CN101260390B - High specific activity phytase gene and application thereof - Google Patents
High specific activity phytase gene and application thereof Download PDFInfo
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- CN101260390B CN101260390B CN2007100378246A CN200710037824A CN101260390B CN 101260390 B CN101260390 B CN 101260390B CN 2007100378246 A CN2007100378246 A CN 2007100378246A CN 200710037824 A CN200710037824 A CN 200710037824A CN 101260390 B CN101260390 B CN 101260390B
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Abstract
The invention discloses a novel AppA-M protein and application of the same. The AppA-M protein is a phytase with high specific activity and can be used for decomposition of phytic acids or phytates. The invention also discloses a gene for encoding the AppA-M protein, and a carrier and a host cell which comprise the gene. The AppA-M protein has wide application prospect in fields such as feedstuff additives and so on.
Description
Technical field
The invention belongs to biological technical field, more specifically, the present invention relates to Sumizyme PHY and application thereof that a kind of height ratio is lived.
Background technology
Phosphorus is the essential mineral element of animal body, though the phosphorus major part that is present in the animal feedstuff can be absorbed by animal body, because it costs an arm and a leg, has limited the use in feed.The phosphorus major part that is present in the plant feed exists with the form of phytic acid (being phytinic acid) or phytate; And lack the Sumizyme PHY (Phytase, the enzyme of catalysis phytic acid and phytate hydrolysis) that decomposes phytic acid in the monogastric animal body, cause the utilization ratio of phosphorus in the feed only to have 1/3 or lower; In order to replenish the deficiency of available phosphorus; Must in feed, add inorganic phosphate, certainly will cause the waste in phosphorus source like this, cause the excessive drainage of phosphorus.Through adding Sumizyme PHY, can improve the utilization ratio of phytate phosphorus in the feed in the monogastric animal feed, reduce the pollution of the discharge of phosphorus environment.Sumizyme PHY belongs to phosphoric acid monoester lytic enzyme, be a kind of can degrading plant the phosphoesterase of phytic acid and its esters in the forage.Sumizyme PHY can be decomposed into inositol and phosphoric acid with phytic acid, and most of Sumizyme PHY (85%) plays a role at stomach, and small portion works at the small intestine front end, and the small intestine rear end does not have the Sumizyme PHY activity.Sumizyme PHY only acts on phytic acid, has only when having the phytic acid of capacity in the feed, and adding Sumizyme PHY just has actual value.Along with after biotechnology particularly adopts the DNA recombinant technology, make Sumizyme PHY extensive application aborning become possibility.
The optimum pH of vegetalitas Sumizyme PHY is between 4.8-6.0, and less than 3.0 o'clock, activity significantly descended in the pH value, even inactivation.The pH value scope that microbial phytase tolerated is wideer than most plants property Sumizyme PHY, and the general pH value is between 2.5-6.0.The optimum temperuture of most Sumizyme PHY performance enzymic activitys is 45-55 ℃.
And in the various Sumizyme PHYs of finding at present, the enzymic activity of most of Sumizyme PHYs is desirable not enough, and activity is the highest about 8000U/ml (fermentation titer).Therefore, this area needs further to seek the new wide and active high Sumizyme PHY of applicable pH value, to improve the absorption of animal to phosphorus, saves cost effectively.
Summary of the invention
The Sumizyme PHY that the object of the present invention is to provide novel height ratio to live, this Sumizyme PHY has very high enzymic activity, and has good PH stability.
In first aspect of the present invention, a kind of isolating albumen is provided, this albumen is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence; Or
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have with the polypeptide function of the same race (promptly having the function of decomposing phytic acid or phytate) of SEQ ID NO:2 aminoacid sequence by (a) polypeptides derived.
In second aspect of the present invention, a kind of isolating polynucleotide are provided, these polynucleotide are selected from down group:
(i) the described proteic polynucleotide of coding; Or
(ii) with (i) in polynucleotide complementary polynucleotide.
In another preference of the present invention, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.
In another preference of the present invention, these polynucleotide have the nucleotide sequence shown in the SEQ ID NO:1.
In the third aspect of the invention, a kind of carrier is provided, it contains described polynucleotide.
In fourth aspect of the present invention, a kind of genetically engineered host cell is provided,
It contains described carrier; Or
Be integrated with described polynucleotide in its genome.
Aspect the of the present invention the 5th, described proteic purposes is provided, be used to decompose phytic acid or phytate.
Aspect the of the present invention the 6th, provide a kind of preparation described proteic method, said method comprises: cultivate described host cell, collect and obtain described albumen.
Aspect the of the present invention the 7th, a kind of compsn is provided, contain the described albumen of significant quantity in the said compsn, and bromatology or the last acceptable carrier of feed.
In another preference of the present invention, contain Mg2+, Ca2+, EDTA or stomach en-in the described compsn.
In another preference of the present invention, do not contain Zn2+, Cr3+, Cu2+, Fe2+ or Fe3+ in the described compsn.
In eight aspect of the present invention, the method that provides a kind of preparation to contain described proteic transgenic plant, described method comprises step: described polynucleotide are imported in the vegetable cell, cultivate described vegetable cell, the regeneration plant.
