CN1241634A - Phytase formulation - Google Patents

Abstract

A stabilized enzyme formulation is disclosed which comprises phytase and at least one stabilizing agent selected from the group consisting of: a) C5 sugars such as xylitol and ribitol, b) polyethylene glycol having a molecular weight of 600 to 4000 Da, c) the disodium salts of malonic, succinic and glutaric acid, and d) carboxymethylcellulose, and e) sodium alginate. Alternatively, phytase may be stabilized by chemical crosslinking with either a) glutaraldehyde, or b) oxidation of phytase carbohydrate residues with sodium periodate and subsequent addition of adipic acid dihydrazide.

Description

Phytase formulation

The present invention relates to liquid and exsiccant phytase formulation, it has by the interpolation of stablizer or by the crosslinked enhanced stability that obtains, thermostability preferably.

Although there is the phosphoric acid salt of a large amount of phytinic acid phosphorus forms in feed, monogastric animal resembles pig and poultry lacks the phosphatic ability of utilizing this form.The alkali of phytinic acid or alkaline earth salt be natural being present in the cereal mainly.Because monogastric animal can not utilize the phosphoric acid salt of this form, so usually phosphoric acid salt is added in the animal-feed.

On the other hand, known a kind of enzyme (flesh phytinic acid phosphohydrolase) that is called phytase is present in plant and the certain micro-organisms.Phytase can produce by fermentation, and use phytase known in the art is as a kind of animal feedstuff additive, with the nutritive value by the inorganic phosphate release from phytinic acid (flesh phytinic acid) raising vegetable material.By in animal-feed, adding phytase, can reduce the level that environment phosphorus pollutes, because the phosphoric acid that the animal capable utilization discharges from phytinic acid by the application of phytase.

For feed applications, a kind of stable preferably heat-staple phytase is by common concern, with avoid can generation in preparation (for example spraying drying, granulation) and the feed course of processing (for example granulation, extrude, expanded) problem, temporary transient therebetween high temperature (up to 80-120 ℃) and shear-stress can influence proteinic structure and cause undesirable loss of activity.

The International Patent Application WO 93/16175 of Gist-Brocades has been described the stable liquid preparation of phytase.Suggestion uses urea and water miscible polyvalent alcohol as stablizer, mentions Sorbitol Powder, glycerine and molecular weight thus and be 6000 polyoxyethylene glycol.

The stability that an object of the present invention is to improve phytase is thermostability preferably, and stability is defined in the ability of retentive activity under the different condition.This stability aspect relates to the complete life cycle of enzyme, comprises generation (fermentation of feed, downstream processing, preparation and thermal treatment), distributes (transportation and storage) and final the application.For commercial interested enzyme such as phytase, importantly the different feed courses of processing such as granulation, extrude with expanded in the high temperature (up to 80-120 ℃) that reached of tolerance, and stable in long storage.

The term that uses among the present invention " stability " relates to all characteristics of a kind of industrial enzyme, comprises as aspects such as activity, specificity, package stability, mechanical stability, microbially stable, toxicity, chemical constitution and physical parameter such as density, viscosity, deliquescence and color, smell and dust.A preferred aspect of the present invention relate to phytase preparation and the feed course of processing such as granulation, extrude with expanded in tolerate heat-killed stability.

A major obstacle of phytase widespread use is these enzymes tolerate the required thermostability of deactivation (80-120 ℃) in the feed course of processing restriction.Current obtainable industrialization phytase all derives from aspergillus niger (A.niger), and heat inactivation is had low intrinsic tolerance.As a kind of selectivity or the method except that molecular biology method, the present invention is by the interpolation of different additive, and is oligomer by the chemically crosslinked of enzyme monomer on the other hand, improved preferably thermostability of proteinic stability.

Also cause experiment of the present invention with so-called total phytase, this enzyme is the phytase according to a kind of theoretical molecular biology method exploitation, compare with aspergillus (Aspergillus) phytase and to have higher inherent stability, see the open text 897 985 of european patent application.In enforcement of the present invention, also can use the total phytase of in embodiment 3-13, describing in detail.

The invention discloses the application of different additive, they as stablizer to the stability of enzyme preferably thermostability work.

About the temperature dependency of the non-preparation phytinic acid specific enzyme activity that can preferably use in the present invention, can form three not on the same group according to their maximum activity.When following temperature, reach maximum activity: for Aspergillus fumigatus (A.fumigatus) and aspergillus niger phytase at 55 ℃, terreus (A.terreus) CBS and Aspergillus nidulans (A.nidulans) phytase are at 45 ℃, and total phytase is at 65 ℃.Select temperature (the phytase complete deactivation of the non-preparation at this moment) conduct of the above 10-15 of the top temperature of surveying ℃ of institute to screen point, be used to study the effect of stablizer to the phytinic acid enzyme heat stability, it is 60 ℃ promptly for Aspergillus nidulans and terreus CBS phytase, aspergillus niger and Aspergillus fumigatus phytase are 65 ℃, and total phytase is 75 ℃.

The invention provides a kind of stable, heat-staple zymin preferably, it comprises phytase and at least a stablizer, and this stablizer is selected from:

A) comprise the polyvalent alcohol of 5 carbon atoms, preferably C ₅Sugar more preferably is Xylitol and ribitol,

B) molecular weight is 600 to 4000Da polyoxyethylene glycol,

C) disodium salt of propanedioic acid, pentanedioic acid and succsinic acid,

D) carboxymethyl cellulose and

E) sodiun alginate

The present invention also provides a kind of stable, heat-staple zymin preferably, and it comprises crosslinked by the following method phytase;

A) by with the chemical reaction of glutaraldehyde; Or pass through

B) oxidation of sodium periodate and the adding of adipic dihydrazide subsequently

Although use is possible from other phytase that other source outside the microorganism obtains, preferably use the phytase of microorganisms.Preferably using mycetogenetic phytase in the present invention, more preferably is to be selected from Aspergillus fumigatus, Aspergillus nidulans, terreus and aspergillus niger.The preferred another kind of phytase that uses is so-called total phytase among the present invention.Yet, it also is possible producing these phytases by genetic engineering, method be the transgenosis that will obtain from fungi to host living beings such as bacterium (for example intestinal bacteria), yeast or another kind of fungi, further details is seen as open text 684313 of european patent application and the open text 867010 of european patent application.

The term zymin comprises all liquid and drying agent, and wherein enzyme phytase can be commercial.Preferably, the source of the phytase of this preparation is the thick flowing product that obtains from fermentation culture.Add selected stablizer or crosslinked phytase carries out the preparation of liquid phytase formulation.For obtaining a kind of stable, heat-staple drying agent preferably, phytase is spraying drying or granulating in the presence of selected stablizer a), or b) chemically crosslinked.

In a kind of preferred embodiment, liquid enzyme formulation comprises the polyoxyethylene glycol as stablizer, and polyoxyethylene glycol is present in the whole preparation with the concentration of 10-50% (w/w).

Preferably zymin comprises the polyoxyethylene glycol that molecular weight is 1000-3350Da.The particularly preferred molecular weight that is to use is about 1450 polyoxyethylene glycol.The molecular weight polyoxyethylene glycol outside preferable range (being respectively 600Da and 4000Da) slightly still demonstrates the effect that conforms with convention, but is not preferred.The stabilization of polyoxyethylene glycol is shown as (the seeing Fig. 2 and Fig. 3) that molecular weight relies on.

In another kind of preferred embodiment of the present invention, stablizer is Xylitol or ribitol.They all are the sugar alcohols with 5 atomic structure of carbon.Preferably use Xylitol and ribitol with the concentration of 20-60% (w/w) in final liquid preparation.Astoundingly, Xylitol and ribitol have improved specific activity 65 ℃ of mensuration as for example stablizer of Aspergillus fumigatus phytase, when the polyvalent alcohol of 12.5% concentration, bring up to 11-12U/mg, when 25% concentration, bring up to the 51-90U/mg (see figure 4).

In another embodiment of the invention, liquid enzyme formulation comprises the disodium salt of pentanedioic acid, succsinic acid or propanedioic acid as stablizer, and the concentration range of salt is 10-30% (w/w) in the whole preparation.As shown in Figure 6, the adding of the malonate of 25% concentration, succinate and glutarate causes the remarkable increase of Aspergillus fumigatus phytinic acid enzyme heat stability, still can detect considerable activity for malonate at 70 ℃, still can detect at 65 ℃ for succinate and glutarate.

In addition, carboxylate salt stimulates the Aspergillus fumigatus phytase activity of measuring in the time of 37 ℃, and observing phytase activity for malonate increases about 4 times, increases by 2 times for succinate, for glutarate less effect is arranged then.Studies show that of the malonate of different concns (5,10 and 25%), the thermally-stabilised of Aspergillus fumigatus phytase is that concentration relies on, and the stimulation of enzymic activity in this concentration range, is not the (see figure 7) that concentration relies at least.Opposite with these discoveries, the monocarboxylate sodium acetate of different concns (5,10 and 25%) causes that the increase of Aspergillus fumigatus phytinic acid specific enzyme activity can reach 2 times in the time of 37 ℃, but has only less effect (see figure 8) for proteinic thermostability.Therefore, can infer that carboxyl is responsible for active the adjusting, and difunctional dicarboxylate may be stablized phytase by ionic interaction.Sodium malonate and sodium succinate make the thermostability of Aspergillus nidulans, terreus CBS, aspergillus niger and total phytase improve 5-15 ℃ usually.On the other hand, only the stimulation that Aspergillus nidulans with quite low specific activity and Aspergillus fumigatus phytase are observed phytase activity is not then observed (seeing Fig. 9 and Figure 10) for terreus CBS, aspergillus niger and total phytase.

In another embodiment of the invention, zymin comprises polymer carboxymethyl cellulose and/or sodiun alginate as stablizer, and their concentration in whole liquid preparation is 1-20%, 1-10% (w/w) preferably.Adding these polymers in the Aspergillus fumigatus phytase formulation causes the phytinic acid enzyme heat stability significantly to increase 5-10%.

In another embodiment of the invention, zymin comprises alginate, preferably sodiun alginate is as stablizer, most preferably with the concentration of 1-10% (w/w) in the whole liquid preparation.

In another embodiment of the present invention, zymin comprises crosslinked phytase.Preparation for this stable phytase form adds glutaraldehyde with the concentration that can cause protein oligomerization in phytase.

In another embodiment, zymin comprises by the crosslinked phytase of its sugar chain.The crosslinked periodate oxidation that comprises the first step saccharide residue is the aldehyde radical of generation and the reaction of adipic dihydrazide subsequently.

According to used condition, crosslinking reaction can produce the multiple derivative of enzyme, promptly

A) only with the enzyme molecule of the modification of hydrazide group reaction of adipic dihydrazide,

B) contain or do not contain the enzyme of the intramolecular crosslinking of intermolecular cross-linking, and

C) intermolecular cross-linking, soluble oligomer or insoluble polymer.

In most cases, this reaction produces the mixture of several forms.The Aspergillus fumigatus of in multiple-shaped nuohan inferior yeast (Hansenula polymorpha), all expressing and the crosslinked formation that causes the oligomerization form of total phytase.Can control crosslinked degree effectively by the degree that changes oxydasis.The optimal heat of observing phytase when the sodium periodate of 50mM concentration is applied to the phytinic acid enzyme solution of 5mg/ml is stable.For two kinds of phytases, observe the increase (see Figure 12) of thermostability between 10 ℃ to 15 ℃.Should be pointed out that even not add adipic dihydrazide that the phytase of oxidation can form dimer, tripolymer and the tetramer (seeing Figure 11 A) of significant quantity.

Another aspect of the present invention relates to listed stablizer and is used for exsiccant/solid phytase formulation production as additive.In this embodiment of the present invention, the adding of stablizer (Xylitol/ribitol of 1-20% (w/w); molecular weight preferably the dicarboxylate of the polyoxyethylene glycol of the 1-20% between 1000-3350Da (w/w) and/or 1-20% (w/w) (as malonate; succinate and glutarate); and/or polymer carboxymethyl cellulose and/or the alginate of 1-10% (w/w); preferably be dissolved in the sodiun alginate in the 100-200ml phytase liquid (crosslinked or noncrosslinking)) or in the standard particle mixture, add solid chemical compound and (comprise the sulfonated lignin as tackiness agent; silicon-dioxide and gypsum (gipsum) as carrier).This preparation can cause the recovery of the phytase activity measured behind the high-shear granulation process to improve (up to 20%), and this method comprises particle 45 ℃ of drying step of 15 minutes on fluidized bed dryer.In addition, when these particles that comprise stablizer mix with feed, compare with the particle that does not contain these additives, they show that in feed processing (for example 85 ℃ prilling process) back the enzymic activity that reclaims increases.

