to the selected pH before adding the polyanion.
  • the pH is set to the selected value after adding the polyanion.
  • the pH is maintained near the selected value until the end of precipitation.
  • the pH is set and optionally maintained at range 3-5, such as 3-4.9, 3-4.8, 3-4.7, 3-4.6, 3-4.5, 3-4.4, 3-4.3, 3-4.2, 3-4.1, 3-4, 3-3.9, 3-3.8, 3.1-4.9, 3.1-4.8, 3.1-4.7, 3.1-4.6, 3.1-4.5, 3.1-4.4, 3.1-4.3, 3.1-4.2, 3.1-4.1, 3.1-4, 3.1-3.9, 3.1-3.8, 3.2-4.9, 3.2-4.8, 3.2-4.7, 3.2-4.6, 3.2-4.5, 3.2-4.4, 3.2-4.3, 3.2-4.2, 3.2-4.1, 3.2-4, 3.2-3.9, 3.2-3.8, 3.3-4.9, 3.3-4.8, 3.3-4.7, 3.3-4.6, 3.3-4.5, 3.3-4.4, 3.3-4.3, 3.3-4.2, 3.3-4.1, 3.3-4, 3.3-3.9, or 3.3-3.8, or at about 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,
  • the polyanion is a polyanion salt, preferably a Na salt of the polyanion.
  • the polyanion is a salt with another monovalent, such as a K salt.
  • the polyanion is alginic acid, pectic acid, hyaluronic acid, phytic acid, or a salt of such a polyanionic acid, or any combination thereof.
  • the polyanion is a polyanionic salt.
  • the polyanion is Na alginate, Na polypectate, Na hyaluronate, Na phytate or a combination thereof, or a combination of the polyanionic salt with at least one polyanion.
  • the salt of the polyanionic acid is added in a solid form or as an aqueous solution to the aqueous solution comprising phytase.
  • polyanion salt is added in a solid form to the aqueous solution comprising phytase.
  • the method comprises controlling pH by keeping it in the range 3-5, preferably in the range 3.1-4, after adding the polyanion.
  • the precipitated phytase is washed.
  • the precipitated, optionally washed, phytase is dehydrated to obtain a dry product.
  • the dry product is in the form of a pellet, extrudate, granule, powder or a coated product.
  • dehydration is done by protein drying technique known in prior art including but not limited to spray drying, freeze drying, vacuum drying, evaporating, spray coating, granulation or extrusion.
  • the precipitated phytase is supplemented with a drying additive before drying.
  • Suitable drying additives include but are not limited to sugars like trehalose, sugar alcohols, salts and polymers like polyethylene glycol, such as PEG 4000.
  • the precipitate formed by the present method is suitable for drying also without a drying additive, because of the good stability of the precipitated complex.
  • the precipitated, optionally washed, phytase is suitable to be used in compositions that are cross-linked, immobilized or encapsulated using techniques known in prior art.
  • the precipitated, optionally washed, phytase is suitable to be used as such or at least partially dissolved form in dilute or compressed solid or liquid compositions including but not limited to solutions, suspensions, emulsions, semi-solids, solids, pastes, pellets, cakes, gels, tablets, films or coatings having certain targeted properties like for example controlled rheology, viscosity or enzyme release using techniques known in prior art.
  • the method comprises dissolving the precipitated phytase to obtain a liquid product.
  • a liquid product thus comprises phytase and the selected polyanion used in the precipitation.
  • the method comprises dissolving the dry product. This is advantageous to obtain a reconstituted product having a high concentration.
  • the ratio of phytase to polyanion expressed as FTU:mg polyanion is selected from the range 500-15000.
  • the ratio expressed as FTU:mg polyanion is selected from the range 500-15000, 1000-14000, 1500-13000, or 2000-12000.
  • the amount of the added polyanion is 0.001-2% w/w based on dry matter content.
  • the polyanion is added in an amount of 0.001-2, 0.005-2, 0.01-2, 0.1-2, 0.001-1.5, 0.005-1.5, 0.01-1.5, 0.1-1.5, 0.001-1, 0.005-1, 0.01-1, or 0.1-1% w/w based on dry matter content.
  • the polyanion is an alginic acid, a pectic acid, a hyaluronic acid, a phytic acid or a salt of such an acid, preferably a salt with a monovalent cation.