In another preference of the present invention, said method comprises step:
(s1) Agrobacterium of carrying expression vector is provided, described expression vector contains described polynucleotide;
(s2) vegetable cell or tissue or organ are contacted with Agrobacterium in the step (s1), thereby make described polynucleotide change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(s3) select vegetable cell or tissue or the organ that changes described polynucleotide over to; And
(s4) vegetable cell in the step (s3) or tissue or neomorph are become plant.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Embodiment
The inventor finds the Sumizyme PHY that a kind of new height ratio is lived, inventor general's called after AppA-M through extensive and deep research.Test confirms that this Sumizyme PHY has very high enzymic activity, and (enzyme work reaches 3.19 * 10 under normal temperature (37 ℃)
3U/mg); And this Sumizyme PHY all can be kept enzyme work more than 80% between pH2～10, has good pH stability.It is thus clear that this Sumizyme PHY is with a wide range of applications.Accomplished the present invention on this basis.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification like polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating AppA-M albumen or polypeptide " is meant that described AppA-M albumen does not contain natural relative other albumen, lipid, carbohydrate or other material basically.Those skilled in the art can use the purified technology of protein purifying AppA-M albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell), to produce.The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises the proteic fragment of AppA-M, verivate and analogue.As used herein, term " fragment ", " verivate " are meant with " analogue " and keep identical biological function of natural A ppA-M albumen of the present invention or active polypeptide basically.Polypeptide fragment of the present invention, verivate or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue); And so substituted amino-acid residue can be also can not encoded by genetic code; Or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical; Or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period; Polyoxyethylene glycol for example) merges formed polypeptide; Or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (like leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying or fusion rotein).These fragments of definition, verivate and analogue according to this paper belong to the known scope of those skilled in the art.
In the present invention, term " AppA-M albumen " refers to have the SEQ ID NO:2 polypeptide of sequence of AppA-M protein-active.This term also comprises having and variant form AppA-M albumen identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50; Preferably 1-30, more preferably 1-20,1-10 best; Also better for 1-8,1-5) amino acid whose disappearance, insertion and/or replacement; And at C-terminal and/or N-terminal interpolation one or several (being generally in 20, preferably is in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of AppA-M and reactive derivative.
The variant form of polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with coded albumen of the DNA of AppA-M protein D NA hybridization and polypeptide or the albumen that utilizes the proteic antiserum(antisera) of anti-AppA-M to obtain.The present invention also provides other polypeptide, as comprises AppA-M albumen or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the proteic soluble fragments of AppA-M.Usually; This fragment have the AppA-M protein sequence at least about 20 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids; More preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of AppA-M albumen or polypeptide.These analogues and the proteic difference of natural A ppA-M can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps have both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain through various technology, as through radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(like D-amino acid), and has non-natural analogue that exist or synthetic amino acid (like β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to above-mentioned representational polypeptide of giving an example.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modify and also comprise glycosylation.Modified forms also comprises have the phosphorylated amino acid residue sequence of (like Tyrosine O-phosphate, Serine O-phosphate, phosphothreonine).Thereby also comprise and modified the polypeptide that has improved its anti-proteolyze performance or optimized solubility property.
In the present invention; " AppA-M albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8; More preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.For example, these conservative propertys variation polypeptide can carry out the amino acid replacement according to table 1 and produce.
Table 1
| Amino-acid residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
The present invention also provides the polynucleotide sequence of code book invention AppA-M albumen or its conservative property variation polypeptide.
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the verivate of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it possibly be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide according to the invention.In the present invention, " stringent condition " is meant: (1) than hybridization under LIS and the comparatively high temps and wash-out, like 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, like 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (like PCR) of nucleic acid to confirm and/or the proteic polynucleotide of separation coding AppA-M.
AppA-M pyrenoids thuja acid full length sequence of the present invention or its fragment can use the method for pcr amplification method, recombination method or synthetic to obtain usually.For the pcr amplification method; Can be disclosed according to the present invention about nucleotide sequence; Especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually need carries out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can come to obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell again over to, from the host cell after the propagation, separates obtaining relevant sequence then through ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, through first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) through chemosynthesis.Can this dna sequence dna be introduced in various existing dna moleculars as known in the art (or like carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention through chemosynthesis.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or AppA-M albumen coded sequence, and produce the method for polypeptide according to the invention through recombinant technology.
Recombinant DNA technology (Science, 1984 through routine; 224:1431), the AppA-M albumen of reorganization is expressed or produced to polymerized nucleoside acid sequence of the present invention capable of using.In general following steps are arranged:
(1). with the proteic polynucleotide of coding AppA-M of the present invention (or varient), or with recombinant expression vector conversion that contains these polynucleotide or transduction proper host cell;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, AppA-M albumen polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell is viral or other carriers.In a word, as long as can in host, duplicate and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains AppA-M encoding histone dna sequence dna and suitable transcribing/the translate expression vector of wave.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition; Expression vector preferably comprises one or more selected markers; To be provided for selecting the phenotypic character of transformed host cells; Cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness like eukaryotic cell, or be used for colibacillary kantlex or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, like bacterial cell; Or eukaryotic cell such as low, like yeast cell; Or higher eucaryotic cells, like vegetable cell.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; Fungal cell such as yeast; Vegetable cell etc.