Another aspect of the present invention relates to the method that preparation is used for the feed composition of monogastric animal, and method is to add a kind of heat-staple drying or liquid enzyme formulation according to any claim (1-13) in feed.Can carry out several feed making methods to the feed of adding phytase, as extrude, expanded and granulation, wherein temporary transient high temperature can appear, thermally-stabilised then is favourable part.

Stable zymin of the present invention can be used for the example of feed pill.Available tap water dilutes heat-staple liquid enzyme formulation, has the solution of the phytase activity (100-200 phytase unit/g solution) of hope with generation.The feed pill is transferred to mechanical mixer, and the zymin with dilution in stirring is injected on the feed pill, and to produce a kind of product of homogeneous, it has the phytase activity of adding, as 500 units phytase/kg feed pill.In addition, in that being carried out, exsiccant or liquid enzyme formulation directly can be mixed with the ground feed in this mixture as granulation, the expanded or first being processed extruded.

The dietetically desirable a kind of method that phosphorus is provided to monogastric animal that relates in one aspect to again of the present invention wherein uses feed according to the present invention to raise this animal, does not add other phosphorus in feed.

Experimental result of the present invention is shown in the following accompanying drawing.

Fig. 1. the temperature dependency of Aspergillus fumigatus, Aspergillus nidulans, terreus CBS, aspergillus niger and the total phytase activity of measuring under embodiment 1 described standard test condition is relatively.

Fig. 2. different polyoxyethylene glycol are to the influence of Aspergillus fumigatus phytinic acid specific enzyme activity in the time of 65 ℃.

Fig. 3. 50% different molecular weight polyethylene glycol solution is to the influence of the thermostability of aspergillus niger, total, terreus CBS and Aspergillus nidulans phytase.Measure specific activity for terreus CBS and Aspergillus nidulans phytase at 60 ℃, the aspergillus niger phytase is 65 ℃ of mensuration, and total phytase is 75 ℃ of mensuration.

Fig. 4. in the time of 65 ℃ 25% with the influence of 50% different polyhydric alcohol solutions to Aspergillus fumigatus phytinic acid specific enzyme activity.

Fig. 5. in the presence of 50% Xylitol as additive, the temperature dependency of aspergillus niger (A), total (B), Aspergillus nidulans (C) and terreus CBS (D) phytase activity.

Fig. 6. in the presence of propanedioic acid disodium, disodium succinate and the pentanedioic acid disodium of 25% concentration, the temperature dependency of Aspergillus fumigatus phytase activity.

Fig. 7. in the presence of 5%, 10% and 25% propanedioic acid disodium, the temperature dependency of Aspergillus fumigatus phytase activity.

Fig. 8. in the presence of 5%, 10% and 25% sodium acetate, the temperature dependency of Aspergillus fumigatus phytase activity.

Fig. 9. in the presence of 25% propanedioic acid disodium, the temperature dependency of aspergillus niger (A), total (B), terreus CBS (C) and Aspergillus nidulans (D) phytase activity.

Figure 10. in the presence of 25% disodium succinate, the temperature dependency of aspergillus niger (A), total (B), terreus CBS (C) and Aspergillus nidulans (D) phytase activity.

Figure 11.

A) with behind the sodium periodate incubation of different concns, the SDS-PAGE of Aspergillus fumigatus phytase sample.

B) use adipic dihydrazide crosslinked subsequently after, (A) in the SDS-PAGE of the different samples of Aspergillus fumigatus phytase of oxidation.

Figure 12. after reaching before usefulness Periodic acid/adipic dihydrazide is crosslinked, the temperature dependency of Aspergillus fumigatus phytase and total phytase activity.

Figure 13. the design of total phytinic acid enzyme sequence.Letter is represented amino-acid residue with the form of one-letter code.Use following sequence to carry out series arrangement: the phyA of terreus 9A-1 (people such as Mitchell, 1997; From amino acid (aa) 27), phyA (people such as van Loon, 1998 of terreus cbs116.46; From aa 27), phyA (people such as Piddington, 1993 of aspergillus niger awamori mutation; From aa 27), phyA (people such as van Hartingsveldt, 1993 of phyA (deriving from aa 27), the aspergillus niger strain NRRL3135 of aspergillus niger T213; From aa 27), phyA (people such as Pasamontes, 1993 of Aspergillus fumigatus ATCC 13073; From aa 25), phyA (people such as van Loon, 1998 of Aspergillus fumigatus ATCC3 2722; From aa 27), phyA (people such as van Loon, 1998 of Aspergillus fumigatus ATCC58128; From aa 27), phyA (people such as van Loon, 1998 of Aspergillus fumigatus ATCC 26906; From aa 27), phyA (people such as van Loon, 1998 of Aspergillus fumigatus ATCC 32239; From aa 30), phyA (people such as Pasamontes, the 1997a of Emericella nidulans; From aa 25), phyA (people such as Pasamontes, the 1997a of Talaromyces thermophilus; From aa 24) and phyA (people such as Mitchell, 1997 of Myceliophthora thermophila; From aa 19).The service routine PILEUP sequence of calculation is arranged.The manual position of modifying breach.The capitalization amino-acid residue of specific position belongs to the amino acid combination of setting up total residue in the series arrangement.The aminoacid sequence that under the consensus sequence that calculates, shows the final total phytase (Fcp) that makes up with black matrix.According to the manual breach of filling in the consensus sequence that calculates of embodiment 3 described principles.

Figure 14. the dna sequence dna of total CONSENSUS PHYTASE-1 gene (fcp) and the dna sequence dna that is used for gene constructed primer.The codon frequency table (GCG routine package, 9.0) of the yeast genes of service routine BACKTRANSLATE people such as (, 1984) Devereux and high expression level is converted to dna sequence dna with the aminoacid sequence (Figure 13) that calculates.The signal peptide of the phytase of terreus cbs.116.46 and N-are terminal to be merged.The representative of black matrix base is used to produce the oligonucleotide sequence of gene.In addition on the sequence or under pointed out oligonucleotide title separately.The base that marks with underscore is represented the initial sum terminator codon of gene.The base of writing out with italic shows the EcoRI site of two introducings.

Figure 15. the series arrangement and the consensus sequence of five kinds of basidiomycetes (Basidiomycetes) phytase.Letter is represented amino-acid residue with one-letter code.Start from paxillus involutus (Paxillus involutus) phyA (aa21) and the phyA2 (aa21 of amino-acid residue shown in the parenthesis, WO 98/28409), (aa 24 for fine hair bolt bacterium (Trametes pubescens), WO 98/28409), flat the edge of a field eats (Agrocybe pediades) (aa 19, WO 98/28409) and Peniophora lycii (aa 21, WO 98/28409) the aminoacid sequence of phytase, be used for carrying out series arrangement, and be called the calculating (embodiment 4) of the corresponding consensus sequence of " Basidio ".Carry out series arrangement with program PILEPUP.The manual position of modifying breach.Calculate consensus sequence with program PRETTY.When 0.5 weight (vote weight) was distributed to two kinds of paxillus involutus phytases, using weight was that all other genes of 1.0 carry out the calculating of consensus sequence.Can not determine the position that has residue in program, the Basidio sequence comprises a dash.The capitalization amino-acid residue of specific position belongs to the amino acid combination of setting up total residue in the series arrangement.

Figure 16. the design of total CONSENSUS PHYTASE-10 aminoacid sequence.The consensus sequence of the phytase of the phytinic acid enzyme sequence of Thermomyceslanugiosa people such as (, 1998) Berka and five kinds of basidiomycetes is added in the series arrangement of Figure 13, with the consensus sequence of program PRETTY computed improved.In addition, omit the aminoacid sequence of aspergillus niger T213, therefore, the aspergillus niger phytinic acid enzyme sequence that keeps is used 0.5 weight.Further information is seen embodiment 14.

Figure 17. the DNA and the aminoacid sequence of total CONSENSUS PHYTASE-10.On corresponding DNA sequence, write out aminoacid sequence with one-letter code.Be used for assembled base because of oligonucleotide sequence be bold-faced letter.Oligonucleotide and the amino acid whose mark of comparing change with total CONSENSUS PHYTASE-1 mark with underscore, and with outstanding its corresponding triplet code of small letter.Assemble the fcp10 gene by following oligonucleotide: CP-1, CP-2, CP-3.10, CP-4.10, CP-5.10, CP-6, CP-7.10, CP-8.10, CP-9.10, CP-10.10, CP-11.10, CP-12.10, CP-13.10, CP-14.10, CP-15.10, CP-16.10, CP-17.10, CP-18.10, CP-19.10, CP-20.10, CP-21.10, CP-22.10.New synthetic oligonucleotide numeral 10 other marks.Phytase comprises following 32 exchange: Y54F, E58A, D69K, D70G, A94K, N134Q, I158V, S187A, Q188N, D197N, S204A, T214L, D220E, L234V, A238P, D246H, T251N, Y259N, E267D, E277Q, A283D, R291I, A320V, R329H, S364T, I366V, A379K, S396A, G404A, Q415E, A437G, A463E.The sudden change of emphasizing with bold-faced letter shows that this is as single mutation is measured in the total CONSENSUS PHYTASE-1 to having a kind of stabilization of CONSENSUS PHYTASE-1.

Figure 18. be used for the series arrangement of total CONSENSUS PHYTASE-1 1 design.Opposite with the design of total CONSENSUS PHYTASE-10, use 0.2 the weight of distributing for every kind of basidiomycetes sequence, use all basidiomycetes phytases to have the design of the aminoacid sequence of CONSENSUS PHYTASE-1 1 as sequence independently.In addition, in series arrangement, reuse the aminoacid sequence of aspergillus niger T213.

Figure 19. DNA and the aminoacid sequence of total CONSENSUS PHYTASE-1-Re [8]-Q50T-K91A.On corresponding DNA sequence, write out aminoacid sequence with one-letter code.The amino-acid residue of replacing marks with underscore.The terminator codon of gene is with asterisk (*) mark.

Figure 20. DNA and the aminoacid sequence of total CONSENSUS PHYTASE-10-Re [3]-Q50T-K91A.On corresponding DNA sequence, write out aminoacid sequence with one-letter code.The amino-acid residue of replacing marks with underscore.The terminator codon of gene is with asterisk (*) mark.

Figure 21. the DNA and the aminoacid sequence of Aspergillus fumigatus ATCC 13073 phytase α-mutant.On corresponding DNA sequence, write out aminoacid sequence with one-letter code.The amino-acid residue of replacing marks with underscore.The terminator codon of gene is with asterisk (*) mark.

Figure 22. the DNA and the aminoacid sequence of total phytase-7.On corresponding DNA sequence, write out aminoacid sequence with one-letter code.Be used for assembled base because of oligonucleotide sequence be bold-faced letter.Marked the oligonucleotide and the amino acid that exchange with underscore, and with outstanding its corresponding triplet code of small letter.Assemble the fcp7 gene by following oligonucleotide: CP-1, CP-2, CP-3, CP-4.7, CP-5.7, CP-6, CP-7, CP-8.7, CP-9, CP-10.7, CP-11.7, CP-12.7, CP-13.7, CP-14.7, CP-15.7, CP-16, CP-17.7, CP-18.7, CP-19.7, CP-20, CP-21, CP-22.New synthetic oligonucleotide numeral 7 other marks.Compare with original total phytase, this phytase comprises following 24 exchange: 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.

