CN1632110A - Modified high specific activity phytase, gene encoding same and high expression thereof - Google Patents

Abstract

The invention has supplied an advanced high specific activity plant enzyme and its coding gene, compared with the former one, it has higher enzyme, activity in acid and neutral PH. Also, the recombinant yeast cell with the plant enzyme gene is also supplied. Its expressing quantity of plant enzyme can be 6*106u/ml, and the plant enzyme can be widely applied in feed and food industries.

Description

A kind of high-specific-activity phytase of improvement and encoding gene thereof and efficiently express

Technical field

The present invention relates to a kind of phytase and encoding gene thereof of improvement.The invention still further relates to and contain recombinant yeast cell this phytase gene, that can efficiently express phytase.

Background technology

Phytase (EC.3.1.3.8) is a kind of enzyme of energy hydrolysis phytic acid.It can be degraded to phytate phosphorus inositol and phosphoric acid.Phytase extensively is present in the microorganism, as the aspergillus in the fungi (Yamada K.et al.Agric.Biol.Chem.32:1275-1282,1986; VanGorcom R.F.M.et al., US Patent No.5436156,1995), yeast (NayiniN.R.et al.Lebensm Wiss.Technol.17:24-26,1984); Subtilis in the bacterium (Paver, V.K.J., Bacteriol.151:1102-1108,1982), false monospore bacillus (Cosgrove D.J., Austral.J.Biol.Sci.23:1207-1220,1970), lactobacillus (Shirai K., Letters Appl.Biol.Sci.19:366-369,1994), intestinal bacteria (Greiner R., Arch Biochem.Biophys.303:107-113,1993) etc.

Phytase can be widely used in (Ware J.H.et al., USPatent No.3297548,1967 in the monogastric animal feed; Nelson T.S.et al., J.Nutrition101:1289-1294,1971; Nelson T.S.et al., Poult Sci.47:1842-1848,1968).It can make the utilization ratio of phosphorus in the plant feed improve 60%, and the phosphorus excretion reduces 40% in the ight soil, also can reduce the anti-oxidant action of phytate phosphorus.Therefore significant to improving the livestock industry productivity effect and reducing phytate phosphorus to the pollution of environment.

By genetic engineering means phytase gene being efficiently expressed in recombinant bacterial strain is the effective way that phytase is able to extensive cheap production and practical application.Recombinate acid phytase gene phyA in the aspergillus niger (Van Gorcom R.F.M.et al., 1995) such as nineteen ninety-five Van Gorcomd, makes the expression amount of phytase in recombinant bacterial strain reach 2.8 * 10 ⁵U/mL produces bacterial strain with natural phytase and compares and have increased significantly, and greatly reduces the production cost of phytase.The expression amount of gene engineering yeast its acid phytase on the lab scale level of structures such as Yao Bin in 1997 reaches 5 * 10 ⁵U/mL (being equivalent to express in every milliliter of fermented liquid 5mg phytase albumen) (Yao Bin etc., Chinese invention patent 97121731.9,2000) brings up to 1 * 10 with expression amount on the pilot scale level ⁶The U/mL fermented liquid, higher 3000 times than the phytase expression amount among the natural strains A spergillus niger963 of primary, than abroad being used for commercialization produces the genetically engineered aspergillus of acid phytase (Van Gorcom R.F.M.et al., US Patent 5436156,1995) double above.Employings such as nearest Yao Bin derive from colibacillary high specific activity phytase gene and have made up recombination yeast, and the expression level of phytase is brought up to 6 * 10 ⁶More than (Yao Bin, biotechnology journal, Vol20, No1,2004).

The pH characteristic of phytase is one of key factor that influences the phytase practical application effect.The effect place of phytase is the gi tract of animal, and GI pH value is to be elevated to 6.5～7.5 gradually from about 1.5～2.0, if phytase all can be kept high reactivity in the entire area of pH2.0～7.0, just can give full play to the efficient of phytase, reach better feeding effect.Deriving from colibacillary phytase APPA and have the advantage of high specific acitivity, is the phytase of at present tool application potential.1999, (Rodriguez E.Arch Biochem Biophys, 365:262～267 such as Rodriguez, the intestinal bacteria of the phytase generating that 1999) from pig manure, screens, and be cloned into phytase gene appA, this full length gene 1299bp, 432 amino acid of encoding.The appA gene does not have homology except having 47% homology with Bacillus sp.DS11 phytase gene substantially with other source phytase gene, has than big-difference on the zymologic property yet.This phytase is the 6-phytase, and the ratio that is so far the to be reported the highest phytase (Golovan S.Can JMicrobiol, 46:59～71,2000) of living can reach 3,000 than work, about 000IU/mg.