  • the polyanion is a combination of phytic acid or its salt together with a polyanion selected from alginic acid, pectic acid, hyaluronic acid, or a salt of such an acid. It is preferable to use a further polyanion when phytic acid, or its salt, is used to keep the phytase-polyanion complex stable for a longer time. This is particularly useful when precipitating phytase with phytic acid or its salt in conditions where the phytase has enzyme activity.
  • the salt is an alginate salt, a polypectate salt, a hyaluronate salt, a phytate salt or any combination thereof.
  • said salt is a Na salt, K salt, NH 4 salt or Ca salt, preferably a Na salt.
  • composition is a liquid product or a dry product.
  • the precipitated phytase is harvested by sedimentation, decantation, centrifugation, filtration, or a combination thereof.
  • the precipitated phytase is preferably harvested in a small volume. This allows recovering the precipitated phytase in a high concentration and specific activity.
  • Advantageously precipitating and/or concentrating phytase by the present method offers a simple way to improve cost efficiency in the production of concentrated phytase products compared to the current concentrating methods by ultrafiltration that does not remove high molecular weight impurities.
  • the precipitate can optionally be washed and then dissolved by increasing ionic strength of the medium above 0.25 M and adjusting pH above 4. This is particularly useful to manufacture compositions in which the final formulation is liquid.
  • Such liquid of dissolved precipitate or precipitate suspension without dissolving step are both equally useful to de dried e.g. with spray drying if dry product is the desired final formulation.
  • the ionic strength of the aqueous medium comprising phytase is below 0.25 M before adding polyanion.
  • ionic strength is kept below 0.25 M after adding polyanion to keep phytase in non-soluble form.
  • dissolving the precipitated phytase is carried out in the presence of CaCl 2 ).
  • Adding of CaCl 2 ) to precipitated phytase is advantageous because it dissolves the phytase-alginate complex in below 0.25M concentrations, as shown in Example 24.
  • CaCl 2 ) is added to dissolve the precipitated phytase preferably in an amount of 70 mM, 40 mM or less, and more preferably in an amount of at least 10 mM, 20 mM or 30 mM.
  • CaCl 2 ) is an advantageous agent compared other salts such as sodium chloride or sodium sulphate, because the other salts are needed in much higher concentrations such as 250 mM to solubilise the precipitated phytase.
  • the phytase is a recombinant phytase.
  • the phytase is E. coli phytase, Aspergillus phytase or Buttiauxella phytase, preferably a phytase having phytate degrading activity.
  • At least one component of the phytase composition preferably the phytase enzyme, has a different structural or physical characteristic compared to a corresponding natural component from which at least one component is derived from.
  • the characteristic is uniform size, homogeneous dispersion in the composition, non-native glycosylation, non-native stability, production level, or purity.
  • the phytase composition comprises a phytase-polyanion complex. Such a complex does not occur in a natural environment of phytases. In the present invention such a complex is however possible to achieve.
  • Factors contributing to the complex formation are concentration of phytase and polyanion, pH, and ionic strength and temperature.
  • the phytase is a bacterial phytase, preferably a bacterial recombinant phytase expressed in a heterologous host cell.
  • the phytase is a 6-phytase having enzyme activity for 6-phos, preferably a protein engineered variant, chimeric or hybrid phytase.
  • the hybrid phytase comprises phytases that are engineered to contain elements of two or more phytases.
  • the phytase is a fungal phytase, preferably a fungal recombinant phytase expressed in a heterologous host cell.
  • the phytase is a 3-phytase, preferably a protein engineered variant, chimeric or hybrid phytase.
  • the hybrid phytase comprises phytases that are engineered to contain elements of two or more phytases.
  • the polyanion is selected such that it is capable of forming a reversible phytase-polyanion complex.
  • suitable polyanions are alginic acid, a pectic acid, a hyaluronic acid, and phytic acid or a salt of the acid or any combination thereof.
  • These and other polyanions can also be found in for example naturally occurring polysaccharides, gums or hydrogels like xanthan gum, gum arabic, heparin or carrageenan, or synthetic compounds having corresponding functions or characteristics.
  • pH of the aqueous solution of phytase is set to 3-5 before adding the polyanion.
  • pH is controlled after adding polyanion by setting and/or keeping it at 3-5.