Figure 23. the differential scanning calorimetric (DSC) of total CONSENSUS PHYTASE-1 and total CONSENSUS PHYTASE-10.With the about 50-60mg/ml of protein example simmer down to, and to the 10mM sodium acetate, pH5.0 fully dialyses.The constant heat rate of using 10 ℃/minute is up to 95 ℃.The DSC (upper diagram) of total CONSENSUS PHYTASE-10 produces 85.4 ℃ melting temperature (Tm), and it is higher 7.3 ℃ than the fusing point (78.1 ℃, bottom graph) of total CONSENSUS PHYTASE-1.

Figure 24. the differential scanning calorimetric (DSC) of total CONSENSUS PHYTASE-10-Re-Q50T and total CONSENSUS PHYTASE-10-Re-Q50T-K91A.With the about 50-60mg/ml of protein example simmer down to, and to the 10mM sodium acetate, pH5.0 fully dialyses.The constant heat rate of using 10 ℃/minute is up to 95 ℃.Total CONSENSUS PHYTASE-10-Re-DSC (upper diagram) of Q50T produces 88.6 ℃ melting temperature (Tm), and finds that the fusing point of total CONSENSUS PHYTASE-10-Re-Q50T-K91A is 89.3 ℃.

Figure 25. the comparison of optimum temperuture between total CONSENSUS PHYTASE-1, total CONSENSUS PHYTASE-10 and the total CONSENSUS PHYTASE-10-Re-Q50T.For the mensuration of optimum temperuture, the series of temperature between 37 ℃ and 86 ℃ is carried out the phytase standard test.Supernatant liquor with the dilution of the yeast saccharomyces cerevisiae strain that transforms carries out this mensuration.Other component in the supernatant liquor shows that the mensuration to optimum temperuture does not influence: ^, total CONSENSUS PHYTASE-1; ☆, total CONSENSUS PHYTASE-10; ■, total CONSENSUS PHYTASE-10-Re-Q50T.

Figure 26. profile of activity and substrate specificity that the pH-of total CONSENSUS PHYTASE-10 and variant heat-Q50T and heat-Q50T-K91A relies on.Use the standard test method in the suitable damping fluid (seeing embodiment 11) of different pH values, to measure the activity of phytase.Figure is the profile of activity of the pH-dependence of the total CONSENSUS PHYTASE-10 () of demonstration, total CONSENSUS PHYTASE-10-Re-Q50T (.) and total CONSENSUS PHYTASE-10-Re-Q50T-K91A (^) a).Figure b) shows that corresponding substrate specificity, its measuring method are to replace phytinic acid with specified compound in standard test; Blank bar, total CONSENSUS PHYTASE-10 (grey bar, total CONSENSUS PHYTASE-10-Re-Q50T; Black bar, total CONSENSUS PHYTASE-10-Re-Q50T-K91A).Digital corresponding to following compound: 1, phytinic acid; 2, p-nitrophenyl phosphate; 3, phenyl-phosphate; 4, fructose-1,6-diphosphate; 5, fructose-6-phosphate; 6, G-6-P; 7, ribose-5-phosphoric acid; 8, the DL-glycerol-3-phosphate; 9, glycerine-2-phosphoric acid; 10,3-phoshoglyceric acid; 11, phosphoenolpyruvic acid; 12, AMP; 13, ADP; 14, ATP.

Figure 27. profile of activity and substrate specificity that the pH-of total CONSENSUS PHYTASE-1-Re [8]-Q50T and total CONSENSUS PHYTASE-1-Re [8]-Q50T-K91A relies on.Use the standard test method in the suitable damping fluid (seeing embodiment 11) of different pH values, to measure phytase activity.Figure a) shows the profile of activity of the pH-dependence of Q50T (■) and Q50T-K91A-variant (.).Figure b) shows that corresponding substrate specificity, its measuring method are to replace phytinic acid (blank bar, total CONSENSUS PHYTASE-1-Re [8]-Q50T with specified compound in standard test; Packing, total CONSENSUS PHYTASE-1-Re [8]-Q50T-K91A).Substrate is listed in the legend of Figure 26.

Figure 28. the differential scanning calorimetric (DSC) of total CONSENSUS PHYTASE-1-Re [8]-Q50T and total CONSENSUS PHYTASE-1-Re [8]-Q50T-K91A.With the about 50-60mg/ml of protein example simmer down to, and to the 10mM sodium acetate, pH5.0 fully dialyses.The constant heat rate of using 10 ℃/minute is up to 95 ℃.Total CONSENSUS PHYTASE-1-Re [8]-DSC (last figure) of Q50T shows 84.7 ℃ melting temperature (Tm), and finds that the fusing point of total CONSENSUS PHYTASE-1-Re [8]-Q50T-K91A is 85.7 ℃.

Figure 29. the comparison of optimum temperuture between total CONSENSUS PHYTASE-1, total CONSENSUS PHYTASE-1-Re [3] and the total CONSENSUS PHYTASE-1-Re [8].In order to measure optimum temperuture, under the series of temperature between 37 ℃ and 86 ℃, carry out the phytase standard test.Protein with purifying from the supernatant liquor of the yeast saccharomyces cerevisiae strain that transforms carries out this mensuration.Zero, total CONSENSUS PHYTASE-1; , total CONSENSUS PHYTASE-1-Re [3]; ▲, total CONSENSUS PHYTASE-1-Re [8].

Figure 30. the profile of activity that the pH-of the phytase of total CONSENSUS PHYTASE-1, total phytase-7 and black mold NRRL3135 relies on and the comparison of substrate specificity.Use the standard test method in the suitable damping fluid (seeing embodiment 11) of different pH values, to measure phytase activity.Figure a) shows the profile of activity of the pH-dependence of the phytase (zero) of total CONSENSUS PHYTASE-1 (■), black mold NRRL 3135 and total phytase-7 (▲).Figure b) shows that corresponding substrate specificity, its measuring method are to replace phytinic acid (black bar, black mold NRRL 3135 phytases with specified compound in standard test; Grey bar, total CONSENSUS PHYTASE-1; Dashed bars, total phytase-7).Substrate is listed in the legend of Figure 26.

Figure 31. the differential scanning calorimetric (DSC) of the phytase of Aspergillus fumigatus ATCC 13073 and stable α-mutant thereof, this mutant comprise following amino acid exchange: F55Y, V100I, F114Y, A243L, S265P, N294D.With the about 50-60mg/ml of protein example simmer down to, and to the 10mM sodium acetate, pH5.0 fully dialyses.The constant heat rate of using 10 ℃/minute is up to 95 ℃.The DSC (upper diagram) of total Aspergillus fumigatus 13073 phytases shows 62.5 ℃ melting temperature (Tm), and finds that the fusing point of α-mutant is 67.0 ℃.

Figure 32. Aspergillus fumigatus 13073 wild-types, Aspergillus fumigatus α-mutant and a kind of further comparison of the optimum temperuture of stable α-mutant (E59A-S126N-R329H-S364T-G404A).In order to measure optimum temperuture, under the series of temperature between 37 ℃ and 75 ℃, carry out the phytase standard test.Supernatant liquor with the dilution of the yeast saccharomyces cerevisiae strain that transforms carries out this mensuration.Other component of supernatant liquor shows the not influence of mensuration to optimum temperuture.Zero, Aspergillus fumigatus ATCC 13073 phytases; ▲, Aspergillus fumigatus ATCC 13073 α-mutant; , Aspergillus fumigatus ATCC 13073 α-mutant-(E59A-S126N-R329H-S364T-G404A)-Q27T; ■, Aspergillus fumigatus ATCC13073 α-mutant-(E59A-S126N-R329H-S364T-G404A)-Q27T-K68A.Q27T and K68A correspond respectively to total CONSENSUS PHYTASE-1 Q50T and K91A.

Figure 33. the aminoacid sequence of total phytase 12 (consphy12), it comprises many avtive spot residues of transferring to total CONSENSUS PHYTASE-10-Re-Q50T-K91A from " basidio " consensus sequence.

Embodiment 1

A) material

Phytinic acid (ten disodium salts) and polyoxyethylene glycol, polyvalent alcohol, dicarboxylic acid sodium, sodium periodate, adipic dihydrazide and other additive are available from goods providers.Other all pharmaceutical chemicalss are the analytical pure level at least.5ml HiTrap desalting column obtains from Pharmacia.SDS-PAGE gel (4-12%NuPAGE Bis-Tris Pre-Cast) and damping fluid are supplied by NOVEX.

B) expression of phytase and purifying

Aspergillus fumigatus, terreus CBS phytase and total phytase overexpression in multiple-shaped nuohan inferior yeast.Aspergillus niger and Aspergillus nidulans phytase overexpression in aspergillus niger.The former clone who describes these phytases of people such as Pasamontes [application and environmental microbiology (Appl.Environ.Microbiol.) (1997), 63,1696-1700 page or leaf], purifying and sign.Have structure, clone and the purifying of phytase according to the open text 897 985 of european patent application.The total phytase of non-preparation is because amino acid exchange (50 usefulness L replace Q in the site) has the enhanced thermostability up to 70 ℃, and compares with the Aspergillus fumigatus phytase and to have three times of high specific activities.

C) phytase activity is measured

In order to carry out the mensuration of thermostability, by with the enzyme of purifying at the 0.2M sodium acetate, dilution is 0.05U/ml (activity of 37 ℃ of mensuration) among the pH5.0 (containing or do not contain the additive of %w/w), carries out the mensuration of enzymic activity with phytinic acid in different temperature.One equal portions protein soln (250 μ l) the temperature preincubation of hope 5 minutes, is added isopyknic 0.2M sodium acetate that contains 1% phytinic acid subsequently, pH5.0 solution (with the 10ml equal portions uniform temp preincubation 10 minutes).Sample is at temperature (for example at 60 or 65 ℃, being used for the screening of the additive effect) incubation of hope after 15 minutes, by the adding termination reaction of 0.5ml 15% trichoroacetic acid(TCA).The mensuration of the inorganic phosphate that discharges with standard method.

D) evaluation of thermal stabilization additive

Usually, polyvalent alcohol is dissolved in the 0.2M sodium acetate with the concentration of 25% or 50% (w/w), among the pH5.0.Except that molecular weight is 4000,8000 and 10000 PEG uses with 25% concentration, PEG is with 50% concentration dissolving.For the screening of PEG and other polyvalent alcohol, select 60 ℃ preincubation and temperature of reaction for Aspergillus nidulans and terreus CBS phytase, Aspergillus fumigatus and aspergillus niger phytase are selected 65 ℃, and total phytase is selected 75 ℃.

With 5%, 10% and 25% concentration dissolving propanedioic acid disodium, disodium succinate and pentanedioic acid disodium, add additive and substrate (on seeing) at enzyme and measure phytase activity after with following temperature preincubation: 37,45,50,55,60,65,70,75,80 and 85 ℃.With the same manner, detected in the presence of 25% Xylitol and ribitol the temperature dependency of different phytase activities.The concentration that should be pointed out that substrate adding back additive reduces by half.

E) sugar chain is crosslinked

[organic chemistry research 47: the stability of enzyme is with stable as described in saccharase as people such as Cesi, the international academic meeting paper collection of holding at Dutch Maastricht in 1992, Elsevier SciencePublications B.V., Amsterdam, Holland] carry out the crosslinked of phytase sugar chain.At the 0.2M sodium acetate, under the existence of different concns among the pH5.0 (0,5,10,20,30,40 and 50mM) sodium periodate, (5mg protein/ml) is 30 ℃ of incubations 2 hours, and spends the night in 4 ℃ of storages with the phytase sample.Use the 0.2M sodium acetate, pH5.0 goes up each sample desalination at the 5ml HiTrap desalting column (Pharmacia) that is connected with AktaExplorer system (Pharmacia) as elution buffer.Realize that crosslinked method is that 100 μ l 0.5M are dissolved in the 0.2M sodium acetate, the adipic dihydrazide among the pH5.0 adds in the oxidation products of 900 μ l desalinations.Carrying out the phytase activity of sample after oxidation and cross-linking step measures and gel electrophoresis.

F) the high-shear granulation of heat-staple phytase

100-250ml phytinic acid enzyme solution (the crosslinked or noncrosslinking phytase of 2500-5000 unit altogether) is added 5-10% calcium lignin sulphonate (Borregard, Norway), 5-20% silicon-dioxide (Sipernat 50S, Degussa, Germany), in the 1kg drying composite of 0-20% thermo-stabilizer and gypsum.In the high-shear granulation process, add entry and have the particle of wishing character up to formation.With particle 45 ℃ of dryings 15 minutes in fluidized bed dryer, use natural palm fat (palm 46, Florin, Basel, Switzerland) fat bag quilt subsequently.