The optimal pH of APPA is 4.5, but the pH scope of its effect is narrower, when pH2.5 and pH6, enzymic activity only be the suitableeest enzymic activity 50%, like this, it can not bring into play high-level efficiency in the whole gi tract of animal.

Summary of the invention

General purpose of the present invention is that appA suddenlys change to phytase gene by DNA shuffing technology, makes the phytase after the sudden change obtain improvement in nature at pH, can keep higher enzymic activity than protoenzyme in the scope of pH2～7.Further, utilize bio-reactor pichia spp (Pichia pastoris) to efficiently express this mutator gene, make phytase be able to cheap production.

One of purpose of the present invention is that phytase APPA is carried out molecular improvement, makes it have higher enzymic activity in the scope of pH2～7.The phytase (name and be APPA-m) of improvement, compare with former phytase, 7 amino acid whose differences are arranged, respectively by original+47A ,+56P ,+169R ,+210C ,+251M ,+252P and+347D is mutated into+47D ,+56Q ,+169G ,+210F ,+251I ,+252R and+347N.Phytase APPA-m among the present invention has higher enzymic activity in the scope of pH2～7.It 2.5 and enzymic activity during pH6.0 more than 70% of the suitableeest enzymic activity is arranged, and protoenzyme only has about 50%.Phytase of the present invention has aminoacid sequence shown in Figure 4.

Another object of the present invention provides a kind of dna molecular of the phytase of the present invention of encoding.Described dna molecular can have the nucleotide sequence of Fig. 3.

Three of purpose of the present invention provides the method that the appA-m gene after the improvement efficiently expresses in expression system.Comprise phytase expression method in the screening of genetic transforming method, recon of structure, the recipient bacterium of a whole set of recombinant expression vector and molecular assay method and the recon.The present invention adopts pichia spp (P.pastoris) as appA-m expression of gene acceptor, and it is laid a good foundation for utilizing recombination yeast large-scale industrialization, low-cost fermentative production phytase.

Technical solution

Technological approaches of the present invention is as follows:

1. the adoptable transgenation method of transgenation has rite-directed mutagenesis, PCR to cause wrong sudden change and DNA shuffling etc.Using DNA shuffling technology (Zhou Jiahai etc., biological chemistry and biophysics progress, 27:655～657,2002) in this patent suddenlys change to phytase gene.Phytase gene appA through the small pieces that DNAase I is digested to 10～200bp, is obtained a large amount of mutating molecules after no primer PCR, primer PCR amplification.

2. (Maniatis T., et al.Molecularcloning.New York:Cold Spring harbor laboratory, 1982) are cloned in gene clone according to a conventional method.Above-mentioned phytase mutating molecule is cloned on the escherichia coli vector pET-22b (+), and same method also can be cloned on escherichia coli cloning carrier or the expression vector, and as pPUC18, pPGEM etc., transformed into escherichia coli obtains recon.

3. the screening of improvement phytase is carried out property testing to the phytase of expression of recombinant e. coli, filters out the phytase gene that obtains improveing at pH, and carries out sequencing.

4. the structure of recombination high efficiency expression system is selected the bio-reactor of efficient expression system-P.pastoris as the Expressing Recombinant Phytase gene.By reorganization in the body phytase gene is incorporated on the zymic genome, filters out positive recombinant.The insect expression system of other eukaryotic expression system such as baculovirus, mould expression system, plant expression system etc. also are adapted to efficiently expressing of this gene.