When polynucleotide of the present invention are expressed in some hosts, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.Transform plant and also can use methods such as Agrobacterium-mediated Transformation or particle gun conversion, for example leaf dish method, rataria conversion method etc.Can use ordinary method regeneration plant for plant transformed cell, tissue or organ, thereby obtain genetically modified plant.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (like temperature transition or chemically induced), cell is cultivated for some time again.
The extracellular can expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating through various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, ultraly handle, ultra centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) is technological with other various LCs and the combination of these methods.
As one embodiment of the present invention, the proteic encoding sox of AppA-M of the present invention capable of using prepares transgenic plant, and the transgenic plant of acquisition are expressed AppA-M albumen.With the forage component of described transgenic plant, can make to have stable source in the feed, thereby not need extra interpolation Sumizyme PHY as animal.
In addition, also can import the proteic encoding sox of AppA-M in animal body, thereby animal self can be expressed AppA-M albumen, make AppA-M albumen become a kind of endogenous enzyme.This has just saved the interpolation work of a lot of external sources.
The present invention also provides a kind of compsn, and it contains on AppA-M albumen of the present invention and the bromatology of safe and effective amount or feed is learned and gone up acceptable carrier or vehicle.This type carrier comprises (but being not limited to): damping fluid, glucose, water, glycerine, ethanol, acetate, wheat bran, corn cob or their combination.Composite preparation should be complementary with administering mode.Compsn of the present invention can be made into the form of granule, and for example the aqueous solution with corn cob or wheat bran and other assistant agents prepares through ordinary method.
When feeding animals, the proteic compsn of AppA-M that will contain safe and effective amount usually gives animal, in preparation during granulated feed, regulates in the feed the proteic enzyme work of AppA-M usually at 0.005-50U/g; 0.05-5U/g preferably; Be more preferably 0.1-1U/g.Certainly, specifically give dosage also the considered feed other nutritive ingredient prescription, animal from factors such as body situations, these all are well-known to those skilled in the art.
As an instance of the present invention; The invention provides a kind of isolating polynucleotide; Described polynucleotide are through the sample the cud of gathering a large amount of soil samplings or animal from the woods zone of accumulating dead leaf all the year round; Therefrom choose the flora that phytic acid is had very strong metabolic capacity, and carried out codon optimized the acquisition according to the zymic codon-bias.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 1233 bases, and the coding total length is 410 amino acid whose AppA-M albumen (SEQ ID NO:2).This albumen can add animal-feed to or other need replenish Sumizyme PHY with in the material that decomposes phytic acid or phytate, is used to decompose phytic acid or phytate.
Major advantage of the present invention is:
Separate obtaining the Sumizyme PHY that a kind of new height ratio is lived first, this Sumizyme PHY has very high activity, and (specific activity is 3.19 * 10
3U/mg), in the fermentor tank level, expression amount reaches 2.5mg/mL behind methanol induction 120h, and phytase activity (fermentation titer) reaches 1.2 * 10
4More than the U/mL; This Sumizyme PHY all can be kept enzyme work more than 80% between pH2～10, have good pH stability; This Sumizyme PHY also has the ability of extraordinary antipepsin hydrolysis ability and part anti-trypsin hydrolysis; Therefore visible, Sumizyme PHY of the present invention is with a wide range of applications.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or according to the method for announcing in the following document: Carl W.Dieffenbach and Gabriela S.Devksler eds.PCRPrimer:A Laboratory Manual.Cold Spring Harbor Laboratory Press, 1995.Or the condition of advising according to manufacturer.
Embodiment 1: the clone of high specific activity phytase gene
1. sample collecting
Sample from the cud of regional a large amount of soil samplings of collection of the woods that accumulate dead leaf all the year round or animal; These are local owing to have a large amount of vegetal phytic acid; Therefore can obtain phytic acid is had the flora of very strong metabolic capacity through screening, thereby isolate phytase gene with height ratio characteristic alive.
2. adopt and exempt from the drowning flat total DNA of cultural method isolates from pedotheque
Take by weighing sample 2 grams, adding 0.6g micro glass beads (d 0.11mm), 4000 rev/mins vibrate 2 times.Add 300 μ l2%SDS+12% phenol Tris damping fluid (pH8.0) solution 1 hour on ice, and added equivalent phenol Tris damping fluid, pH8.0 (about 700ml), abundant mixing, through 4 ℃, 13, centrifugal 5 minutes of 000rpm.Upper solution adds the 3M NaAc pH5.2 of 0.1 times of volume, adds 0.6 times of volume Virahol mixing behind the mixing.The DNA deposition is dissolved in 200 μ l1 * TE (thick DNA).Claim that the 100mg cesium chloride places a new 1.5ml Epp. centrifuge tube, add the thick DNA of 100 μ l mixing gently, left standstill under the room temperature dark condition 1-3 hour.Room temperature, 13,000rpm, centrifugal 20 minutes.Add 400 μ l aseptic deionized waters and 300 μ, 1 Virahol in the supernatant, room temperature left standstill 30 minutes.Room temperature, 13,000rpm, centrifugal 20 minutes.Deposition is dissolved in 100 μ l1 * TE and 40 μ l8M Potassium ethanoates (KAc), and room temperature left standstill 15 minutes.4 ℃, 13, centrifugal 15 minutes of 000rpm.Supernatant adds 0.6 times of volume Virahol mixing.Room temperature left standstill 30 minutes.Room temperature, 15, centrifugal 20 minutes of 000rpm.The DNA deposition is dissolved in 100 μ l1 * TE.Adopt Wizard spin column clean-up separating kit purify DNA sample.Purify DNA is dissolved in the 10mMTris-EDTA that TV is 100 μ l (pH8.0) damping fluid.