G) the granulation stability of heat-staple drying and liquid phytase formulation

Heat-staple drying and liquid phytase formulation (as mentioned above) are mixed with feed, subsequently granulation under 85 ℃ of steam conditions.By measuring the phytase activity in the pill that the juice neutralization transports before the granulation, measure the granulation stability of phytase.Embodiment 2

Studies show that as embodiment 1 the temperature dependent of described different fungi phytase activities, maximum activity is in following temperature: Aspergillus fumigatus phytase and aspergillus niger phytase are at 55 ℃, terreus CBS phytase and Aspergillus nidulans phytase are at 45 ℃, and total phytase is at 65 ℃.Select institute to survey on the top temperature 10-15 ℃ temperature, be used to study the influence of polyvalent alcohol, polyoxyethylene glycol, dicarboxylate, carboxymethyl cellulose and sodiun alginate the phytinic acid enzyme heat stability as screening point.

A) adding of different molecular weight polyethylene glycol

The adding of 50% or 25% (between the reaction period be 25% and 12.5% final concentration) polyoxyethylene glycol has strengthened specific activity at the Aspergillus fumigatus phytase of 65 ℃ of mensuration in molecular weight dependence mode, observes maximum value (specific activity 80U* (mg protein) with PEG 1450 ^-1), PEG 1000 (50U* (mg protein) ^-1) and PEG 3350 (42U* (mg protein) ^-1) considerable activity also arranged.Fig. 2 has summarized this result of experiment.

Molecular weight is 600,1000,1450,3350 with the PEG of 4000Da other phytase of being surveyed to be shown similar effect.This result of experiment is shown among Fig. 3.

B) adding of polyvalent alcohol

Concentration is 25% and 50% polyvalent alcohol ribitol, Xylitol (C ₅Sugared) and Sorbitol Powder (C ₆Sugar) significantly improved the thermostability of Aspergillus fumigatus phytase.This is shown among Fig. 4.

Erythritol, N.F,USP MANNITOL, mannoheptulose and mannoheptose can not be dissolved in the 0.2M sodium acetate with the concentration of 50% (w/w), among the pH5.0, therefore only show 25% value.Be respectively 11,21 and 11U* (mg protein) at the specific activity of 65 ℃ of mensuration in the presence of ribitol 25%, Xylitol and the Sorbitol Powder ^-1, specific activity is respectively 51,90 and 74U* (mg protein) in the presence of 50% ribitol, Xylitol and sorbitol solution ^-1

Comprise more than 6 or be less than the polyvalent alcohol such as the glycerine (C of 5 carbon atoms ₃Sugar), erythritol (C ₄Sugar), mannoheptose and mannoheptulose (C ₇Sugar) effect lower to the thermally-stabilised demonstration of Aspergillus fumigatus phytase.

The Xylitol of 50% concentration also makes the optimum temperuture of Aspergillus nidulans, terreus CBS, aspergillus niger and total phytase improve 10-15 ℃.The results are shown among Fig. 5.

C) adding of dicarboxylic acid

Malonate, succinate and the glutarate of 25% concentration (12.5% final concentration in the determination of activity) cause significantly improving of Aspergillus fumigatus phytinic acid enzyme heat stability, still detect considerable activity at 70 ℃, succinate and glutarate at 65 ℃ for malonate.The results are shown among Fig. 6.

In addition, dicarboxylate stimulates the Aspergillus fumigatus phytase activity 37 ℃ of mensuration, approximately improves 4 times for the malonate phytase activity, and succinate improves 2 times, and glutarate has less effect.Studies show that of different concns (5%, 10% and 25%) malonate, the thermally-stabilised of Aspergillus fumigatus phytase is that concentration relies on, and the stimulation of enzymic activity is not the concentration dependence at least in this concentration range.This is shown among Fig. 7.

Opposite with these discoveries, the sodium acetate of different concns (5%, 10% and 25%), a kind of monocarboxylic acid causes 2 times of raisings of 37 ℃ of specific activities of Aspergillus fumigatus phytase, but this proteinic thermostability is only had less effect.This can find out in Fig. 8.

Propanedioic acid disodium and disodium succinate make the thermostability of Aspergillus nidulans, terreus CBS, aspergillus niger and total phytase improve 5-15 ℃ usually.On the other hand, only Aspergillus nidulans and Aspergillus fumigatus phytase are observed the stimulation of phytase activity, the both has quite low specific activity, but terreus CBS, aspergillus niger and total phytase are not then observed.This obtains proof in Fig. 9 and Figure 10.

D) crosslinked effect

In a preliminary experiment, by using the crosslinked Aspergillus fumigatus phytase of glutaraldehyde incubation monomer.The final thermally-stabilised of 60 ℃ of mensuration reached maximum value after 1 hour reaction times, but caused activity to lose (37 ℃ of mensuration).In another group experiment, Aspergillus fumigatus phytase monomer is crosslinked by its sugar chain.Realize this type crosslinked only with specific activity than small loss (＜10%), and when surpassing the sodium periodate concentration of 15mM, cause the formation of oligomerization form.This can be as seen from Figure 11.

Heat-staple scope depends on the concentration of periodate, and reaches maximum value at 50mM, and the high specific acitivity that observe this moment is up to 75 ℃ (seeing Figure 12).For also observing of the remarkably influenced of phytase oligomerization to thermostability by the crosslinked total phytase of sugar chain.This can be as seen from Figure 12.

In this work, we are with making great efforts to concentrate on the heat stabilization (this is used for the stable of industrial enzyme by highly recommended) of low Mr additive and the heat stabilization of chemically modified, though because technology and economic reasons it has been generally acknowledged that a kind of method in back does not have magnetism.

We have found C5 sugar thermally-stabilised to many different phytases of expressing in filamentous fungus (aspergillus niger) or yeast (multiple-shaped nuohan inferior yeast).The raising of thermostability has the difference of some degree between different phytases, but all about 10 ℃.The effect of PEG is that molecular weight relies on.With molecular weight 1000 and 3350Da between PEG to obtain the optimal heat of all phytases stable.

Sodium acetate, a kind of monocarboxylic acid and be the main ingredient that the standard phytase activity is measured causes the enhancing that the concentration of Aspergillus fumigatus phytase activity relies on, but for the not effect of thermostability of phytase.Therefore, carboxyl may be responsible for active the adjusting, and difunctional dicarboxylate may be stablized phytase by ionic interaction.Embodiment 3

The design of the aminoacid sequence of total CONSENSUS PHYTASE-1

The arrangement of aminoacid sequence

Use the program PILEUP and canonical parameter (breach produces and penalizes 12, and breach extends the penalizes 4) sequence of calculation of sequential analysis bag 9.0 editions people such as (, 1984) Devereux to arrange.Use text editor to modify the position of breach.Table 1 shows the sequence (seeing Figure 13) that does not contain signal sequence, and this sequence is used for the carrying out of series arrangement, arranges to start from the described amino acid of table 1 (aa).

Table 1: be used for the source of phytinic acid enzyme amino acid sequence of total CONSENSUS PHYTASE-1 design and weight-the derive from phyA of terreus 9A-1, aa 27, weight 0.5 (people such as Mitchell, 1997)-derive from the phyA of terreus cbs116.46, aa 27, weight 0.5 (people such as van Loon, 1998)-derive from the phyA of aspergillus niger awamori mutation, aa 27, weight 0.33 (people such as Piddington, 1993)-derive from the phyA of aspergillus niger T213, aa 27, weight 0.33-derives from the phyA of aspergillus niger strain NRRL3135, aa 27, weight 0.33 (people such as van Hartingsveldt, 1993)-derive from the phyA of Aspergillus fumigatus ATCC 13073, aa 26, weight 0.2 (people such as Pasamontes, 1997)-derive from the phyA of Aspergillus fumigatus ATCC 32722, aa 26, weight 0.2 (people such as van Loon, 1998)-derive from the phyA of Aspergillus fumigatus ATCC 58128, aa 26, weight 0.2 (people such as van Loon, 1998)-derive from the phyA of Aspergillus fumigatus ATCC 26906, aa 26, weight 0.2 (people such as van Loon, 1998)-and deriving from the phyA of Aspergillus fumigatus ATCC 32239, aa 30, weight 0.2 (people such as van Loon, 1998)-derive from the phyA of Emericella nidulans, aa 25, and weight 1.0 (people such as Pasamontes, 1997a)-derive from the phyA of Talaromyces thermophilus ATCC 20186, aa 24, weight 1.0 (people such as Pasamontes, 1997a)-deriving from the phyA of Myceliophthora thermophila, aa 19, the calculating of the total CONSENSUS PHYTASE-1 aminoacid sequence of weight 1.0 people such as (, 1997) Mitchell

As input, the program PRETTY by sequential analysis bag 9.0 editions people such as (, 1984) Devereux calculates consensus sequence with the series arrangement of modifying.PRETTY prints sequence with the perpendicular row of its arrangement, and can show the consensus sequence of arranging.The weight allocation of similarity is given all sequences between the phytinic acid enzyme amino acid sequence that will be referred to arrange.Set weight, for example set a sequence subgroup (identical kind, but derive from different strains) the combined effect of all phytases (as the aminoacid sequence of all phytases of Aspergillus fumigatus) to selecting, its meaning is to provide 1 value (seeing Table 1) divided by strain sequence quantity for each sequence.In this way, the closely similar aminoacid sequence that can prevent the phytase of for example different Aspergillus fumigatus strains is preponderated in the consensus sequence that calculates.

Program PRETTY starts from following parameter: great majority have illustrated the quantity of power, do not have under it and are set to 2.0.Determine that the matrix-valued threshold value of score is set to 2, amino-acid residue can not weighting be used for the residue combination under it.For peptide PRETTY use PrettyPep.Cmp total sub matrix.

Can not determine (the position 46,66,82,138,162,236,276,279,280,308,10 positions in the series arrangement of total residue according to the manual to-fill procedure of following principle; Figure 13):, then select this residue (138,236,280) if there is modal residue; If a group of having advantage similar or system identical residue takes place, then select modally, if perhaps can not obtain modally, then select the residue (46,66,82,162,276,308) among this group.If both there be not the advantage residue not have the advantage group, then according to one of the residue of the common hypothesis of protein stability influence being selected occur (279) yet.Other 8 positions (132,170,204,211,275,317,384,447; Figure 13) amino-acid residue of program selection of no use is filled, but generally uses the amino acid that occurs with the selected residue same frequency of program to fill.In most of the cases, eliminated three kinds of aspergillus niger sequences (weight sum: slightly underestimating 0.99) by this correction.Total CONSENSUS PHYTASE-1 aminoacid sequence is to the conversion of dna sequence dna

, and therefore merge as signal peptide with preceding 26 amino-acid residues of terreus cbs116.46 phytase with the N-of all total phytases is terminal.For this prolongation, we use a kind of special methods to calculate corresponding DNA sequence.People such as Purvis (1987) suggestion, in the gene rare codon mix Protein Folding efficient influential.Therefore, shift in the new signal sequence of most in yeast saccharomyces cerevisiae, expressing and producing to the distribution of major general's rare codon in terreus cbs116.46 signal sequence, the signal sequence of terreus cbs116.46 is used for total phytase, for proteinic secretion is very important, but is converted into the codon that yeast saccharomyces cerevisiae uses.For proteinic remainder, we use from the codon frequency table of the high expression level genes of brewing yeast of GCG routine package acquisition, are dna sequence dna with the amino acid sequence translation that calculates.

The sequence of the fcp gene that produces is shown among Figure 14.The structure and the clone of total CONSENSUS PHYTASE-1 gene

Be used alternatingly the justice and the sequence of antisense strand, the dna sequence dna of the total CONSENSUS PHYTASE-1 (fcp) of calculating be divided into the oligonucleotide of 85bp.Each oligonucleotide with its before and the overlapping 20bp of oligonucleotide of afterwards relative chain.In Figure 14, shown available from Microsynth the position of Balgach (Switzerland) and all primers of obtaining with the PAGE purified form.The PCR reaction

In three PCR reactions, the synthetic oligonucleotide is formed complete gene.Use the high fidelity test kit of Boehringer Mannheim (Boehringer Mannheim, Mannheim, Germany) and the thermal cycler Protokol of AMS biotechnology (Europe) company limited (Lugano, Switzerland) ^TMCarry out PCR.