Description of drawings

The physical map of Fig. 1 intestinal bacteria recombinant vectors pBY

The pH characteristic series 1 of phytase APPA-m after Fig. 2 sudden change and former phytase AppA effect is AppA; Series 2 is APPA-m

Fig. 3 appA-m gene DNA sequence

The appA-m amino acid sequence coded that Fig. 4 derives

The physical map of Fig. 5 recombinant yeast expression vector pFF02

The SDS-PAGE of Fig. 6 phytase that different induction time accumulated in the 5L fermentor tank

1 is the standard protein molecule; 2-10 is respectively and induced the back 0,12,24,36,48,60,72,96,108 hour;

Embodiment

Experiment condition

1. bacterial strain and carrier coli strain E.coli DH _5a, plasmid pET-22b (+) etc. is available from Promega company, yeast strain Pichia pastoris GS115 (His ^-Mut ⁺), plasmid pFF01 is so kind as to give by Canadian Alberta doctor D.Luo of university.

2. enzyme and test kit restriction enzyme, ligase enzyme, Taq enzyme, DNAaseI are Boehringer company product.PCR Kit all purchases the company in Promega.

3. biochemical reagents DNA synthetic agent is a Milipore company product.Primer is synthetic with the ABI Cyclone of company dna synthesizer.IPTG, X-Gal, SDS and sodium phytate are Sigma company product.TEMED, ammonium persulphate, acrylamide and methylene diacrylamide are Promega company product.

Substratum intestinal bacteria substratum be LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).The yeast perfect medium is YPD (1% yeast extract, 2% peptone, 2% glucose); The yeast conversion substratum is RDB[18.6% sorbyl alcohol, 2% glucose, 1.34%Yeast Nitrogen Base W/O amino acids (YNB), 0.00004%Biotin, 0.005% L-glutamic acid, 0.005% methionine(Met), 0.005% Methionin, 0.005% leucine, 0.005% Isoleucine, 2% agarose]; It is MM (1.34%YNB, 0.00004%Biotin, 0.5% methyl alcohol, 1.5% agarose) and MD (1.34%YNB, 0.00004%Biotin, 2% glucose, 1.5% agarose) that yeast is selected substratum; Yeast inducing culture BMGY[1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 1% glycerine (V/V)] and BMMY (replace glycerine divided by 0.5% methyl alcohol, all the other compositions are identical with BMGY).The recombination yeast fermention medium is 10 * Basal Salts (2.67% phosphoric acid, 0.093% calcium sulfate, 1.82% vitriolate of tartar, 1.49% sal epsom, 0.413% potassium hydroxide, 4% glycerine or a glucose); Used trace salt solution PTM1 (0.6% copper sulfate, 0.008% sodium iodide, 0.3% manganous sulfate, 0.02% Sodium orthomolybdate, 0.002% boric acid, 0.05% cobalt chloride, 2% zinc chloride, 6.5% ferrous sulfate, 0.025% vitamin H, 0.5% sulfuric acid) in the fermentation.

Experiment one

This description of test carries out the program of phytase gene sudden change.

Get the DNA of 1 μ g phytase gene appA, at room temperature handled 5,10,15,20 minutes,, determine best enzymolysis time, make the enzymolysis product size concentrate on 20～100bp by electrophoresis observation dna segment size with the DNAase I of 0.05U.Utilize DNA glue to reclaim test kit reclaims 20～100bp from running gel dna segment.The segment that reclaims is carried out pcr amplification under the situation of no primer, electrophoresis reclaims the dna segment greater than former appA mrna length, carries out the primer PCR amplification again.The primer that uses synthetic according to 5 ' and 3 ' terminal sequence of appA gene, 2 primer sequences are respectively: 5 ' GAATTCATGAAAGCGATCTTAATC 3 ' and 5 ' GAATTCTTACAAACTGCACGC 3 ' (underscore is the EcoRI restriction enzyme site for designing partly) carries out gel electrophoresis after the amplification, reclaim size and the identical dna segment of former appA gene.The phytase gene molecule that has just comprised a large amount of sudden changes in these dna segments.

Experiment two

Suddenly change clone's program of phytase gene molecule of this description of test.

The dna molecular that obtains in the experiment one after cutting with the EcoRI enzyme, is connected in 15 ℃ with the carrier pET-22b (+) that cuts through the EcoRI enzyme and spends the night transformed into escherichia coli E.coli DH _5a, select positive recombinant behind the coated plate on the LB substratum, the extraction plasmid carries out enzyme and cuts evaluation, obtains recon.Contain a kind of phytase gene molecule in each recon.Recombinant plasmid pBY collection of illustrative plates is seen Fig. 1.