3. the structure of the total DNA cosmid library of group's level
Adopt clay SuperCos1 Cosmid Vector Kit (available from Stratagene company) to make up the cosmid library of the total DNA of group's level.The a large amount of enzymes of Sau3A I enzyme amount of employing 0.006u/ μ g DNA are cut the total DNA of group's level of purifying, and conventional freeze-thaw method reclaims the partially digested fragment about 40kb, is connected with carrier S uperCos1/BamH1 fragment.The test kit specification sheets that provides by Stratagene company makes up cosmid library.With after being connected product and packing of the total dna fragmentation of group's level and carrier, change in the e. coli jm109 (ATCC) with phage packaging albumen.The e. coli jm109 of transfection is coated on the tetracyclin resistance LB solid medium; The bacterium colony that grows put respectively in tetracyclin resistance and tsiklomitsin add on the LB solid medium of kalamycin resistance; The transformant of on two kinds of resistance substratum, growing is that cosmid vector is from connecting conversion; The subtracting background colony count, rule of thumb formula calculating titre (pfu/ml) is 4 * 10
6(greater than 10
6), show that library construction meets the requirements.
4. screen phytic acid ca hydrolytic activity transformant
The transfection bacterium is pressed every plate 10
3Individual bacterium colony is coated on the 0.5% phytic acid ca solid medium; The bacterium colony of growing two days later; The bacterium colony point that will have transparent hydrolysis circle with aseptic toothpick is on the solid medium that contains phytic acid ca; 50 of average every plates are picked out the maximum several bacterium colonies of hydrolysis circle after the cultivation, ordinary method is extracted these clones' plasmid.
5. the analysis of the dna fragmentation of high-specific-activity phytase and active checking
Adopting EcoR I restriction endonuclease to carry out enzyme the plasmid of aforementioned extraction cuts; Be cloned into afterwards in the MCS of pUC18 carrier (Huamei Bio-Engrg Co.), order-checking is through the means of information biology; Analyze the protein sequence of these genes, find homologous gene with it.Experimental result shows, in 3 clone's that obtained, is same gene, and this gene and colibacillary Sumizyme PHY appa gene have higher similarity (homology is 96%).
Embodiment 2: the transformation of high specific activity phytase gene and synthetic
Gene order according to coding Escherichia coli Sumizyme PHY maturation protein; Preferences according to the pichia spp codon; Under the prerequisite that does not change its aminoacid sequence, carry out the sequence transformation, the appearance of in sequence, avoiding being rich in AT sequence (ATTTA, AATAA, AATTAA etc.) simultaneously.And the MCS of pressing used cloning vector designs, adds suitable restriction endonuclease sites; Full gene is divided into A (161bp), B (287bp), C (325bp), D (225bp), 5 parts of E (235bp), again each part is divided into the strand nucleotide fragments that length is no more than 60 Nucleotide and carries out synthetic.
After gene removes the signal coding sequence of N-end 66bp, total length 1233bp.Press pichia spp codon deflection; Under the prerequisite that does not change aminoacid sequence, transform; Change 262 bases altogether and related to 229 codons; G+C content becomes 49.1% by original 53.7%, and the G+C content of codon the 3rd bit base becomes 48.4% by original 54.8%, meets the content characteristics of pichia spp G+C basically.