(mixture 1 Figure 14) is mixed into the concentration of every kind of oligonucleotide 0.2pmol/ μ l to CP-10 with oligonucleotide CP-1.Prepare second kind of oligonucleotide mixture (mixture 2) with CP-9 to CP-22 (every kind of oligonucleotide 0.2pmol/ μ l).In addition, in the PCR reaction, use 4 kinds of short primers:

CP-a：????????????EcoRI

5’-TATATGAATTCATGGGCGTGTTCGTC-3’

CP-b：

5’-TGAAAAGTTCATTGAAGGTTTC-3’

CP-c：

5’-TCTTCGAAAGCAGTACAAGTAC-3’

CP-e：????????????????EcoRI

5 '-TATATGAATTCTTAAGCGAAAC-3 ' PCR reaction a:10 μ l mixture, 1 (every kind of oligonucleotide 2.0pmol)

2 μ l Nucleotide (every kind of Nucleotide 10mM)

2 μ l primer CP-a (10pmol/ μ l)

2 μ l primer CP-c (10pmol/ μ l)

10.0 μ l PCR damping fluid

0.75 μ l polysaccharase mixture

73.25 μ l H ₂OPCR reaction b:10 μ l mixture 2 (every kind of oligonucleotide 2.0pmol)

2 μ l Nucleotide (every kind of Nucleotide 10mM)

2 μ l primer CP-b (10pmol/ μ l)

2 μ l primer CP-e (10pmol/ μ l)

10.0 μ l PCR damping fluid

0.75 μ l polysaccharase mixture (2.6U)

73.25 μ l H ₂The reaction conditions of OPCR reaction a and b:

-45 ℃ of steps 12 minutes

-72 ℃ of steps 2 30 seconds

-94 ℃ of steps 3 30 seconds

-52 ℃ of steps 4 30 seconds

-72 ℃ of steps 51 minute

Step 3 to 5 repeats 40 times.

By agarose gel electrophoresis (0.9% agarose) and gel extracting subsequently (QIAEX II gel extraction agent box, Qiagen, Hilden, Germany) purified pcr product (670 and 905bp).Dna fragmentation with purifying carries out PCR reaction c.The PCR product (≈ 50ng) of PCR reaction c:6 μ l reaction a

The PCR product (≈ 50ng) of 6 μ l reaction b

2 μ l primer CP-a (10pmol/ μ l)

2 μ l primer CP-e (10pmol/ μ l)

10.0 μ l PCR damping fluid

0.75 μ l polysaccharase mixture (2.6U)

73.25 μ l H ₂The reaction conditions of OPCR reaction c:

-94 ℃ of steps 12 minutes

-94 ℃ of steps 2 30 seconds

-55 ℃ of steps 3 30 seconds

-72 ℃ of steps 41 minute

Step 2 to 4 repeats 31 times.

The PCR product (1.4kb) of purifying generation with EcoRI digestion, is connected to also dephosphorylized pBsk (-) carrier (Stratagene, La Jolla, CA, the U.S.) of EcoRI digestion as mentioned above.Connect mixture transformed into escherichia coli XL-1 competent cell (Stratagene, La Jolla, CA, the U.S.) with 1 μ l.All standard methods are carried out as described in people such as Sambrook (1987).Total phytase gene (fcp, dna sequence dna Figure 14) by order-checking control structure known in the art.The design of embodiment 4 improved total phytases (total CONSENSUS PHYTASE-10) aminoacid sequence

Use the program PILEUP and the canonical parameter (breach produces and penalizes 12, and breach extends penalizes 4) of sequential analysis bag 9.0 editions people such as (, 1984) Devereux to calculate the series arrangement that is used for total CONSENSUS PHYTASE-10 design.Use text editor to modify the position of breach.Use following sequence to carry out the series arrangement of basidiomycetes phytase, it starts from the described amino acid of table 2 (aa): table 2: be used for the source of 5 kinds of basidiomycetes phytases that corresponding amino acid consensus sequences (basidio) calculates and weight-the derive from phyA1 of paxillus involutus NN 005693, aa 21, weight 0.5 (WO 98/28409)-the derive from phyA2 of paxillus involutus NN 005693, aa 21, weight 0.5 (WO 98/28409)-the derive from phyA of fine hair bolt bacterium NN 9343, aa 24, weight 1.0 (WO 98/28409)-the derive from phyA of flat the edge of a field mushroom NN 009289, aa 19, weight 1.0 (WO 98/28409)-the derive from phyA of Peniophora lycii NN 006113, aa 21, and weight 1.0 (WO98/28409) series arrangement is shown among Fig. 3.

In table 3, arranged and be used to carry out the gene that ultimate sequence is arranged.First amino acid (aa) of the sequence of using in arrangement is mentioned after biological name.Table 3: be used for the source of phytinic acid enzyme sequence of total phytase 10 designs and weight-the derive from phyA of terreus 9A-1, aa 27, weight 0.5 (people such as Mitchell, 1997)-derive from the phyA of terreus cbs116.46, aa 27, weight 0.5 (people such as van Loon, 1998)-derive from the phyA of aspergillus niger awamori mutation, aa 27, weight 0.5 (people such as Piddington, 1993)-derive from the phyA of aspergillus niger strain NRRL3135, aa 27, weight 0.5 (people such as van Hartingsveldt, 1993)-derive from the phyA of Aspergillus fumigatus ATCC 13073, aa 26, weight 0.2 (people such as Pasamontes, 1997)-derive from the phyA of Aspergillus fumigatus ATCC 32722, aa 26, weight 0.2 (people such as van Loon, 1998)-derive from the phyA of Aspergillus fumigatus ATCC 58128, aa 26, weight 0.2 (people such as van Loon, 1998)-the derive from phyA of Aspergillus fumigatus ATCC 26906, aa 26, weight 0.2 (people such as van Loon, 1998)-derive from the phyA of Aspergillus fumigatus ATCC 32239, aa 30, weight 0.2 (people such as van Loon, 1998)-and deriving from the phyA of Emericella nidulans, aa 25, weight 1.0 (people such as Pasamontes, 1997a)-derive from the phyA of Talaromyces thermophilus ATCC 20186, aa 24, and weight 1.0 (people such as Pasamontes, 1997a)-derive from the phyA of Myceliophthora thermophila, aa 19, weight 1.0 (people such as Mitchell, 1997)-derive from the phyA of Thermomyces lanuginosa, aa 36, weight 1.0 (people such as Berka, 1998) consensus sequence of kind of basidiomycetes phytase-5, and weight 1.0 (Basidio, Figure 15)

Corresponding series arrangement is shown among Figure 16.The calculating of total-ten amino acid sequence

In order to improve series arrangement, we add the original consensus sequence of 5 kinds of phytases that derive from 4 kinds of different basidiomycetes in a bigger arrangement, it is called as Basidio, still comprise undetermined sequence location (seeing Figure 15), add the nearly all phytinic acid enzyme sequence of original total phytase calculating and new phytinic acid enzyme sequence of a kind of ascomycetes Thermomyces lanuginosa of being used for.Use the consensus sequence of basidiomycetes phytinic acid enzyme sequence, can not take the difference between the 5 seed amino acid sequences into account, but take common between ascomycetes and the basidiomycetes phytase and different amino-acid residues into account.

We are set to 2.0 with great majority (plurality), set the threshold to 3.The weight of using is listed in the table 3.Series arrangement and corresponding consensus sequence are shown among Figure 16.Compare with the total phytase of primary, new total phytinic acid enzyme sequence has 32 different amino acid.Can not calculate the position of total amino-acid residue according to embodiment 3 described principle to-fill procedure PRETTY.The residue none of program suggestion is replaced.

In addition, we comprise all basidiomycetes phytases as the monamino acid sequence, but with 0.2 weight allocation in series arrangement.Corresponding series arrangement is shown among Figure 18.The consensus amino acid sequences that calculates (total CONSENSUS PHYTASE-1 1) has following difference with the sequence of total CONSENSUS PHYTASE-10.The letter X meaning is the total amino acid that program can not be calculated; Amino acid in the parenthesis is corresponding to the amino acid that finally is contained among the total CONSENSUS PHYTASE-10.

D35X, X (K) 69K, X (E) 100E, A101R, Q134N, X (K) 153N, X (H) 190H, X (A) 204S, X (E) 220D, E222T, V227A, X (R) 271R, H287A, X (D) 288D, X (K) 379K, X (I) 389I, E390X, X (E) 415E, X (A) 416A, X (R) 446L, E463A, and its numbering such as Figure 17.

We have also checked the influence of the monamino acid replacement of improved consensus sequence 10 and 11 suggestions to original total phytinic acid enzyme stability.Its method is described among the embodiment 5.Total CONSENSUS PHYTASE-10 aminoacid sequence is to the conversion of dna sequence dna

, and therefore merge as signal peptide with preceding 26 amino-acid residues of terreus cbs116.46 phytase with the N-of total CONSENSUS PHYTASE-10 is terminal.The method of using is further described among the embodiment 3.

The sequence of the fcp10 gene that produces is shown among Figure 17.The structure and the clone of total CONSENSUS PHYTASE-10 gene (fcp10)

Be used alternatingly the justice and the sequence of antisense strand, the dna sequence dna of the fcp10 of calculating be divided into the oligonucleotide of 85bp.Each oligonucleotide with its before and the overlapping 20bp of oligonucleotide of afterwards relative chain.Shown available from Microsynth the position of Balgach (Switzerland) and all primers of obtaining with the PAGE purified form among Figure 17.The PCR reaction

In three PCR reactions, the synthetic oligonucleotide is formed complete gene.Use the high fidelity test kit of Boehringer Mannheim (Boehringer Mannheim, Mannheim, Germany) and the thermal cycler Protokol of AMS biotechnology (Europe) company limited (Lugano, Switzerland) ^TMCarry out PCR.Following oligonucleotide uses with the concentration of 0.2pmol/ml.

Mixture 1.10:CP-1, CP-2, CP-3.10, CP-4.10, CP-5.10, CP-6, CP-7.10, CP-8.10, CP-9.10, CP-10.10

Mixture 2.10:CP-9.10, CP-11.10, CP-12.10, CP-13.10, CP-14.10, CP-15.10, CP-16.10, CP-17.10, CP-18.10, CP-19.10, CP-20.10, CP-21.10, CP-22.10

With the new synthetic oligonucleotide of digital 10 marks.Compare with original total phytase, this phytase comprises following 32 kinds of exchanges, and these line out below exchanging in Figure 17: Y54F, E58A, D69K, D70G, A94K, N134Q, I158V, S187A, Q188N, D197N, S204A, T214L, D220E, L234V, A238P, D246H, T251N, Y259N, E267D, E277Q, A283D, R291I, A320V, R329H, S364T, I366V, A379K, S396A, G404A, Q415E, A437G, A463E.

Use 4 kinds short PCR primers to carry out the assembling of oligonucleotide:

CP-a：?????????????????Eco?RI

5’-TATATGAATTCATGGGCGTGTTCGTC-3’

CP-b：

5’-TGAAAAGTTCATTGAAGGTTTC-3’

CP-c.10：

5’-TCTTCGAAAGCAGTACACAAAC-3’

CP-e：?????????????Eco?RI

5 '-TATATGAATTCTTAAGCGAAAC-3 ' PCR reaction a:10 μ l mixture, 1.10 (every kind of oligonucleotide 2.0pmol)

2 μ l Nucleotide (every kind of Nucleotide 10mM)

2 μ l primer CP-a (10pmol/ml)

2 μ l primer CP-c.10 (10pmol/ml)

10.0 μ lPCR damping fluid

0.75 μ l polysaccharase mixture

73.25 μ l H ₂OPCR reaction b:10 μ l mixture 2.10 (every kind of oligonucleotide 2.0pmol)

2 μ l Nucleotide (every kind of Nucleotide 10mM)

2 μ l primer CP-b (10pmol/ml)

2 μ l primer CP-e (10pmol/ml)

10.0 μ l PCR damping fluid

0.75 μ l polysaccharase mixture (2.6U)

73.25 μ l H ₂The reaction conditions of OPCR reaction a and b:

-45 ℃ of steps 12 minutes

-72 ℃ of steps 2 30 seconds

-94 ℃ of steps 3 30 seconds

-52 ℃ of steps 4 30 seconds

-72 ℃ of steps 51 minute

Step 3 to 5 repeats 40 times.