Experiment three

The suddenly change screening procedure of phytase of this description of test.

1000 recons that obtain in the experiment two are inoculated into respectively in the 5mL LB nutrient solution, and 37 ℃ are cultured to OD value 0.6, and adding IPTG is 0.5mmol/L to final concentration, and continuation was cultivated 2 hours, the abduction delivering phytase.Centrifugal collection thalline, thalline are resuspended in the 5mL 0.25mol/L acetate buffer solution (pH5.0), and ultrasonic disruption thalline, 15000rpm went cell debris, supernatant liquor to be used to carry out phytase activity mensuration in centrifugal 15 minutes.Measuring method is: the enzyme diluent of 0.2mL adds the sodium phytate of 0.8mL 1.25mmol/L, and 37 ℃ of insulation 30min add 1mL 10%TCA and stop enzyme reaction alive, add 2mL ferrous sulfate-ammonium molybdate colour developing liquid then, and 700nm measures content of inorganic phosphorus.Contrast makes enzyme-deactivating for add 1mL 10%TCA earlier in the enzyme diluent of 0.2mL, adds the substrate insulation with volume again.A unit of enzyme activity (U) is defined as: under certain condition, it is a unit of enzyme activity that per minute discharges the required enzyme amount of 1nmol inorganic phosphorus.At first under the condition of pH4.5, measure enzymic activity (A), activated with this understanding recon has 520, further under the condition of pH2.0, measure enzymic activity (B), filter out in the B/A value, obtain 8 of recons altogether greater than 60% recon (the B/A value of protoenzyme is about 50%).Further under the condition of pH6.0, measure enzymic activity (C), filter out in the C/A value greater than 60% recon (the B/A value of protoenzyme is about 50%), obtain 1 recon, name and be EappA-m, preliminary screening is to can keep highly active sudden change phytase in wide pH scope.The phytase that EappA-m produced is carried out complete pH property testing, at different pH values (pH2.0,2.5,3.0,3.5 HCl-Gly damping fluid; PH4.0,4.4,5.0,5.5,5.8 HAc-NaAc damping fluid; PH6.0, imidazoles-HCl of 6.5; PH7.0,7.5,8.0,9.0 Tris-HCl damping fluid) measure its enzymic activity down, the result shows (Fig. 2), it can keep high reactivity in the scope of pH2～7, apparently higher than protoenzyme APPA.

Experiment four

The sequence of this description of test sudden change phytase APPA-m.

Phytase gene appA-m among the recon EappA-m is carried out sequencing, and Fig. 3 is its nucleotide sequence, and Fig. 4 is its aminoacid sequence.The result shows, compare with protoenzyme, there are 8 bases to obtain sudden change, its position respectively+140 ,+167 ,+505 ,+629 ,+753 ,+755 ,+1039 and+1044, the sudden change of wherein preceding 7 bases has caused amino acid whose sudden change, respectively by original+47A ,+56P ,+169R ,+210C ,+251M ,+252P and+347D is mutated into+47D ,+56Q ,+169G ,+210F ,+251I ,+252R and+347N.Just because of these sudden changes, make its pH character obtain improvement.

Experiment five

The construction procedures of this description of test appA-m on Yeast expression carrier.

The plasmid that is used to make up Yeast expression carrier is pFF01 (having α-factor secretion signal).At first phytase gene is inserted into the downstream of the signal peptide sequence of above-mentioned expression vector, form correct reading frame with signal peptide, make the goal gene stable integration to yeast chromosomal by the homologous recombination incident between carrier and the yeast P.pastoris chromogene group then.Concrete process is: after the phytase gene appA-m that will remove signal coding sequence goes up enzyme and downcuts from plasmid pBY with EcoRI, electrophoresis reclaims the dna fragmentation of about 1.3Kb, again they are inserted into the EcoRI site on the carrier pFY01, have obtained being used for the recombinant expression vector-pFF02 (Fig. 5) of yeast conversion.The phytase gene that so just will have α-factor secretion signal has been cloned into AOX1 promotor downstream.