Nucleotide sequence (SEQ ID NO:1):
cagagtgagc?ctgagttgaa?actggaatcc?gttgtcatcg?tctctagaca?tggtgttaga 60
gcaccaacca?aggccaccca?acttatgcaa?gatgtcaccc?cagacgcttg?gccaacctgg 120
ccagtcaagc?tgggttggtt?gacacctaga?ggtggtgagc?tcattgctta?cttgggtcac 180
taccaaagac?agcgtcttgt?tgccgacgga?ttggtggcca?agaagggttg?tccacaatct 240
ggtcaagtag?ctattattgc?tgacgtcgac?gaaagaaccc?gtaagacagg?tgaagccttc 300
gccgccggtc?ttgctcctga?ctgtgccatt?tctgttcaca?cccaagttga?cacttcttct 360
ccagatccat?tgttcaaccc?tttgaagact?ggtgtttgcc?aattggacaa?cgctaacgtt 420
actgacgcta?tcttgtccag?agctggagga?tccattgctg?acttcaccgg?tcacagacag 480
actgccttca?gagagttgga?aagagttctt?aacttcccac?aatccaactt?gtgccttaag 540
cgtgagaagc?aagacgaatc?ctgttccttg?actcaagcat?taccatctga?gttgaaggtc 600
tccgccgaca?acgtctcttt?gaccggtgct?gtcagcttgg?cttccatgtt?gactgaaatc 660
tttcttctgc?aacaagctca?aggtatgcct?gagccaggtt?ggggtagaat?caccgactct 720
caccaatgga?acaccttgtt?gtccttgcac?aacgctcaat?tctacttgct?gcagagaact 780
ccagaggttg?ctagatccag?agccacccca?ttgttggact?tgatcaagac?tgctttgact 840
cctcacccac?ctcaaaagca?agcctacggt?gttaccttgc?ccacttctgt?cttgttcatt 900
gccggtcacg?atactaactt?ggcaaatctc?ggcggtgctt?tggagttgaa?ctggactctt 960
cctggtcaac?ctgataacac?tccaccaggt?ggtgagctcg?ttttcgaaag?atggcgtaga 1020
ctatctgata?actctcaatg?gattcaggtt?tcgttggtct?tccaaacttt?gcagcagatg 1080
agagacaaga?ctccactgtc?tttgaacacg?cctccaggag?aagtcaaatt?gaccttggct 1140
ggatgtgaag?agagaaatgc?tcagggtatg?tgttccttgg?ctggtttcac?tcaaatcgtt 1200
aacgaagcta?gaatcccagc?ttgttccgtg?tag 1233
Protein sequence (SEQ ID NO:2):
QSEPELKLES?VVIVSRHGVR?APTKATQLMQ?DVTPDAWPTW?PVKLGWLTPR?GGELIAYLGH 60
YQRQRLVADG?LVAKKGCPQS?GQVAIIADVD?ERTRKTGEAF?AAGLAPDCAI?SVHTQVDTSS 120
PDPLFNPLKT?GVCQLDNANV?TDAILSRAGG?SIADFTGHRQ?TAFRELERVL?NFPQSNLCLK 180
REKQDESCSL?TQALPSELKV?SADNVSLTGA?VSLASMLTEI?FLLQQAQGMP?EPGWGRITDS 240
HQWNTLLSLH?NAQFYLLQRT?PEVARSRATP?LLDLIKTALT?PHPPQKQAYG?VTLPTSVLFI 300
AGHDTNLANL?GGALELNWTL?PGQPDNTPPG?GELVFERWRR?LSDNSQWIQV?SLVFQTLQQM 360
RDKTPLSLNT?PPGEVKLTLA?GCEERNAQGM?CSLAGFTQIV?NEARIPACSV 410
Add the EcoRI site at 5 of gene (SEQ ID NO:1) ' end, 3 ' end adds Not I and HindI II site, so that gene clone is on transfer vector pUC19 and expression vector pPIC9.According to gene synthetic ordinary method, the synthetic gene each several part is cloned respectively on carrier pUC19, further splicing obtains the full gene appA-m of complete transformation, recombinant plasmid called after pUC19-appA-m again.The appA-m gene confirms that behind sequencing this gene is consistent with the sequence of design.
Embodiment 3: the efficient induction of high specific activity phytase gene in yeast expressed and analyzed
1. the structure of synthetic segmental splicing and recombinant expression vector
Utilize EcoR I/Not I restriction enzyme site, appA-m is downcut from pUC19-phyA-m, be cloned on the yeast expression vector pPIC9 (Invitrogen), obtain recombinant vectors pPIC9-appA-m through EcoRI/Not I site.The reorganization operation of DNA is mainly according to people such as Sambrook, and molecular cloning: lab guide carries out.
2. the zymic electric shock transforms and screening
Recombinant vectors pPIC9-appA-m cut with the BglII enzyme make it linearizing, electric shock transforms pichia spp GS115 (Invitrogen).The picking positive transformant.Electricity conversion and screening method are referring to Invitrogen company operational manual.
3. the abduction delivering of recombination yeast
Recombination yeast is cultivated 48h in 30 ℃ of shaking tables in 5mL BMGY substratum; Centrifugal collection thalline; Add 1mLBMMY methanol induction substratum suspension thalline; Continuation is at 30 ℃ of following inducing culture 48h, and sampling detects the phytase activity in each bacterial strain supernatant, therefrom filters out the transformant of Expressing Recombinant Phytase.
Unit of enzyme activity is defined as: under certain condition, it is a unit of enzyme activity (U) that PM discharges the required enzyme amount of 1 μ mol inorganic phosphorus.
Activity determination method is referring to embodiment 5, and the enzymatic determination system pH is 5.0.
4. the expression of the horizontal Sumizyme PHY of fermentor tank
Recombination yeast ferments in 5L fermentor tank (BIOSTAT B5 type).The fermentation of recombination yeast is the high-cell density fed-batch fermentation, and fermenting process is divided into the strain culturing stage, carbon source is fed stage and abduction delivering stage, and concrete grammar is seen the Invitrogen operational manual.The SDS-PAGE that every 12h takes a sample and once measures the accumulation volume of the Sumizyme PHY of expressing and carry out expressing protein in inducing process.
Experimental result shows: select the high pichia spp recombinant bacterial strain P.pastorispPIC9-appA-m74 of shaking table horizontal expression amount to carry out the research of 5L ferment tank.Detect less than phytase activity in the thalli growth stage fermentation supernatant before inducing.Along with inducing of methyl alcohol, phytase activity power significantly increases in the supernatant, and zymoprotein constantly accumulates.Expression amount reaches 2.5mg/mL behind methanol induction 120h, and Sumizyme PHY (AppA-M) active (fermentation titer) reaches 1.2 * 10
4More than the U/mL, and the activity of the most of Sumizyme PHYs of prior art is the highest only at 8000U/ml.