By agarose gel electrophoresis (0.9% agarose) and gel extracting subsequently (QIAEX II gel extraction agent box, Qiagen, Hilden, Germany) purified pcr product (670 and 905bp).Dna fragmentation with purifying carries out PCR reaction c.The PCR product (≈ 50ng) of PCR reaction c:6 μ l reaction a

The PCR product (≈ 50ng) of 6 μ l reaction b

2 μ l primer CP-a (10pmol/ml)

2 μ l primer CP-e (10pmol/ml)

10.0 μ l PCR damping fluid

0.75 μ l polysaccharase mixture (2.6U)

73.25 μ l H ₂The reaction conditions of OPCR reaction c:

-94 ℃ of steps 12 minutes

-94 ℃ of steps 2 30 seconds

-55 ℃ of steps 3 30 seconds

-72 ℃ of steps 41 minute

Step 2 to 4 repeats 31 times.

The PCR product (1.4kb) of purifying generation with EcoRI digestion, is connected to also dephosphorylized pBsk (-) carrier (Stratagene, La Jolla, CA, the U.S.) of EcoRI digestion as mentioned above.Connect mixture transformed into escherichia coli XL-1 competent cell (Stratagene, La Jolla, CA, the U.S.) with 1 μ l.All standard methods are carried out as described in people such as Sambrook (1987).The dna sequence dna of the gene (fcp10) that makes up by order-checking inspection known in the art.The introducing of the single mutation that embodiment 5 advises by the aminoacid sequence of total CONSENSUS PHYTASE-10 and total CONSENSUS PHYTASE-1 1 improves the thermostability of total CONSENSUS PHYTASE-1

In order to improve homogenic thermostability, detect the stabilization of amino-acid residues different between the consensus sequence of target protein and calculating and also be possible all stable sudden change composition target proteins.We use total phytase as target protein, and detect its effect to the protein stability of 34 seed amino acid residues, and this is different from the total phytase 10 and/or 11 for single mutation.

For structure is used for the mutein of expressing at aspergillus niger, yeast saccharomyces cerevisiae or multiple-shaped nuohan inferior yeast, use the corresponding expression plasmid that comprises total phytase gene template (seeing embodiment 8-10) as site-directed mutagenesis.Use " the quick exchange of Stratagene according to working specification ^TMThe site-directed mutagenesis test kit " (La Jolla, CA, the U.S.) and use corresponding primer to introduce sudden change.All sudden changes carried out and corresponding primer thereof are summarized in the table 4.Identify the plasmid of sudden change with hope by dna sequence analysis known in the art.Table 4: the primer that is used for total phytase site-directed mutagenesis

(with the base of the outstanding exchange of black matrix.Being introduced on the sequence of restriction site marks.When restriction site write in the parenthesis, described site was destroyed by the introducing of sudden change.)

The mutant primer group

Kpn?I-

Q50T????5’-CACTTGTGGGGTACCTACTCTCCATACTTCTC-3’

5’-GAGAAGTATGGAGAGTAGGTACCCCACAAGTG-3’

Y54F????5’-GGTCAATACTCTCCATTCTTCTCTTTGGAAG-3’

5’-CTTCCAAAGAGAAGAATGGAGAGTATTGACC-3’

E58A????5’-CATACTTCTCTTTGGCAGACGAATCTGC-3’

5’-GCAGATTCGTCTGCCAAAGAGAAGTATG-3’

Aat?II

D69K????5’-CTCCAGACGTCCCAAAGGACTGTAGAGTTAC-3’

5’-GTAACTCTACAGTCCTTTGGGACGTCTGGAG-3’

Aat?IID70G????5’-CTCCAGACGTCCCAGACGGCTGTAGAGTTAC-3’

5’-GTAACTCTACAGCCGTCTGGGACGTCTGGAG-3?’K91A????5’-GATACCCAACTTCTTCTGCGTCTAAGGCTTACTCTG-3’

5’-CAGAGTAAGCCTTAGACGCAGAAGAAGTTGGGTATC-3’

ScaIA94K?????????5’-CTTCTAAGTCTAAGAAGTACTCTGCTTTG-3’

5’-CAAAGCAGAGTACTTCTTAGACTTAGAAG-3’A101R???5’-GCTTACTCTGCTTTGATTGAACGGATTCAAAAGAACGCTAC-3’

5’-GTAGCGTTCTTTTGAATCCGTTCAATCAAAGCAGAGTAAGC-3’N134Q???5’-CCATTCGGTGAACAGCAAATGGTTAACTC-3’

5’-GAGTTAACCATTTGCTGTTCACCGAATGG-3’

Nru?IK153N???5’-GATACAAGGCTCTCGCGAGAAACATTGTTC-3’

5’-GGAACAATGTTTCTCGCGAGAGCCTTGTATC-3’

Bss?HII158V???5’-GATTGTTCCATTCGTGCGCGCTTCTGGTTC-3’

5’-GAACCAGAAGCGCGCACGAATGGAACAATC-3’

Bcl?ID197N????5’-CTCCAGTTATTAACGTGATCATTCCAGAAGG-3’

5’-CCTTCTGGAATGATCACGTTAATAACTGGAG-3’

Apa?IS187A????5’-GGCTGACCCAGGGGCCCAACCACACCAAGC-3’

5’-GCTTGGTGTGGTTGGGCCCCTGGGTCAGCC-3’

Nco?IT214L????5’-CACTTTGGACCATGGTCTTTGTACTGCTTTCG-3’

5’-CGAAAGCAGTACAAAGACCATGGTCCAAAGTG-3’

Avr?IIE222T????5’-GCTTTCGAAGACTCTACCCTAGGTGACGACGTTG-3’

5’-CAACGTCGTCACCTAGGGTAGAGTCTTCGAAAGC-3’V227A????5’-GGTGACGACGCTGAAGCTAACTTCAC-3’

5’-GTGAAGTTAGCTTCAGCGTCGTCACC-3’

Sac?IIL234V????5’-CTAACTTCACCGCGGTGTTCGCTCCAG-3’

5’-CTGGAGCGAACACCGCGGTGAAGTTAG-3’A238P????5’-GCTTTGTTCGCTCCACCTATTAGAGCTAGATTGG-3’

5’-CCAATCTAGCTCTAATAGGTGGAGCGAACAAAGC-3’

Hpa?IT251N????5’-GCCAGGTGTTAACTTGACTGACGAAG-3’

5’-TTCGTCAGTCAAGTTAACACCTGGC-3’

Aat?IIY259N????5’-GACGAAGACGTCGTTAACTTGATGGAC-3’

5’-GTCCATCAAGTTAACGACGTCTTCGTC-3’

Asp?IE267D????5’-GTCCATTCGACACTGTCGCTAGAACTTC-3’

5’-GAAGTTCTAGCGACAGTGTCGAATGGAC-3’E277Q????5’-CTGACGCTACTCAGCTGTCTCCATTC-3’

5’-GAATGGAGACAGCTGAGTAGCGTCAG-3’A283D????5’-GTCTCCATTCTGTGATTTGTTCACTCAC-3’

5’-GTGAGTGAACAAATCACAGAATGGAGAC-3’

Ksp?IH287A??????????5’-GCTTTGTTCACCGCGGACGAATGGAG-3’

5’-CTCCATTCGTCCGCGGTGAACAAAGC-3’

Bam?HIR291I????5’-CACGACGAATGGATCCAATACGACTAC-3’

5’-GTAGTCGTATTGGATCCATTCGTCGTG-3’

Bsi?WIQ292A????5’-GACGAATGGAGAGCGTACGACTACTTG-3’

5’-CAAGTAGTCGTACGCTCTCCATTCGTC-3’

Hpa?IA320V????5’-GGTGTTGGTTTCGTTAACGAATTGATTGC-3’

5’-GCAATCAATTCGTTAACGAAACCAACACC-3’

(Bgl?II)R329H????5’-GCTAGATTGACTCACTCTCCAGTTCAAG-3’

5’-CTTGAACTGGAGAGTGAGTCAATCTAGC-3’

EcoRVS364T????5’-CTCACGACAACACTATGATATCTATTTTCTTC-3’

5’-GAAGAAAATAGATATCATAGTGTTGTCGTGAG-3’

Nco?II366V????5’-CGACAACTCCATGGTTTCTATTTTCTTCGC-3’

5’-GCGAAGAAAATAGAAACCATGGAGTTGTCG-3’

Kpn?IA379K?????????5’-GTACAACGGTACCAAGCCATTGTCTAC-3’

5’-GTAGACAATGGCTTGGTACCGTTGTAC-3’S396A????5’-CTGACGGTTACGCTGCTTCTTGGAC-3’

5’-GTCCAAGAAGCAGCGTAACCGTCAG-3’G404A????5’-CTGTTCCATTCGCTGCTAGAGCTTAC-3’

5’-GTAAGCTCTAGCAGCGAATGGAACAG-3’Q415E????5’-GATGCAATGTGAAGCTGAAAAGGAACC-3’

5’-GGTTCCTTTTCAGCTTCACATTGCATC-3’

Sal?IA437G????5’-CACGGTTGTGGTGTCGACAAGTTGGG-3’

5’-CCCAACTTGTCGACACCACAACCGTG-3’

Mun?IA463E????5’-GATCTGGTGGCAATTGGGAGGAATGTTTCG-3’

5’-CGAAACATTCCTCCCAATTGCCACCAGATC-3’

Therefore and be used for other sudden change.

Summarize as embodiment 11, be determined at the optimum temperuture of the phytase of the purifying of expressing (embodiment 9) in the yeast saccharomyces cerevisiae.Table 5 shows that each sudden change of introducing is to having the influence of phytinic acid enzyme stability.

Table 5: the stability action that single amino acids is replaced in the total CONSENSUS PHYTASE-1

(+or-represent can reach 1 ℃ positive and negative influence respectively to protein stability, ++ and--represent respectively the positive and negative influence of protein stability between 1 ℃ and 3 ℃; The total phytinic acid enzyme sequence that numeral 10 or 11 is replaced corresponding to suggestion amino acid.)

Stablize medium instability

Sudden change influence sudden change influence sudden change influence

E58A(10)?????????????+ D69K(11)?????????????+ D197N(10)????????????+ T214L(10)????????????++ E222T(11)????????????++ E267D(10)????????????+ R291I*???????????????+ R329H(10)????????????+ S364T(10)????????????++ A379K(11)????????????+ G404A(10)????????????++

D69A????????????????????± D70G(10)????????????????± N134Q(10)???????????????± G186H???????????????????± S187A(10)???????????????± T214V???????????????????± T251N(10)???????????????± Y259N(10)???????????????± A283D(10)???????????????± A320V(10)???????????????± K445T???????????????????± A463E(10)???????????????±

Y54F(10)???????????????- V73I???????????????????- A94K(10)???????????????- A101R(11)??????????????- K153N(11)??????????????- I158V(10)??????????????-- G203A??????????????????-- G205S??????????????????- A217V??????????????????- V227A(11)??????????????-- L234V(10)??????????????- A238P(10)??????????????--

E277Q(10)????- H287A(11)????- Q292A(10)????- I366V(10)????- S396A(10)????-- Q415E(11)????- A437G(10)????-- E451R????????--

*: this amino acid replacement is found in another and takes turns in the sudden change.

We use these primers and the 8 kinds of positive sudden changes of combination (E58A, D197N, E267D, R291I, R329H, S364T, A379K, G404A) in total phytase of above-mentioned technology in the present embodiment.In addition, introduce sudden change Q50T and K91A, it mainly influences the catalysis characteristics (seeing open text 897 985 of european patent application and embodiment 11) of phytase.(DNA and the aminoacid sequence of total phytase-Re [8]-Q50T-K91A) are shown among Figure 19 the phytase gene that produces.By this way, 7 ℃ (seeing Figure 27,28,29) of the optimum temperuture of total phytase and fusing point rising.