Experiment six

This experiment is the program of explanation yeast conversion and screening recombination yeast strain system.

The DNA of plasmid pFF02 shocks by electricity after the DraI enzyme is cut behind the transformed yeast cell, and by recombinating in the body, goal gene will be incorporated in the acceptor yeast genes group.Under the condition that exogenous induction material methyl alcohol exists, the AOX1 promotor can start the expression of its downstream gene, and signal peptide can instruct expression product to enter the zymic Secretory Pathway, through cutting, the foreign protein product is finally secreted to born of the same parents, and the phytase aminoacid sequence that is produced should be identical with naturally occurring ripe phytase by design.Foreign protein can carry out posttranslational modification through such pathways metabolism, for example glycosylation etc., thus obtain the protein product of biologically active.

At first use the DNA of 2～3 times of excessive restriction endonuclease DraI digested plasmid pFF02, make it linearizing, whether the electrophoresis detection enzyme is cut complete.Use the phenol extracting, ethanol sedimentation, 70% ethanol washes twice, lyophilize, sterilized water dissolving is got 1～5 μ g DNA and is transformed the pichia spp cell, coated plate on the RDB solid medium, every plate is coated with 0.1mL, culture dish is inverted under 30 ℃ to be cultured to transformant and to occur.

Transformant can be gone up growth at minimum medium RDB (not containing His), but not transformant can not be grown, this is because recipient bacterium GS115 is the histidine defect type, though and have the his4 gene on the carrier, but do not have the yeast replicon, so the his4 gene on the carrier must be integrated in the yeast genes group and could express.In addition, because the AOX1 gene is damaged in the yeast cell of reorganization, so it just can not utilize methyl alcohol as carbon source again.Like this, with methyl alcohol as the substratum of sole carbon source on transformant just can not grow (perhaps growth is extremely slow), show as methyl alcohol and utilize defective type (mut ^-).

Go up picking his with aseptic toothpick from transforming dull and stereotyped RDB ⁺Recon at first is inoculated on the MM solid medium, inoculates on the MD solid medium, so picking his ⁺Recon was cultivated 2 days for 30 ℃.Screening is at the clone's (his normal but that some growth is arranged on the MM flat board or do not grow fully that grows on the MD flat board ⁺Mut ^-) be positive colony.

In order to screen the restructuring yeast strains that obtains high expression level, directly detect the expression of phytase in the inducing culture.With his ⁺Mut ^-Transformant is at first cultivated in the BMGY substratum, treats that it grows to state of saturation, and the centrifugal BMGY that abandons changes to inducing culture BMMY, gets supernatant liquor and carry out the phytase activity analysis behind inducing culture 36h.By the enzyme assay of Expressing Recombinant Phytase, preliminary screening is to the recon of 66 strain Expressing Recombinant Phytase from 350 strain recombination yeasts, and wherein 1 the highest strain recon of expression amount is named the appA-m-36 into P.pastoris.

Experiment nine

Present embodiment is the program of explanation recombination yeast at 5 liters of fermentor tank middle-high density fermentative production phytases.

Fermenting process is divided into three phases.Specific as follows: 1) the strain culturing stage.Adding 28% ammoniacal liquor before fermention medium 10 * Basal Salts inoculation earlier makes the pH of substratum reach 5.0, add 4.37mL PTM1 by every liter of substratum again, 5-10% inoculates seed liquor, 18～24h is cultivated in aeration-agitation, in culturing process along with the growth of bacterial strain, dissolved oxygen amount in the substratum reduces gradually by 100%, and dissolved oxygen amount will be increased to more than 80% once again after carbon source runs out of, and this moment, the thalline weight in wet base reached 90～110g/L.2) carbon source is fed the stage.Stream adds 25% glucose (containing 12mLPTM1 in every liter), and the stream dosage is 28mL/h/L, cultivates 4h.Adjusting air flow makes dissolved oxygen amount all the time greater than 20%.The thalline weight in wet base reaches 180～220g/L during this EOS.3) the abduction delivering stage.Add inductor methyl alcohol (containing 12mL PTM1 in every liter), make the methyl alcohol final concentration maintain 0.3%, dissolved oxygen amount is all the time greater than 20%.The SDS-PAGE that every 12h takes a sample and once measures the accumulation volume of the phytase of expressing and carry out expressing protein in inducing process.

