CN100340662C - High specific activity phytase gene and its efficient expression - Google Patents
High specific activity phytase gene and its efficient expression Download PDFInfo
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- CN100340662C CN100340662C CNB2004100886593A CN200410088659A CN100340662C CN 100340662 C CN100340662 C CN 100340662C CN B2004100886593 A CNB2004100886593 A CN B2004100886593A CN 200410088659 A CN200410088659 A CN 200410088659A CN 100340662 C CN100340662 C CN 100340662C
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Abstract
The present invention provides phytase with high specific activity and a coded gene thereof. The phytase for coding a phytase gene (the name is PHYQ.) is screened, and the specific activity is as high as 4.1*10<6> IU/mg. Compared with the phytase APPA (from colibacillus) with maximum specific activity, which is reported at present, the present invention has the advantage that the specific activity is as high as 37%. The gene phyQ is long, the total length of the gene is 1299 bp, and 432 amino acids are coded. Compared with the reported phytase, the phytase with maximum homology is phytase APPA from colibacillus. The present invention also provides a recombined yeast cell containing the phytase gene of the present invention. The expression quantity of the phytase in recombined yeast of the present invention can reach 5.5*10<6> U/ml. The phytase of the present invention can be used as a fodder additive applied to fodder industry.
Description
Technical field
The present invention relates to a kind of high-specific-activity phytase and encoding gene thereof.The invention still further relates to and contain recombinant yeast cell this phytase gene, that can efficiently express phytase.
Background technology
50～70% phosphorus exist (Lolas M.Etal.Food Sci.42:1094-1097,1977 with the form of phytate phosphorus (phytinic acid) in the plant; Nelson T.S.Poultry Sci.47:862-871,1967), can not directly be utilized (Cromwell G.L.Biotechnology in the Feed Industry, Lyons T.P.ed.Alltech TechnicalPublication, Nicholasville by the simple stomach animal and human, KY, 133-145), cause the waste of phosphorus source, feed cost to increase, simultaneously, phytate phosphorus can not be utilized and directly excrete by animal, also can cause the phosphorus in soil and waters to pollute.In addition, phytic acid can also with necessary trace element of animal and protein chelating, these nutritive elements can not effectively be utilized, caused reduction (the Sharma C.B.et al. of plant feed (or food) nutritive value, Phytochemistry.17:201-204,1978).The phytase (Phytase, phytase) that extensively is present in the microorganism is inositol and phosphoric acid with hydrolysis of phytic acid.Phytase is fed and is raised the existing conclusive evidence of effect, can make the utilization ratio of phosphorus in the feed improve 60%, and ight soil phosphorus excretion reduces 40%, also can reduce the anti-oxidant action of phytate phosphorus.Therefore add phytase in the feed, can reduce the inorganic phosphate add-on, significant to improving the livestock industry productivity effect and reducing phytate phosphorus to the pollution of environment.
Though phytase has good feeding effect (Ware J.H.et al., US PatentNo.3297548,1967; Nelson T.S.et al., J.Nutrition 101:1289-1294,1971; Nelson T.S.et al., Poult Sci.47:1842-1848,1968), but up to the present it also is not used widely on fodder industry, its basic reason is, the expression amount of phytase is too low in natural microbial, thereby be difficult to a large amount of cheap phytase products that obtain, can not satisfy requirement (the Han Y.W. of feed industrial development, Animal Feed Sci.Technol., 24:345-350,1989). along with biotechnology, development of high-tech such as genetically engineered, people recognize by engineered means, utilize bio-reactor to efficiently express phytase gene, be expected to reach and increase substantially phytase output, the purpose that reduces production costs (Conneely O.M., Biotechnologyin the Feed Industry, T.P.Lyons (Ed), Alltech Technical Publications.Nicholasville, K Y.57-66,1992).
Several microbe-derived phytase genes have obtained separation, as yeast Saccaromyces cerevisiae (Bajwa W., Nucleic Acids Res.12:7721-7739,1984), Aspergillus niger (MacRae W.D., Gene, 71:339-348,1988; Yao Bin, Journal of Agricultural Biotechnology, Vol.6, No.1,1-6,1998), A.terreus and Myceliophthorathermophila (van Loon, Patent NO.EP 0684313A2,1995), A.niger (ficuum) (van Hartimgsveldt et al., Gene, 127:87-94,1993; Ehrlich K.C., Biocem.Biophys.Res.Comm.195:53-57,1993), A.niger var.awamori (Piddington C.S.Gene, 133:55-62,1993) etc.
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 niger 963 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, 20 (1): 78～84,2004).