Use the result of table 5, we have further improved the thermostability of total phytase 10, and method is that reverse mutation K91A, V158I and A396S are carried out in the sudden change of the stability demonstration reinforcing yin essence effect of total phytase.The protein that produces is CONSENSUS PHYTASE-10-Re [3].Gained protein is phytase 10-heat [3].In addition, we introduce sudden change Q50T and K91A, and it mainly influences the catalysis characteristics (seeing the open text 897 985 of patent application EP and embodiment 11 and Figure 26 and Figure 27) of total phytase.The DNA and the aminoacid sequence that produce are shown among Figure 20.Best phytase shows to have high 4 ℃ optimum temperuture and fusing point (Figure 24 and Figure 25) than total phytase 10.In addition, this phytase is the specific activity 250U/mg (Figure 26) that substrate also has when pH5.5 to be increased greatly with the phytinic acid.Embodiment 6

By being replaced with corresponding total CONSENSUS PHYTASE-1 and total CONSENSUS PHYTASE-10 residue, amino-acid residue stablizes Aspergillus fumigatus ATCC 13073 phytases

In the arrangement of the Figure 13 that does not comprise corresponding total phytase amino-acid residue, Aspergillus fumigatus 13073 are 6 exemplary position of unique or almost unique phytase, replace non-total phytase amino-acid residue with total phytase amino-acid residue.In the first round, in Aspergillus fumigatus 13073 phytases, replace following amino acid, comprise that Q27T replaces and the signal sequence (seeing Figure 21) of terreus cbs.116.46 phytase:

F55(28)Y、V100(73)I、F114(87)Y、A243(220)L、S265(242)P、N294(282)D。

Numeral in the parenthesis has provided the numbering of Figure 13.

Take turns second, 4 (E59A, R329H, S364T, G404A) during 7 stable amino acid exchange in the total CONSENSUS PHYTASE-10 sequence are single sudden changes (table 5) in total CONSENSUS PHYTASE-1 after testing, and they are introduced Aspergillus fumigatus α-mutant in addition.In addition, introduce the amino acid that shows reduction phytinic acid protease enzyme susceptibility and replace S126N.

As introducing sudden change (seeing Table 6) as described in the embodiment 5, and as expression as described in the embodiment 8 to 10.Aspergillus fumigatus 13073 inositol six-phosphatase variants that produce are called as α-mutant and α-mutant-E59A-S126N-R329H-S364T-G404A.

Compare with the value of wild-type (optimum temperuture: 55 ℃, Tm:60 ℃), and the optimum temperuture of Aspergillus fumigatus 13073 phytase α-mutant (60 ℃, Figure 32) and fusing point (67.0 ℃ Figure 31) raise 5 ℃.Other 5 amino acid are replaced and are further made 3 ℃ (Figure 32) of optimum temperuture rising.

Table 6: be used for the stable mutant primer of Aspergillus fumigatus phytase ATCC 13073

Mutant primer

F55Y????5’-CACGTACTCGCCATACTTTTCGCTCGAG-3’

5’-CTCGAGCGAAAAGTATGGCGAGTACGTG-3’

(Xho?I)

E58A????5’-CCATACTTTTCGCTCGCGGACGAGCTGTCCGTG-3’

5’-CACGGACAGCTCGTCCGCGAGCGAAAAGTAGG-3’V100I????5’-GTATAAGAAGCTTATTACGGCGATCCAGGCC-3’

5’-GGCCTGGATCGCCGTAATAAGCTTCTTATAC-3’F114Y????5’-CTTCAAGGGCAAGTACGCCTTTTTGAAGACG-3’

5’-CGTCTTCAAAAAGGCGTACTTGCCCTTGAAG-3’A243L????5’-CATCCGAGCTCGCCTCGAGAAGCATCTTC-3’

5’-GAAGATGCTTCTCGAGGCGAGCTCGGATG-3’S265P????????5’-CTAATGGATGTGTCCGTTTGATACGGTAG-3’

5’-CTACCGTATCAAACGGACACATGTCCATTAG-3’N294D????5’-GTGGAAGAAGTACGACTACCTTCAGTC-3’

5’-GACTGAAGGTAGTCGTACTTCTTCCAC-3’

(Mlu?I)R329H????5’-GCCCGGTTGACGCATTCGCCAGTGCAGG-3’

5’-CCTGCACTGGCGAATGCGTCAACCGGGC-3’

Nco?IS364T????5’-CACACGACAACACCATGGTTTCCATCTTC-3’

5’-GAAGATGGAAA CCATGGTGTTGTCGTGTG-3’

(Bss?HI)G404A????5’-GTGGTGCCTTTCGCCGCGCGAGCCTACTTC-3’

The avtive spot amino-acid residue of 5 '-GAAGTAGGCTCGCGCGGCGAAAGGCACCAC-3 ' embodiment, 7 aspergillus niger NRRL3135 phytases is to the introducing of total CONSENSUS PHYTASE-1

We use the crystalline structure of aspergillus niger NRRL 3135 phytases to determine all avtive spot amino-acid residues (embodiment and EP 897 010 see reference).We use the series arrangement of Figure 13, following avtive spot residue and other adjacent residues inequality of total phytase are replaced with the residue of aspergillus niger phytase:

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

As described in embodiment 3, total phytase-7 answer of new protein sequence is translated as dna sequence dna (Figure 22).Use following oligonucleotide mixture as the corresponding gene of generation (fcp7) as described in the embodiment 3;

Mixture 1.7:CP-1, CP-2, CP-3, CP-4.7, CP-5.7, CP-6, CP-7, CP-8.7, CP-9, CP-10.7

Mixture 2.7:CP-9, CP-10.7, CP-11.7, CP-12.7, CP-13.7, CP-14.7, CP-15.7, CP-16, CP-17.7, CP-18.7, CP-19.7, CP-20, CP-21, CP-22.

The dna sequence dna of oligonucleotide is shown among Figure 15.New synthetic oligonucleotide numeral 7 other marks.Behind the identical PCR primer assembling oligonucleotide of use as described in the embodiment 3, as described in the embodiment 8-10 with gene clone to expression vector.

The pH distribution plan of measuring behind expression and the purifying in multiple-shaped nuohan inferior yeast moves in the acid range of pH spectrum, shows best (seeing Figure 30) at pH4.5-5.0.This enzyme has the suitableeest spectrum of wide pH, can reach 60% of its maximum activity at least from pH2.5 to pH6.0.Up to pH5.0, its distribution plan is similar to the distribution plan of aspergillus niger NRRL 3135 phytases.Yet under pH5.0, it lacks typical low spot at the pH4.0 place of aspergillus niger phytase distribution plan.The embodiment 8 total expression of phytase gene in multiple-shaped nuohan inferior yeast

Structure is used to transform multiple-shaped nuohan inferior yeast RB11 (people such as Gemssen, 1994) phytase expression vector, method is the EcoRI fragment of pBsk-fcp or its variant to be inserted multiple clone site, hydrogenlyase (FMD) promoter element of this ura3 selected marker based on yeast saccharomyces cerevisiae, multiple-shaped nuohan inferior yeast and methanol oxidase (MO) the terminator element of multiple-shaped nuohan inferior yeast expression vector pFPMT121.5 ' of fcp gene terminal merges with the FMD promotor, 3 ' terminally merges (people such as Gellissen, 1996 with the MOX terminator; EP 0,299 108 B).The expression vector that produces is called as pFPMTfcp, pFPMTfcp10 and pFPMTfcp7.

The plasmid that makes up is bred in intestinal bacteria.Standard method plasmid DNA purification with prior art.Method as use experience attitude cell preparation as described in the people such as Gelissen (1996) and yeast conversion is converted into Orotidine-5 '-with expression plasmid '-the multiple-shaped nuohan inferior yeast strain RP11 of phosphate decarboxylase (ura3) defective.With each transformation mixture coated plate in the YNB that contains 2% glucose and 1.8% agar (0.14%w/v Difco YNB and 0.5% ammonium sulfate), 37 ℃ of incubations.The single transformant bacterium colony of picking after 4 to 5 days was grown 2 days for 37 ℃ in aforesaid liquid nutrient medium.The fresh phial of the YNB substratum that contains 2% glucose is housed with this culture inoculation of equal portions subsequently.In selective medium, go down to posterity in addition after seven times, expression vector is integrated in the yeast genes group with polymeric form.Subsequently, obtain the stable transformant of mitotic division by two in the non-selective liquid nutrient medium of 3ml (YPD, 2% glucose, 10g yeast extract and 20g peptone) other culturing steps.In order to obtain the recombinant strain of hereditary homogeneous, will finally stablize an equal portions coated plate of culture in selecting on the flat board.Separate single bacterium colony, be used for replacing glucose, making the YNB of 2% glycerine that the fmd promotor derepresses carry out the analysis of phytinic acid expression of enzymes containing.The purifying of total phytase carries out as described in embodiment 9.Embodiment 9 its have the phytase gene in Expression in Saccharomyces Cerevisiae and phytase the purifying from culture supernatant

From corresponding Bluescript plasmid (pBsk-fcp, pBsk-fcp10 and pBsk-fcp7), separate total phytase gene, and be connected to yeast saccharomyces cerevisiae expression vector pYES2 (Invitrogen, San Diego, CA, the EcoRI site of the expression cassette U.S.), or, be subcloned between GAPFL (the Glycerose 3-phosphate dehydrogenase) promotor and pho5 terminator of brachymemma as described in the people such as Janes (1990).Check the correct direction of gene with PCR.Carry out the conversion of yeast saccharomyces cerevisiae strain such as INVSc1 (Invitrogen, San Diego, CA, the U.S.) according to people's such as Hinnen (1978) method.Picking contains single bacterium colony of the phytase gene under the control of GAPFL promotor, selects fiercely to shake (250 rev/mins) in 30 ℃ in the substratum (SD-uridylic, people such as Sherman, 1986) at 5ml and cultivates one day down.Then pre-culture is added in the 500ml YPD substratum people such as (, 1986) Sherman and growth under the same conditions.Carry out inducing of gal1 promotor according to working instructions.Behind the incubation 4 days, centrifugal (7000 rev/mins, GS3 rotary head, 15 minutes, 5 ℃) cell culture fluid is to remove cell, and process is at Amicon8400 order (PM30 film) and ultrafree-15 centrifugal filter device (Biomax-30K, Millipore, Bedford, MA, the U.S.) in the ultrafiltration and concentration supernatant liquor.With the 10mM sodium acetate, pH5.0 goes up concentrated solution (10ml) desalination at the ultra-fine post of 40ml Sephadex G25 (Pharmacia Biotech, Feiburg, Germany) as elutriant.Sample and 2M (NH with desalination ₄) ₂SO ₄Together, directly application of sample is used the 10mM sodium acetate, (the NH from 2M to 0M among the pH5.0 in 1ml butyl Sepharose 4 fast mobile hydrophobic interaction chromatography posts (PharmaciaBiotech, Feiburg, Germany) ₄) ₂SO ₄The linear gradient elution chromatography column.The phytase wash-out is in penetrating liquid (break-throngh), and concentrated also application of sample is in 120ml Sephacryl S-300 gel permeation chromatography post (Pharmacia Biotech, Feiburg, Germany).Total phytase and total phytase-7 wash-out are uniform symmetrical peak, and SDS-PAGE shows that its purity is approximately 95%.The embodiment 10 total expression of phytase gene in aspergillus niger

Bluescript plasmid pBsk-fcp, pBsk-fcp10 and pBsk-fcp7 be as template, is used for the introducing in the downstream, EcoRV site of the upstream, initiator codon BspHI site of gene and terminator codon.Use Expand ^TMHigh fidelity PCR test kit (Boehringer Mannheim, Mannheim, Germany) and following primer:

Primer Asp-1:

Bsp?HI

5’-TATATCATGAGCGTGTTCGTCGTGCTACTGTTC-3’

The primer Asp-2 that is used for fcp and fcp7 clone:

Eco?RV

3’-ACCCGACTTACAAAGCGAATTCTATAGATATAT-5’

The primer Asp-3 that is used for the fcp10 clone:

EcoRV

3’-ACCCTTCTTACAAAGCGAATTCTATAGATATAT-5’

As described in supplier, carry out this reaction.The fcp gene of pcr amplification has a new BspHI site of being introduced by primer Asp-1 at the initiator codon place, it causes second amino-acid residue glycine to be replaced by Serine.Subsequently, with BspHI and EcoRV dna digestion fragment, be connected to the downstream, NcoI site of aspergillus niger glucoamylase promotor (glaA) and the upstream, EcoRV site of Aspergillus nidulans tryptophane C terminator (trpC) people such as (, 1985) Mullaney.After this clone's step, with gene sequencing to detect the mistake that possible PCR introduces.The expression plasmid that corresponds essentially to the pGLAC carrier that produces described in the embodiment 9 of EP 684 313 comprises the Orotidine-5 '-as the coarse chain spore enzyme (Neurospora crassa) of selected marker '-phosphate decarboxylase gene (pyr4).The conversion of aspergillus niger and total phytase expression of gene are carried out as described in EP684 313.As the total phytase of purifying as described in the embodiment 9.The mensuration of embodiment 11 phytase activities and optimum temperuture

Substantially as mensuration phytase activity as described in the people such as Mitchell (1997).At the 200mM sodium acetate that contains 0.5% phytinic acid (≈ 5mM), pH5.0 measures and measures its activity in the mixture.37 ℃ of incubations are after 15 minutes, by the adding termination reaction of isopyknic 15% trichoroacetic acid(TCA).The quantitative phosphoric acid that discharges, method are that 100 μ l are measured mixture and 900 μ l H ₂O and 1ml 0.6MH ₂SO ₄, 2% xitix and 0.5% ammonium molybdate mix.With the standardized solution of potassiumphosphate as reference.Per minute discharged the enzyme amount of 1 μ mol phosphoric acid when one unit enzymic activity was defined as 37 ℃.Use is measured protein concn according to the enzyme optical extinction coefficient at the 280nm place that people such as Pace (1995) calculate: total phytase, 1.101; Total phytase 7,1.068; Total phytase 10,1.039.

For the optimal pH curve, the enzyme of purifying at the 10mM sodium acetate, is diluted among the pH5.0.The protein equal portions of dilution are mixed in a series of different damping fluids with the beginning incubation with isopyknic 1% phytinic acid (≈ 10mM), and damping fluid is: 0.4M glycine/HCl, pH2.5; 0.4M acetate/NaOH, pH3.0,3.5,4.0,4.5,5.0,5.5; 0.4M imidazoles/HCl, pH6.0,6.5; 0.4M Tris/HCl, pH7.0,7.5,8.0,8.5,9.0.Control experiment shows that mixing step only influences pH slightly.Carried out incubation 15 minutes at 37C as previously mentioned.

To measure the phosphate compounds separately that phytinic acid in the mixture replaces with 5mM concentration, carry out the mensuration of phytase substrate specificity.Carry out determination of activity as previously mentioned.

With the preincubation 5 minutes under specific temperature of enzyme (100 μ l) and substrate solution (100 μ l), carry out the mensuration of optimum temperuture.Begin reaction by in enzyme, adding substrate solution.Behind 15 minutes the incubation, use the trichoroacetic acid(TCA) termination reaction, measure the amount of the phosphoric acid that discharges.

The optimal pH of original total phytase is (70U/mg) about pH6.0-6.5.By the introducing of Q50T sudden change, optimal pH changes pH6.0 (130U/mg) into.After K91A introduced, optimal pH oxytropism pH scope moved a pH unit, showed higher specific activity between pH2.5 and pH6.0.The stable mutant and the optimal pH of total CONSENSUS PHYTASE-10 (Figure 26 and Figure 27) have also been shown.

Making up total phytase-7 is used for the catalysis characteristics of aspergillus niger phytase NRRL 3135 is transferred to total phytase, it has the pH distribution plan in the acid range that is transferred to the pH spectrum, shows between pH4.5 and 5.0 best (seeing Figure 31).This enzyme has wide pH spectrum, can reach 60% of its enhanced maximum activity at least from pH2.5 to pH6.0.Compare total CONSENSUS PHYTASE-1, its substrate spectrum also more is similar to aspergillus niger NRRL 3135 phytases.

The optimum temperuture (71 ℃) of total CONSENSUS PHYTASE-1 is than high 16-26 ℃ of the optimum temperuture (45-55 ℃, table 7) of the wild-type phytase that is used to calculate consensus sequence.Improved total CONSENSUS PHYTASE-10 shows that optimum temperuture further is increased to 80 ℃ (Figure 33).Use the supernatant liquor of the yeast saccharomyces cerevisiae strain of excessive generation to find the optimum temperuture (78 ℃) in identical scope of total CONSENSUS PHYTASE-1-Re [8].Be measured to and reach 82 ℃ the highest optimum temperuture for total CONSENSUS PHYTASE-10-Re-Q50T-K91A.

Table 7: the optimum temperuture and the Tm value of the phytase of total phytase and Aspergillus fumigatus, aspergillus niger, E.nidulans and M.Thermophila.As carrying out the mensuration of optimum temperuture as described in the embodiment 11, as described in the embodiment 12 with determine with dsc method Tm value.

Phytase	Optimum temperuture [℃]	The Tm value [℃]
			Total CONSENSUS PHYTASE-10-the Re of the total CONSENSUS PHYTASE-10-Re-Q50T-K91A-total CONSENSUS PHYTASE-1-Re [8] of the total CONSENSUS PHYTASE-10 of Q50T-total CONSENSUS PHYTASE-1-Re [8] of Q50T-total CONSENSUS PHYTASE-1 aspergillus niger NRRL3135 aspergillus fumigatus 13073 aspergillus fumigatus 13073 α of Q50T-K91A-sudden change aspergillus fumigatus 13073 α-sudden changes (best) Aspergillus terreus 9A-1 Aspergillus terreus cbs.116.46 E.nidulans M.Thermophila T.Thermophilus	?82 ?82 ?80 ?78 ?78 ?71 ?55 ?55 ?60 ?63 ?49 ?45 ?45 ?55 ?45	?89.3 ?88.6 ?85.4 ?84.7 ?85.7 ?78.1 ?63.3 ?62.5 ?67.0 ?- ?57.5 ?58.5 ?55.7 ?n.d. ?n.d.

Embodiment 12 usefulness dsies (DSC) are to Measurement of melting point

In order to measure the folding temperature of separating of phytase, the use dsc of having delivered by people such as Brugger (1997).Phytinic acid enzyme solution with the 50-60mg/ml homogeneous detects.The constant heat rate of using 10 ℃/minute is up to 90-95 ℃.

The fusing point of being measured has reflected the result's (table 7) who obtains for optimum temperuture.The most stable total phytase of design is total CONSENSUS PHYTASE-10-Re-Q50T-K91A, and it is presented at the melting temperature (Tm) of 89.3 ℃ selected condition.This fusing point than the wild-type phytase that uses is high 26 to 33.6 ℃.Embodiment 13 basidiomycetes phytase activity sites are to the transfer of total CONSENSUS PHYTASE-10-Re-O50T-K91A

(embodiment 5) as previously mentioned will introduce total phytase 10 from the sudden change of basidiomycetes phytase activity site deutero-.Be prepared as follows 5 kinds of constructs a) to e):

This construct is called total phytase 12, it comprises the avtive spot residue of the selected quantity of basidio consensus sequence, its aminoacid sequence (consphy12) is shown in (preceding 26 amino acid form signal peptide, and underscore marks the position of correction) among Figure 33;

One group of sudden change (group II) is transferred to has 10 sequences, that is: S80Q, Y86F, S90G, K91A, S92A, K93T, A94R, Y95I;

Similarly, shift other one group of sudden change (group III), that is: T129V, E133A, Q143N, M136S, V137S, N138Q, S139A;

Similarly, shift one group of sudden change (group IV), that is: A168D, E171T, K172N, F173W again;

And last, shift again another group sudden change (group V), that is: Q297G, S298D, G300D, Y305T.

As these constructs of expression as described in the embodiment 8 to 10.

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Claims (22)

1. stable drying or liquid enzyme formulation, it comprises phytase and one or more stablizers, and this stablizer is selected from:

A) C ₅Sugar, preferably Xylitol or ribitol,

B) molecular weight 600 to 4000Da, preferably 1000 arrives the polyoxyethylene glycol of 3350Da.

C) propanedioic acid disodium salt, pentanedioic acid disodium salt and disodium succinate salt,

D) carboxymethyl cellulose and

E) alginate, preferably sodiun alginate.

2. stable drying or liquid enzyme formulation, it comprises following crosslinked phytase:

A) use glutaraldehyde, perhaps pass through

B) use sodium periodate oxidation, and react with adipic dihydrazide.

3. according to the zymin of claim 1 or 2, it is characterized in that this phytase is a phytase a kind of originated from fungus or total.

4. according to the zymin of claim 3, it is characterized in that the phytase of this originated from fungus is selected from Aspergillus fumigatus, Aspergillus nidulans, terreus and aspergillus niger phytase.

5. according to any zymin in the claim 1 to 4, it is characterized in that said preparation is a liquid.

6. according to the zymin of claim 5, it is characterized in that this stablizer is a polyoxyethylene glycol, polyoxyethylene glycol is present in the whole preparation with the concentration of 10-50% (w/w).

7. according to the zymin of claim 5 or 6, it is characterized in that this stablizer is Xylitol and/or ribitol, they are present in the whole preparation with the concentration of 20-60% (w/w).

8. according to any zymin in the claim 5 to 7, it is characterized in that this stablizer is pentanedioic acid disodium salt, disodium succinate salt and propanedioic acid disodium salt, the concentration of this salt in whole preparation is 10-30% (w/w).

9. according to any zymin in the claim 5 to 8, it is characterized in that this stablizer is a carboxymethyl cellulose, the concentration of polymer in whole preparation is 1-10% (w/w).

10. according to any zymin in the claim 5 to 9, it is characterized in that this stablizer is a sodiun alginate, the concentration of polymer in whole preparation is 1-10% (w/w).

11., it is characterized in that said preparation is exsiccant/solid according to any zymin among the claim 1-4.

12. according to the zymin of claim 11, it is characterized in that this stablizer is a polyoxyethylene glycol, polyoxyethylene glycol is present in the whole preparation with the concentration of 1-20% (w/w).

13. according to the zymin of claim 11 or 12, it is characterized in that this stablizer is Xylitol and/or ribitol, they are present in the whole preparation with the concentration of 1-20% (w/w).

14. according to any zymin in claim 11 or 13, it is characterized in that this stablizer is pentanedioic acid disodium salt, disodium succinate salt or propanedioic acid disodium salt, the concentration of this salt in whole preparation is 1-20% (w/w).

15. according to any zymin in claim 11 or 14, it is characterized in that this stablizer is a carboxymethyl cellulose, the concentration of this polymer in whole preparation is 1-10% (w/w).

16. according to any zymin in claim 11 or 15, it is characterized in that this stablizer is a sodiun alginate, the concentration of this polymer in whole preparation is 1-10% (w/w).

17., it is characterized in that this phytase monomer is crosslinked by the adding of glutaraldehyde according to any zymin in claim 2-5 or 11.

18. according to any zymin in claim 2-5 or 11, it is characterized in that this phytase monomer is crosslinked, method is to make the saccharide residue oxidation with sodium periodate, adds adipic dihydrazide subsequently.

19. a method for preparing the feed composition that is used for monogastric animal is characterized in that this feed is used according to stable drying or the liquid enzyme formulation of any among the claim 1-18 to handle.

20. a feed composition that is used for monogastric animal, it is a kind of according to any stable drying or liquid enzyme formulation among the claim 1-18 to it is characterized in that this feed comprises.

21. one kind provides phosphorus dietetically desirable method to monogastric animal, it is characterized in that using according to the feed of claim 20 and raises this animal, and do not add other phosphorus in feed.

22. a method for preparing drying or liquid phytase formulation is characterized in that using a kind of stable phytase according to claim 1-18.

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