Fig. 6 is seen in the SDS-PAGE analysis that increases the phytase of expressing accumulation in the fermented liquid with induction time.The result shows that the phytase of expression accumulates with the increase of induction time, peaks when inducing 108 hours, and enzymic activity reaches 6 * 10 ⁶The U/mL fermented liquid.The zymoprotein molecular weight size of expressing is about 45kD.Above result proves that phytase gene has not only obtained expression, effectively secretion, and the phytase of expressing has normal biologic activity.

5’

ATG?AAA?GCG?ATC?TTA?ATC?CCA?TTT?TTA?TCT?CTT?CTG?ATT?CCG?TTA?ACC?CCG?CAA?TCT?GCA

TTC?GCT?CAG?AGT?GAG?CCG?GAG?CTG?AAG?CTG?GAA?AGT?GTG?GTG?ATT?GTC?AGT?CGT?CAT?GGT

GTG?CGT?GCT?CCA?ACC?AAG?GAC?ACG?CAA?CTG?ATG?CAG?GAT?GTC?ACC?CAA?GAC?GCA?TGG?CCA

ACC?TGG?CCG?GTA?AAA?CTG?GGT?TGG?CTG?ACA?CCG?CGA?GGT?GGT?GAG?CTA?ATC?GCC?TAT?CTC

GGA?CAT?TAC?CAA?CGC?CAG?CGT?CTG?GTA?GCC?GAC?GGA?TTG?CTG?GCG?AAA?AAG?GGC?TGC?CCG

CAG?TCT?GGT?CAG?GTC?GCG?ATT?ATT?GCT?GAT?GTC?GAC?GAG?CGT?ACC?CGT?AAA?ACA?GGC?GAA

GCC?TTC?GCC?GCC?GGG?CTG?GCA?CCT?GAC?TGT?GCA?ATA?ACC?GTA?CAT?ACC?CAG?GCA?GAT?ACG

TCC?AGT?CCC?GAT?CCG?TTA?TTT?AAT?CCT?CTA?AAA?ACT?GGC?GTT?TGC?CAA?CTG?GAT?AAC?GCG

AAC?GTG?ACT?GAC?GCG?ATC?CTC?AGC?GGG?GCA?GGA?GGG?TCA?ATT?GCT?GAC?TTT?ACC?GGG?CAT

CGG?CAA?ACG?GCG?TTT?CGC?GAA?CTG?GAA?CGG?GTG?CTT?AAT?TTT?CCG?CAA?TCA?AAC?TTG?TGC

CTT?AAA?CGT?GAG?AAA?CAG?GAC?GAA?AGC?TTT?TCA?TTA?ACG?CAG?GCA?TTA?CCA?TCG?GAA?CTC

AAG?GTG?AGC?GCC?GAC?AAT?GTC?TCA?TTA?ACC?GGT?GCG?GTA?AGC?CTC?GCA?TCA?ATG?CTG?ACG

GAG?ATA?TTT?CTC?CTG?CAA?CAA?GCA?CAG?GGA?ATC?CGG?GAG?CCG?GGG?TGG?GGA?AGG?ATC?ACC

GAT?TCA?CAC?CAG?TGG?AAC?ACC?TTG?CTA?AGT?TTG?CAT?AAC?GCG?CAA?TTT?TAT?TTG?CTA?CAA

CGC?ACG?CCA?GAG?GTT?GCC?CGC?AGC?CGC?GCC?ACC?CCG?TTA?TTA?GAT?TTG?ATC?AAG?ACA?GCG

TTG?ACG?CCC?CAT?CCA?CCG?CAA?AAA?CAG?GCG?TAT?GGT?GTG?ACA?TTA?CCC?ACT?TCA?GTG?CTG

TTT?ATC?GCC?GGA?CAC?GAT?ACT?AAT?CTG?GCA?AAT?CTC?GGC?GGC?GCA?CTG?GAG?CTC?AAC?TGG

ACG?CTT?CCC?GGT?CAG?CCG?AAT?AAT?ACG?CCG?CCA?GGT?GGT?GAA?CTG?GTG?TTT?GAA?CGC?TGG

CGT?CGG?CTA?AGC?GAT?AAC?AGC?CAG?TGG?ATT?CAG?GTT?TCG?CTG?GTC?TTC?CAG?ACT?TTA?CAG

CAG?ATG?CGT?GAT?AAA?ACG?CCG?CTG?TCA?TTA?AAT?ACG?CCG?CCC?GGA?GAG?GTG?AAA?CTG?ACC

CTG?GCA?GGA?TGT?GAA?GAG?CGA?AAT?GCG?CAG?GGC?ATG?TGT?TCG?TTG?GCA?GGT?TTT?ACG?CAA

ATC?GTG?AAT?GAA?GCA?CGC?ATA?CCG?GCG?TGC?AGT?TTG?TAA 3’

MKAILIPFLSLLIPLTPQSAFAQSEPELKLESVVIVSRHGVRAPTKDTQLMQDVTQ

DAWPTWPVKLGWLTPRGGELIAYLGHYQRQRLVADGLLAKKGCPQSGQVAIIA

DVDERTRKTGEAFAAGLAPDCAITVHTQADTSSPDPLFNPLKTGVCQLDNANV

TDAILSGAGGSIADFTGHRQTAFRELERVLNFPQSNLCLRREKQDESFSLTQALPS

ELKVSADNVSLTGAVSLASMLTEIFLLQQAQGIREPGWGRITDSHQWNTLLSLH

NAQFYLLQRTPEVARSRATPLLDLIKTALTPHPPQKQAYGVTLPTSVLFIAGHDT

NLANLGGALELNWTLPGQPNNTPPGGELVFERWRRLSDNSQWIQVSLVFQTLQ