  • polyanion is added as a buffered aqueous solution.
  • the solution is buffered to pH selected from the range 3-5.
  • the precipitated phytase is recovered by microfiltration, belt filtration, centrifugation, dynamic cross flow filtration, sedimentation, or any combination thereof.
  • the precipitated phytase is recovered by microfiltration or centrifugation.
  • the phytase-polyanion complex obtained with the present method is stable, it is possible to wash the precipitated phytase.
  • the method it is possible to use the method to remove other components present e.g. in a spent fermentation broth to which phytase is produced in recombinant production.
  • macromolecules such as proteins other than phytase, carbohydrates and cellular debris can be removed by washing.
  • small molecule components present in the spent fermentation broth can be removed by washing. Washing is advantageous also to effectively remove microbiological material from the phytase composition.
  • the aqueous solution comprising phytase contains recycled supernatant obtained by concentrating phytase and recovering the supernatant to be used as the recycled supernatant.
  • washing is carried out by using a buffer solution having pH and/or ionic strength close to the pH and/or ionic strength used in the precipitation step.
  • ionic strength of the wash solution is lower than used in the precipitation of the phytase.
  • pH of the wash solution having ionic strength below 0.25 M is lower or higher than used in the precipitation step.
  • the pH is higher or lower by at least 1 pH unit, such as 1.5 or 2 pH units.
  • pH is controlled by setting it to a value selected from a suitable range for precipitation of a certain phytase. This can be determined by the skilled person by carrying out the present method at various pH values and calculating precipitation yield and/or specific activity at each pH.
  • Example 2 provides an example of calculating precipitation yield. Suitable range can then be selected based e.g. on the desired yield and/or specific activity obtained at a given pH.
  • phytase enzyme activity is determined using the method disclosed in Example 1.
  • the method is an industrial scale method.
  • the method is carried out in the sequence specified in a claim, aspect or embodiment.
  • FTU is determined according to ISO 30024:2009(E).
  • Enzyme materials used for polyelectrolyte precipitations were different phytase sources, an E. coli phytase, an Aspergillus phytase and a Buttiauxella phytase. All these phytases were expressed in Trichoderma reesei fungus. Precipitations of E. coli phytase or Aspergillus phytase were started using either clarified spent fermentation broths or concentrates of them, containing preservative to prevent microbial contaminations, from several different fermentations of phytase protein. Spent fermentation broths were clarified by filtration and concentrated with 10 kDa ultrafiltration membrane to increase protein concentration.
  • Precipitation of Buttiauxella phytase was done using purified enzyme liquid from dried granule as starting material. Enzyme activity of the protein was measured as the release of inorganic phosphate from sodium phytate (0.98% (w/v) phytate at 37° C. in 250 mM sodium acetate buffer at pH 5.50 in 60 min (FTU activity; ISO 30024:2009(E) Animal feeding stuffs—Determination of phytase activity). Protein concentration was measured with Bio-Rad Protein Assay that is based on the colour change of Coomassie brilliant blue G-250 dye in response to various protein concentrations. The Coomassie blue dye binds to primarily basic and aromatic acid residues, especially arginine.
  • Bio-Rad Protein Assays Dye Reagent Concentrate Bio-Rad No. 500-0006
  • bovine gamma globulin protein standard Bio-Rad Protein Assay Standard I. No. 500-0005
  • Precipitations were let to continue for an hour in cold room. After this samples were taken to analyse enzyme activities. Final precipitation conditions and activity yields are represented in table 1. Needed tap water and concentrate amounts were calculated so that aimed FTU:mg of sodium alginate—ratio and calculated dry matter contents were reached. Precipitation yields were calculated based on soluble phytase activity analysed in supernatants after precipitate separation by centrifugation compared to the total activity. These results show that phytase can be precipitated from concentrate of clarified spent fermentation broth with high yield using alginate as polyanionic precipitant.
  • Phytase precipitation was done using clarified spent fermentation broth of E. coli phytase as enzyme material. Using the same FTU:mg of sodium alginate ratio as in the example 2 the precipitation was performed simultaneous with ultrafiltration step that is normally used to concentrate proteins of clarified spent fermentation broths. This was continued with buffer exchange and two washing steps. Different steps are represented in table 4. First dry sodium alginate was added slowly to clarified spent fermentation broth while stirring properly with magnetic stirrer. Precipitate formation started during sodium alginate addition and it was let to proceed in the next steps. The clarified spent fermentation broth containing phytase-alginate precipitate was concentrated by ultrafiltration.