5. the SDS-PAGE of expression product Sumizyme PHY analyzes and the de-glycosylation processing
Be taken at the nutrient solution behind the shaking table abduction delivering 48h, centrifuging and taking 8 μ L supernatants carry out SDS-PAGE and analyze.
Get 9 μ L supernatants and carry out the de-glycosylation processing, set up following reaction system: 9 μ L supernatants add 1 μ L10 * Glycoprotein sex change damping fluid, add 1.2 μ L10 * G5 damping fluid, 1 μ L Endo H behind 100 ℃ of reaction 10min, 37 ℃ of reaction 1h.
Experimental result shows: identify the proteic expression of Sumizyme PHY through SDS-PAGE; From the SDS-PAGE electrophoresis, can see that the Sumizyme PHY of Pichia anomala expression has three protein bands that vary in size; Its apparent molecular weight is respectively about 50kD, 52kD and 54kD, and is all big than the theoretical molecular of inferring from aminoacid sequence (about 45kD).Analyze the aminoacid sequence of this Sumizyme PHY, finding has three place's potential glycosylation sites, thus might be because of glycosylation causes molecular weight bigger than normal, and glycosylation in various degree causes the difference of expressing protein molecular weight.
6. the Southern trace of recombination yeast and Nortern trace
Choose different transformants and host strain GS115 and be inoculated in respectively in the BMGY substratum, 30 ℃ of shaking table 48h cultivate the back and extract genomic dna; 30 ℃ of shaking tables are cultivated 48h in the BMGY substratum, and behind the centrifugal collection thalline, 30 ℃ of inducing culture 48h among the BMMY extract the total RNA of yeast.With the phytase gene XbalI/PstI enzyme switchback section of taking up (+50～+ 785bp) carry out Southern trace and Nortern engram analysis respectively for probe.
Experimental result shows: phytase gene has obtained transcribing in the recombination yeast, and the mRNA amount of transcribing does not have significant difference in high expression level recon and low express recombinant.
Embodiment 4: the high-specific-activity phytase zymologic property detects
The Sumizyme PHY purification process of Pichia anomala expression is following: at first carry out desalting treatment, again through molecular sieve Superdex_75_HR_10/30 purifying.Substep is collected elution peak, and every pipe 1mL is as the sample of Sumizyme PHY zymologic property research.
Purified Sumizyme PHY carries out enzymatic reaction to measure its ph optimum and optimum temperuture under different pH and condition of different temperatures.Enzyme liquid is handled down 1h and under differing temps (60 ℃, 70 ℃, 80 ℃), handled 30min respectively in 37 ℃ in the damping fluid of different pH values, under 37 ℃, the condition of pH5.0, measure enzymic activity respectively with the pH stability of measuring enzyme and the thermostability of enzyme.
The unit of enzyme activity (U) that is shown than the every mg zymoprotein of being defined as of unit alive of enzyme.Protein content through in the Xylene Brilliant Cyanine G method working sample enzyme liquid records its phytase activity property simultaneously, obtains the specific activity of enzyme thus.
Use the sodium phytate of different concns to be substrate, under pH5.0,37 ℃ of conditions, react 10min mensuration enzymic activity, calculate the K of enzyme
mValue and V
Max.
In enzymatic reaction system, adding final concentration is 1mM different metallic ion and chemical reagent, under 37 ℃, pH5.0 condition, measures enzymic activity, studies its influence to enzymic activity.
With pH2.0Gly-HCl damping fluid preparation 0.1mg/mL stomach en-, pH7.0Tris-HCl damping fluid preparation 0.1mg/mL trypsinase.Get the Sumizyme PHY enzyme liquid behind the 0.5mL purifying, add 0.5mL stomach en-and trypsinase respectively, 37 ℃ of insulations, the different time sampling is measured enzymic activity under 37 ℃, pH5.0 condition.
Experimental result shows that the specific activity of this recombinant phytase AppA-M is 3.19 * 10
3U/mg, Km=0.55mmol/L, Vmax=3.934 * 10
3U/ (mg.min).Ph optimum is 4.5, when pH greater than 6 the time, enzyme is lived and is reduced rapidly; Enzyme is lived and all can be maintained more than 80% between pH2～10, has good pH stability.At pH5.0, the mensuration result of enzymic activity shows under the differing temps, and the optimal reactive temperature of this enzyme is 60 ℃.Be incubated 30min down at 60 ℃, the residual enzyme activity is 40%, 70 ℃ and 80 ℃ and is incubated 30min down that the residual enzyme activity reduces to 15%.
In enzymatic reaction system, add different chemical reagent, measure enzymic activity then respectively, the result shows that Mg2+, Ca2+ and EDTA have activation to AppA-M, and enzymic activity promotes 13%, 12% and 16.9% respectively; Configuration metal ions Zn 2+, Cr3+, Cu2+, Fe2+ and Fe3+ all have restraining effect in various degree to the enzymatic reaction of AppA-M, and SDS has suppressed its phytase activity fully.