QMRDKTPLSLNTPPGEVKLTLAGCEERNAQGMCSLAGFTQIVNEARIPACSL

Claims (10)

1, a kind of phytase of improvement is characterized in that, by the sudden change to the gene of phytase APPA, and express the phytase gene of this sudden change and obtain, and the enzymic activity of the phytase of this improvement when pH2.5 and pH6.0 reaches more than 70% of the suitableeest enzymic activity.

2, the phytase of improvement according to claim 1 is characterized in that ,+47 ,+56 ,+169 ,+210 ,+251 ,+252 and+sudden change taken place in 347 amino acid.

3, the phytase of improvement as claimed in claim 2, it is characterized in that, said amino acid whose sudden change by original+47A ,+56P ,+169R ,+210C ,+251M ,+252P and+347D is mutated into+47D ,+56Q ,+169G ,+210F ,+251I ,+252R and+347N.

4, the phytase of improvement as claimed in claim 1 is characterized in that, this zymoprotein has aminoacid sequence as shown in Figure 4.

5, a kind of gene of phytase of the improvement of encoding, it is characterized in that, this gene is by acquisition that phytase gene appA is suddenlyd change, wherein+140 ,+167 ,+505 ,+629 ,+753 ,+755 ,+1039 and+sudden change taken place, made the enzymic activity of phytase when pH2.5 and pH6.0 of improvement reach more than 70% of the suitableeest enzymic activity in 1044 base.

6, the gene of improvement phytase as claimed in claim 5 is characterized in that, this improvement phytase gene has nucleotide sequence shown in Figure 3.

7, the gene of improvement phytase as claimed in claim 5 is characterized in that, said transgenation is that rite-directed mutagenesis, PCR cause wrong sudden change or DNA shuffing.

8, express the method that efficiently expresses of the gene of improvement phytase as claimed in claim 5, the expression method that comprises phytase gene in the screening of genetic transforming method, recon of structure, the recipient bacterium of a whole set of recombinant expression vector and molecular assay method and the recon, it is characterized in that, utilize the bio-reactor of efficient expression system pichia spp as the Expressing Recombinant Phytase gene.

9, the high-efficiency expression method of the gene of expression improvement phytase as claimed in claim 8, it is characterized in that other the eukaryotic expression system such as insect expression system, mould expression system, plant expression system that also can utilize baculovirus is as the bio-reactor of Expressing Recombinant Phytase gene

10, the high-efficiency expression method of the gene of expression improvement phytase as claimed in claim 8 is characterized in that, intestinal bacteria are used as the clone and express the carrier of this improvement phytase gene.

Images