  • the diafiltration step was started by adding 40 mM sodium acetate buffer (nominal pH 3.6) slowly to the retentate while stirring properly with magnetic stirrer. Diafiltration was continued until 1.3 times concentration was reached. In the next step double washing was made using tap water. In the last step double washing was made by microfiltration. The aim of this step was to wash out other proteins and high molecular weight compounds and keep phytase inside the membrane as a solid complex. Calculated phytase yield of this combined precipitation, concentration and harvesting test was 72% analysed based on activity in the retentate after microfiltration. Specific phytase activity was 40% higher in the final retentate than in the clarified spent fermentation broth. These results show that alginate precipitation can be made simultaneous with ultrafiltration.
  • Material for harvesting tests was prepared using the method in example 2.
  • a stock solution of 0.4% (w/w) sodium alginate was used.
  • Final precipitation conditions were: 0.19% (w/w) sodium alginate, 0.05 M sodium acetate buffer.
  • Aimed FTU:mg of sodium alginate-ratio was 10 000 and calculated dry matter contents was 5.2% (w/w). Needed tap water and concentrate amounts were calculated so that aimed FTU:mg of sodium alginate-ratio and calculated dry matter contents was reached. Calculated precipitation yield of this batch was 75% analysed based on soluble activity in the filtrate.
  • the precipitated material was a concentrate of E. coli phytase. Used precipitation conditions were the same as in batches 1 and 2 in the example 2. Calculated precipitation yield of this batch was 71% analysed based on soluble activity in supernatant. This precipitated E. coli phytase was used in the precipitate harvesting experiment.
  • Example 8 Material from example 8 harvested by centrifugation was used as a starting material for preparing the stabilized liquid formulations 1 and 2 of phytase-alginate complex.
  • formulation 3 precipitated material was taken from batch 4 of example 2.
  • Precipitate was harvested by sedimentation.
  • Retentates from microfiltration harvesting experiments in example 6 were used as starting material in spray drying tests to produce dry product of phytase-alginate solid complex.
  • Büchi Mini Spray Dryer B-290 was used for sample drying. Six different spray dryings were performed. Drying additive, either PEG 4000 or trehalose, was used in three of these experiments. Additive was added right before spray drying. PEG was added as 50% (w/w) liquid to the harvested precipitate solution while stirring properly. Trehalose was added as dry powder to the precipitate solution while stirring properly. Precipitate solution amounts with or without additives fed to the spray dryer varied from 570 g to 760 g. During spray drying precipitated solution were kept in room temperature under proper stirring. During spray drying inlet temperature was kept near 130° C.
  • Formulations from example 12 were taken under accelerated storage stability study. Storage time was 8 weeks. For stability study each of the formulated liquids was divided in 15 ml falcon tubes closed with caps, 5 ml of liquid per tube. One tube was prepared for each storage time point. All sample tubes were placed into 37° C. climate chamber. From each time point one sample tube was taken to freezer before activity analysis. Phytase activities from each storage time point were compared to the starting point phytase activity. Commercial liquid phytase product without polyelectrolyte precipitation treatment stored in the same manner was used as a reference. Activity concentration levels of the studied formulations varied. Activity concentration of formulation 1 was 1.5 ⁇ higher compared to the reference and activity concentration of formulation 2 was 1.1 ⁇ higher than the reference. Enzyme activity of formulation 3 was at the same level as reference. Stability results are illustrated graphically in FIG. 5 . The results show improved stability when phytase-polyanion complex has been formulated to liquid product compared to the reference product without complex formation.
  • the precipitated material was a concentrate of E. coli phytase. Used precipitation conditions were following: 0.05 M sodium acetate buffer, 0.46% (w/w) sodium alginate, 4000 FTU:mg of sodium alginate—ratio, calculated dry matter 5.2%. After precipitation these batches were combined and excess of liquid part was removed to receive precipitate slurry having dry matter 12.3%. This slurry was spray dried using the method in example 13 in exception that no drying additive was used. During spray drying inlet temperature was kept near 132° C. and pump value near 34% so that outlet temperature would stay near 75° C. Activity and dry matter contents were analysed from precipitate solution and final spray dried powder to calculate drying yields. Powder dry matter was 96.1% and drying yield was 100%.