Sumizyme PHY AppA-M with pepsin 120min after, enzymic activity has improved 17%; Behind trypsin treatment 120min, the enzymic activity of residue 32.8%.Explain that Sumizyme PHY AppA-M has the ability of extraordinary antipepsin hydrolysis ability and part anti-trypsin hydrolysis.
And, through measuring, to compare with colibacillary appa albumen, the enzyme work of high-specific-activity phytase of the present invention has improved about 50%.
Embodiment 5: the phytase activity measuring method
1. method principle
Sumizyme PHY is under certain temperature and pH condition, and the hydrolysis substrate sodium phytate generates ortho-phosphoric acid and inositol derivative, and in acidic solution, handling with vanadium ammonium molybdate can generation xanchromatic (NH
4)
3PO
4NH
4VO
316MoO
3Mixture carries out colorimetric estimation under wavelength 415nm.
2. reagent and solution
Agents useful for same in present method when dated other do not require, all refers to AR and meets three grades of water stipulating among the GB/T6682.And washing test is not used phosphorous clean-out system with container.
2.10.25mol/L acetate buffer (1):
Take by weighing the 34.02g SODIUM ACETATE TRIHYDRATE in the 1000ml volumetric flask, add the 900ml water dissolution, regulate pH to 5.0 ± 0.01, and be settled to 1000ml, deposit 2 months under the room temperature effectively with zero(ppm) water with hydrochloric acid.
2.20.25mol/L acetate buffer (2):
Take by weighing the 34.02g SODIUM ACETATE TRIHYDRATE, 0.5g Triton X-100,0.5g bovine serum albumin (BSA) is in the 1000ml volumetric flask; Add the 900ml water dissolution; Regulate pH to 5.0 ± 0.01 with hydrochloric acid, and be settled to 1000ml, deposit 2 months under the room temperature effectively with zero(ppm) water.
2.37.5mmol/L sodium phytate (substrate) solution
Take by weighing 0.6929g sodium phytate (C
6H
6O
24P
6Na
12) in the 100ml volumetric flask, dissolve and be settled to scale with 0.25mol/L acetate buffer (1), adjust pH to 5.0, existing with join (ultimate density in the real reaction liquid is 5.0mmol/L) at present.
2.4 salpeter solution: the 1:2 aqueous solution.
2.5100g/L ammonium molybdate solution: take by weighing 10g ammonium molybdate [(NH
4)
6Mo
7O
244H
2O] in the 100ml volumetric flask, add 1.0ml ammoniacal liquor (25%) and be settled to scale with water dissolution.
2.62.35g/L Ammonium Vanadate Solution: take by weighing 0.235g ammonium vanadate (NH
4VO
3) in the brown volumetric flask of 100ml, add the 2ml salpeter solution, be settled to scale with water dissolution.Preserved for 1 week under the lucifuge condition effectively.
2.7 color stop buffer: pipette 2 parts of salpeter solutions, 1 part of ammonium molybdate solution, 1 part of Ammonium Vanadate Solution mixes the back to be used, existing with join at present.
2.8 potassium primary phosphate (KH
2PO
4): standard substance.
3 instrument and equipments
3.1 laboratory common instrument equipment.
3.2 water bath with thermostatic control: 37 ± 0.1 ℃.
3.3 spectrophotometer; The 10mm cuvette is arranged, can under 415nm, measure absorbancy.
3.4 magnetic stirring apparatus.
3.5 eddy current type mixing tank.
3.6 acidometer: be accurate to behind the radix point 2.
3.7 whizzer: rotating speed is more than the 4000r/min.
4. specimen preparation
Get the Sumizyme PHY sample, to 200g, need not pulverize by the Sumizyme PHY product with sample divider pass for the quartering of usefulness routine, and mixed feed and additive premix need be pulverized the standard sieve through 0.45mm, and the sealed vessel of packing into prevents that sample constituents from changing.
5. determination step
5.1 absolute method
5.1.1 typical curve
Accurately take by weighing 0.6804g at 105 ℃ of benchmark potassium primary phosphates that dry to constant weight in the 100ml volumetric flask, with 0.25mol/L acetate buffer dissolving, and be settled to 100ml, concentration is 50.0mmol/L.Dilution proportion by following table 2 becomes different concns, with the sample reaction assay, is X-coordinate with the light absorption value, and the amount of inorganic phosphorus in the reaction system (μ mol) is an ordinate zou, lists linear regression equation (y=ax+b).
Table 2
| The standard sequence number | Amount of dilution, ml | Concentration, mol/ml | The amount of inorganic phosphorus in the reaction system, mol |
| 12345 | 0.5→1?0.5→2?0.5→4?0.5→8?0.5→16 | 25 12.5 6.25 3.125?1.5625 | 5 ?2.5 ?1.25 0.625?0.3125 |
5.1.2 the preparation of sample solution:
Take by weighing two parts on sample, every part of 1.0000g is accurate to 0.0001g; Place the 100ml volumetric flask, add about 70ml acetate buffer (2), a magnetic bar; High-speed stirring 30min on magnetic stirring apparatus is settled to scale (deducting the volume of magnetic bar) with acetate buffer (2).Shake up, on whizzer with the centrifugal 10min of 4000r/min.The supernatant of obtaining different volumes makes sample liquid concentration remain on about 0.4U/ml question response with acetate buffer (2) dilution.