The highest phytase of the specific activity that is separated at present is for deriving from colibacillary phytase APPA (Golovan S.Can J Microbiol, 46:59～71,2000).Its specific activity can reach 3,000, about 000IU/mg.
Summary of the invention
The objective of the invention is by making up the method for chitling road microorganism total DNA genomic library, directly screen high specific activity phytase gene, further, utilize bio-reactor pichia spp (Pichia pastoris) to efficiently express this gene, make phytase be able to cheap production.
Another object of the present invention provides a kind of dna molecular of the phytase of the present invention of encoding.Described dna molecular has the nucleotide sequence of Fig. 2 (SEQ ID NO.1).
A further object of the present invention provides the method that phytase gene of the present invention efficiently expresses in expression system.
The inventor screens high specific activity phytase gene from chitling road microorganism total DNA.The phytase of the phytase gene that screens (name and be PHYQ) coding, its specific activity is up to 4.1 * 10
6IU/mg albumen, the phytase APPA (derive from intestinal bacteria) the highest with the specific activity of present report compares, and specific activity is high by 37%.Long this full length gene 1299bp of this gene phyQ, 432 amino acid of encoding, compare with the phytase of report, what homology was the highest is to derive from colibacillary phytase APPA, but nucleotide sequence homology only is 67.7%, amino acid sequence homology is 50.3% only, is a kind of new phytase gene, has the described nucleotide sequence as Fig. 2 (SEQ ID NO.2).
The present invention also provides the phytase with the aminoacid sequence shown in Fig. 3 (SEQ ID NO.2) to have.
The invention provides the method that the phyQ gene efficiently expresses in expression system.Comprise that phytase is expressed in the screening of genetic transformation, recon of structure, the recipient bacterium of a whole set of recombinant expression vector and molecular assay method and the recon, wherein the present invention adopts pichia spp (P.pastoris) as phyQ expression of gene acceptor, and it is laid a good foundation for utilizing recombination yeast large-scale industrialization, low-cost fermentative production phytase.
Above-mentioned phyQ gene is in the method that expression system efficiently expresses, and other operable eukaryotic expression system is that insect expression system such as silkworm-baculovirus expression system, mould expression system such as aspergillus niger are expressed and plant expression system such as corn, soybean etc.
Above-mentioned phyQ gene can use intestinal bacteria as the carrier of cloning and expressing this phytase gene in the method that expression system efficiently expresses.
The present invention also provides and has contained described phyQ expression carrier.
The invention provides the expression vector pPIC9-phyQ of phyQ.
Concrete technical scheme provided by the invention is as follows:
In the chitling road extraction of microorganism total DNA and purifying with reference to from soil, extract, method (Zhang Furui etc., microorganism journal, 2003, the 2:276-282 of the total DNA of purifying; Ogram A etc., J Microb.Methods, 1987,7:57-66).
2. (Maniatis T., et al.Molecular cloning.New York:Cold Spring harbor laboratory, 1982) are cloned in gene clone according to a conventional method.The total DNA enzyme that extracts is cut rear clone to 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 phytase is carried out property testing to the phytase of expression of recombinant e. coli, filters out the encoding gene of the high phytase of specific activity, 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.
The invention provides phytase and encoding gene thereof with high specific acitivity.By genetic engineering means phytase gene is expressed efficiently in recombinant bacterial strain, make phytase cheaply on a large scale to produce, and be widely used in practice.Bring huge economic benefit.
Description of drawings
The physical map of Fig. 1 intestinal bacteria recombinant vectors pWY
Fig. 2 phyQ gene DNA sequence
The phyQ amino acid sequence coded that Fig. 3 derives
The physical map of Fig. 4 recombinant yeast expression vector pPIC9-phyQ
The fermentation diagram of Fig. 5 Expressing Recombinant Phytase in the 5L fermentor tank; Embodiment
Embodiment
Experiment condition
1. bacterial strain and carrier coli strain E.coli DH5a, plasmid pET-22b (+) etc. are available from Promega company, and yeast strain Pichia pastoris GS115 (His-Mut+), plasmid pPIC9 are so kind as to give (Invitrogen company product) by Canadian Alberta doctor D.Luo of university.
2. enzyme and test kit restriction enzyme, ligase enzyme are Boehringer company product.
3. biochemical reagents 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
The extraction and the purifying procedure of this description of test chitling road microorganism total DNA.