  • This dry product was taken under accelerated storage stability study. Storage time was 8 weeks. For stability study the dry powder was divided in 50 ml falcon tubes closed with caps, 8-10 g in each tube. For each storage time points one tube was prepared. All sample tubes were placed to 40° C. climate chamber. From each time point one sample tube was taken to freezer before activity analysis. Phytase activities from each storage time points were compared to the starting point phytase activity. Stability results are illustrated graphically in FIG. 6 . These results show improved stability when phytase-polyan ion complex has been formulated to dry product: curve showing residual activity at different time points was 19% higher level after storage of 8 weeks at 40° C. compared to the reference commercial dry phytase products without precipitation/complex formation studied in the same manner.
  • Dry powder samples 1, 2, 3 and 4 from spray drying experiments in example 13 were used to study heat stability by a standardized pelleting tests at an independent institute (Danish Technological Institute, Kolding, Denmark).
  • Test articles were mixed into mash feeds, that were treated in a conditioner with hot water steam resulting in different mash feed temperatures before pelleting.
  • Recoveries were calculated from enzyme activities analysed in the pellets after heat treatment compared to the enzyme activity in the mash feed without conditioning and pelleting. These recoveries were compared to commercial dry phytase product without precipitation/complex formation tested in the same manner. Recovery improvement percentages are represented graphically in FIG. 7 .
  • Ratio Soluble enzyme FTU:mg of activity of total Precipitation Used sodium alginate pH sodium alginate
  • Activity % yield (%) Fluka (71238) 4.15 10 680 21 79 Sigma (W201502) 4.17 10 859 27 73 Manucol DH (FMC) 4.29 10 794 6 94 Manucol DH MCLDHP 4.15 10 973 26 74 (FMC) Manucol LD (FMC) 4.26 10 842 6 94
  • Phytase concentrate was added to the reagent solutions at room temperature and vortexed properly, whereupon phytase-polyanionic complex was rapidly formed. Needed tap water and concentrate amounts were calculated so that aimed FTU:mg of polyanionic compound-ratio and calculated dry matter contents was reached. Final sodium acetate concentration in all experiments was 0.05 M. Final precipitation conditions and activity yields are represented in table 11 and soluble activity curves are illustrated graphically in FIG. 8 . Precipitation yields were calculated based on soluble phytase activity in supernatants. These results show that phytase can be precipitated with all studied polyanions and precipitate yield can be improved by changing ratio of FTU:mg polyanionic compound.
  • Example 19 Precipitation Using phytic acid as polyanionic Compound Alone and in Combination with sodium alginate
  • Phytic acid substrate of phytase—was studied as polyanionic compound to see its phytase precipitating potential. Also precipitation using combination of sodium alginate and phytic acid was studied. A series of 2 g batch precipitation experiments were performed using concentrate of E. coli phytase. For batch precipitations reagent solutions were prepared in 2 ml Eppendorf tubes by adding 1 M sodium acetate buffer (nominal pH 3.6) and stock solution of selected polyanionic compounds to tap water. Used polyanionic stock solutions were: 2 (w/w) sodium alginate and 5% (w/w) phytic acid.
  • Phytase concentrate was added to the reagent solutions cooled down in ice bath and stirred properly, whereupon phytase-polyanionic complex was rapidly formed. Needed tap water and concentrate amounts were calculated so that aimed FTU:mg of polyanionic compound-ratio and calculated dry matter contents was reached. Final sodium acetate concentration in all experiments was 0.05 M. Final precipitation conditions and yields are represented in table 12. Precipitation yields were calculated based on soluble protein concentration in supernatants compared to the total protein concentration.
  • E. Coli phytase precipitation was done in similar manner as described in the Example 4.
  • Precipitate was harvested by sedimentation.
  • dissolving of the harvested precipitate was done using CaCl 2 ) instead of Na 2 SO 4 that has been used in the previous precipitate dissolving examples.
  • To dissolve the harvested precipitate dry calcium chloride dihydrate was added and mixed to the harvested precipitate with and without other additives to achieve final experiment conditions shown in the table 19 below.

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