Aforementioned Sumizyme PHY purifying is after use acetate buffer (1) to dilute according to above step.
5.1.3 reaction
Get the 10ml test tube and operate by following reaction sequence, in reaction process, begin from adding substrate, the timed interval that in every test tube, adds reagent wants absolute consistent, 37 ℃ of hydrolysis 30min.
Reactions step and reagent, solution usage are seen table 3.
Table 3
5.1.4 sample determination
Reacted sample at room temperature leaves standstill 10min, as becomes turbid and need at the centrifugal 10min of the above 4000rpm of whizzer, and supernatant is with the blank zeroing of typical curve, at the blank (A of spectrophotometer 415nm wavelength working sample
0) and the light absorption value of sample solution (A), A-A
0Be the actual measurement light absorption value.Calculate the activity of Sumizyme PHY with linear regression equation.
6 results calculate and expression
6.1 calculation formula
The activity of Sumizyme PHY in the sample, with the expression of the activity unit " U " in every gram (or mL) sample, calculation formula is following.
In the formula:
C---the y value that calculates by linear regression equation according to the light absorption value of actual appearance liquid;
F---the total extension rate before the sample solution reaction;
0.2---0.2ml enzyme diluent (step 5.1.2);
M---sample quality;
30---the reaction times (minute).
Embodiment 6: protein variant
Adopt conventional method; Add 6 histidine-tagged (His-6) at the proteic N end of AppA-M of the present invention; After using ordinary method to give expression to the N end to carry histidine-tagged AppA-M albumen, use the Ni-NTA affinity chromatography to be further purified by histidine-tagged target peptide.
Wash-out is also collected purified product; Press embodiment 4 and 5 described methods; Measure the proteic the enzyme activity of AppA-M and the PH stability that are obtained; The result shows and describedly carries histidine-tagged AppA-M albumen and the AppA-M protein-active with histidine-tagged is not close, and also has good enzymic activity and PH stability.
Embodiment 7: the feed that contains height ratio Sumizyme PHY alive
Preparation phytase composition (5000 type particulate product) is like table 4:
Table 4
| Composition | Content |
| W-Gum | 90wt％ |
| Microcrystalline Cellulose sodium | 2wt％ |
| Sumizyme PHY | 5000U/g |
| Moisture content | Surplus (8wt%) |
With above-mentioned each composition thorough mixing, process the granulated feed product according to the method for routine.
By following table 5 prescription, with ordinary method prepare feedstuff for laying hens (content: wt%), the phytase composition of employing is 5000 type particulate product of aforementioned preparation:
Table 5
| Raw material | Control group (Kg) | Test 1 group (Kg) |
| Corn | 63 | 63 |
| Dregs of beans | 15 | 15 |
| Expanded soybean | 18 | 18 |
| The dish dregs of rice | 0 | 0.39 |
| Calcium powder | 1.2 | 1.8 |
| Phytase composition | 0 | 0.01 |
| Secondary calcium phosphate | 1.5 | 0.5 |
| Salt | 0.3 | 0.3 |
| Preblend | 1 | 1 |
| Amount to | 100 | 100 |
With 1 group of each raw material thorough mixing of above-mentioned control group and experiment, process feed according to ordinary method.
Embodiment 8: animal experiment
1 group of feed of the test that contains high-specific-activity phytase and control group feed difference feeding laying hen with embodiment 7 preparations.
The result finds, compares with the control group feed, and animal can add about 65% secondary calcium phosphate less in the feed after adding said 5000 type particulate product behind 1 group of feed of edible test, reduced cost significantly; And this kind feed can also improve the speed of growth of laying hen, reduces feedstuff-meat ratio, makes the keeper obtain more economic benefit.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
< 110>Sichuan Heben Bioengineering Co., Ltd.
< 120>high specific activity phytase gene and application thereof
<130>070926
<160>2
<170>PatentIn?version3.3
<210>1
<211>1233
<212>DNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>phytase gene
<400>1
<210>2
<211>410
<212>PRT
< 213>artificial sequence
<220>
<221>MISC_FEATURE
< 223>Sumizyme PHY albumen
<400>2
Claims (8)
1. an isolating albumen is characterized in that, this proteic aminoacid sequence is shown in SEQ ID NO:2.
2. isolating polynucleotide is characterized in that, these polynucleotide are selected from down group:
(i) the described proteic polynucleotide of coding claim 1; Or
(ii) with (i) in polynucleotide complementary polynucleotide.
3. polynucleotide as claimed in claim 2 is characterized in that, these polynucleotide have the nucleotide sequence shown in the SEQ ID NO:1.
4. a carrier is characterized in that, it contains the described polynucleotide of claim 2.
5. the described proteic purposes of claim 1 is characterized in that, is used to decompose phytic acid or phytate.
6. one kind prepares the described proteic method of claim 1, it is characterized in that, this method comprises cultivates the described host cell of claim 5, collects and obtains the described albumen of claim 1.
7. a compsn is characterized in that, contains the described albumen of claim 1 of significant quantity in the said compsn, and bromatology or the last acceptable carrier of feed.
8. one kind prepares the method that contains the described proteic transgenic plant of claim 1, it is characterized in that, described method comprises step: the described polynucleotide of claim 2 are imported in the vegetable cell, cultivate described vegetable cell, the regeneration plant.
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