Get chitling liquid 0.5mL, add 1mL DNA extraction liquid (100mmol/L Tris-HCl, 100mmol/L EDTA, the 100mmol/L sodium phosphate, 1.5mol/L NaCl, 1%CTAB, pH9.0), mixing adds 100mL Proteinase K (10mg/mL), and 37 ℃ are shaken 1hr, the SDS that adds 1.5mL 20%, 65 ℃ of water-bath 2hr, the centrifugal 10min of 6000rpm collects supernatant liquor.Precipitation part adds the 20%SDS of 450mL extracting solution and 50mL again, 65 ℃ of water-bath 2hr, the centrifugal 10min of 6000rpm, collect supernatant liquor and with last time supernatant liquor merge.Add isopyknic chloroform extracting in the supernatant liquor, supernatant liquor precipitates with 0.6 times of volume.Precipitation is dissolved in the 50mL water standby.
The DNA purifying adopts the method for electrophoresis purifying.Total DNA of extracting at 0.5% agarose gel electrophoresis, is cut the DNA band, reclaim with QXII DNA purification kit, recovery method is seen the test kit operation instruction.
Experiment two
Clone's program of this description of test dna molecular.
The dna molecular that obtains in the experiment one, partially digested with Sau3A, make the disconnected 2.0～8.0kb that concentrates on of enzyme section, electrophoresis reclaims, be connected in 15 ℃ with the carrier pET-22b (+) that cuts through the BamHI enzyme and spend the night, transformed into escherichia coli E.coli DH5a selects positive recombinant behind the coated plate on the LB substratum, the extraction plasmid carries out enzyme and cuts evaluation, obtains recon.Contain section of DNA molecule in each recon.
Experiment three
The suddenly change screening procedure of phytase of this description of test.
5000 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.Screen 8 recons and can produce phytase activity, further be determined by experiment the specific activity of phytase.After 8 recons are cultivated, collect somatic cells, carry out ultrasonic disruption, the cytoclasis liquid that contains phytase concentrates with 85% ammonium sulphate precipitation, purifying KTA FPLC fast liquid chromatography instrument (Pharmacia company) purifying.The concentrated solution that contains enzyme is at first used the desalination of HiPrep_26/10_Desalting post, damping fluid is 20mmol/L HAc-NaAc (pH5.0), flow velocity is 5mL/min, collect elution peak, then separate with ion exchange column Hitrip_SP_Sepharose_XL (5mL), the A pump is 15mmol/L HAc-NaAc (pH5.0), the B pump is 1mol/L NaCl, 15mmol/LHAc-NaAc (pH5.0) high-salt buffer, flow velocity is 1.5mL/min, high-salt buffer is from 10 post beds of 0～100% gradient elution, the fraction collection elution peak, further use gel column Superdex_75_HR_10/30 purifying by the elution peak after the enzyme assay, damping fluid is similarly 15mmol/L HAc-NaAc (pH5.0), and flow velocity is 0.8mL/min, collects elution peak and obtains pure protein.The phytase of purifying is carried out the mensuration of enzymic activity, calculate the ratio of enzymic activity and zymoprotein, obtain the specific activity of enzyme.In 8 recons, what the phytase specific activity of generation was the highest reaches 4.1 * 10
6IU/mg albumen is higher than the used phytase of present report, than the highest phytase APPA also high 37% of the specific activity of present discovery.This recombinant plasmid is named and is that pWY, this phytase name and is PHYQ.
Experiment four
The sequence of this description of test phytase PHYQ.
Phytase gene on the recombinant plasmid pWY is carried out sequencing, and Fig. 3 is its nucleotide sequence, and Fig. 4 is its aminoacid sequence.The result shows, long this full length gene 1299bp of this gene phyQ, 432 amino acid of encoding, compare with the phytase of report, what homology was the highest is to derive from colibacillary phytase APPA, but nucleotide sequence homology only is 67.7%, and amino acid sequence homology is 50.3% only, is a kind of new phytase gene.
Experiment five
The construction procedures of this description of test phyQ on Yeast expression carrier.
The plasmid that is used to make up Yeast expression carrier is pPIC9 (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: 5 ' end and 3 ' end according to phytase gene phyQ design a pair of primer, on primer, add the EcoRI site (p1:5 ' TTGAATTCATGATACCCACCGGAAACCC; P2:5 ' GCGAATTCTTACAGATCCCACGCCAAAAT), with recombinant plasmid pWY is template, pcr amplification goes out the phytase gene of 1.3Kb, after cutting with the EcoRI enzyme, electrophoresis reclaims approximately, they are inserted into the EcoRI site on the carrier pPIC9, the direction of insertion by sequencing judgement phyQ has obtained the correct recombinant expression vector that is used for yeast conversion one pPIC9-phyQ (Fig. 4) of direction of insertion again.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 pPIC9-phyQ shocks by electricity after the BglII 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 BglII digested plasmid pPIC9-phyQ, 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 5 μ gDNA 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+ recon with aseptic toothpick from transforming dull and stereotyped RDB, at first be inoculated on the MM solid medium, inoculate on the MD solid medium, so picking his+ recon was cultivated 2 days for 30 ℃.Screening is normal in growth on the MD flat board but clone's (his+mut-) that have any to grow or do not grow fully on the MM flat board is 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.The his+mut-transformant is at first cultivated in the BMGY substratum, treated that it grows to state of saturation, 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 42 strain Expressing Recombinant Phytase from 350 strain recombination yeasts, and wherein 1 the highest strain recon of expression amount is named the phyQ-66 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 12mL PTM1 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 accumulation volume of the phytase of expressing is once measured in every 12h sampling in inducing process.
The result shows that the phytase of expression accumulates with the increase of induction time, peaks when inducing 120 hours, and enzymic activity reaches 5.5 * 10
6U/mL fermented liquid (Fig. 5).The result proves that phytase gene has not only obtained expression, effectively secretion, and the phytase of expressing has normal biologic activity.
Application?Project
-------------------
＜110〉Applicant: Liu Daqing
＜120〉Title: high specific activity phytase gene and efficiently expressing
<210>1
<211>Length：1299
SequenceName：phyQ
<212>Type：DNA
＜213〉the unknown
Sequence
--------
<400>PreSequenceString：SEQ?ID?No.1
atgataccca?ccggaaaccc?acagctatct?catgtgactc?catttaccgc?gcaatctgca 60
tttgcacagc?gagaacctat?gctcatgctc?gtaaatatgg?tgaatctcag?tggacatggg 120
gcgcgtatcc?caactaagga?tacgcaacgc?atgcaggacc?tcacccgaga?cgcatgtcaa 180
acccgcccgg?taaacgtggg?aacgctggcg?ccgtgcggtg?gtttcctaat?ccgctactcc 240
ggtgattacg?gccgcgaacg?tctccaagcc?atgggaaaag?tggcgaacca?gggctatccg 300
cagccgggtc?aggtcttgat?ttctgcaaat?gttcccgagc?gttcccgtag?cacaggaaaa 360
gcctgcgccg?ttgggctccg?accttactgt?gcagggaccg?tccctaccca?cgaagaagag 420
tccagtcggg?atccggcgtt?taatgaacta?acaactgatg?tttcgcaact?taataacaag 480
aacgtgcccg?acgcgggggt?cagcagatca?gtagggtcaa?gggctgactc?caccgaacat 540
aagcaaacgg?tatttcggca?actttcccgg?tcgcttaatt?ggccgctttc?attcttgtat 600
cttacccgtg?ccaaactaga?cgcgagctgt?ttattacggc?acccattgtt?agcggaactc 660
ccggtgattt?ccgaccccgt?ctcaggaact?agtgccctat?acctgacatc?ggtgcgtacg 720
acgatacggc?tcgggcaaca?ttcacagata?atcccggagc?aaaagtgggt?ggggatctgg 780
gatttacata?cgtggagggc?ctccctaaga?gcgcatggag?cgcaaagaga?ttttttactt 840
cgccggccac?cggtcccccg?cgtccgcata?gccccggggt?tagacccgat?caccacagct 900
ttgacttccc?tttcaggcca?aactcaggag?gaagctgtgg?gcctacccgc?ctcagttttg 960
tttataatcg?gacttgatat?ttatctgccc?aatcttaccg?gcggggtgga?attcaacgcg 1020
acgcgcgccg?gagagcctta?taatttgccg?cccggtgcgc?aactgaaatt?tgggggctgc 1080
ggtcttctaa?atgatatttg?ccagaaaatt?caggcgccgc?tccgcttcat?aacttttatg 1140
caggcccgtg?gccaaacgaa?gctagcattt?aaggggcccg?gcggagaatt?gaatctgacc 1200
aaagcaggcc?ctgaagcgcg?aaattttcag?ggttagtgtt?ccccggccgg?ttatgcgccg 1260
ctcgcgcatg?ttgcgaacat?tttggcgtgg?gatctgtaa 1299
<210>2
<211>Length：1211
SequenceName：PHYQ
<212>Type：PRT
＜213〉the unknown
<400>PreSequenceString：SEQ?ID?No.2
MIPTGNPQLS?HVTPFTAQSA?FAQREPMLML?VNMVNLSGHG?ARIPTKDTQR?MQDLTRDACQ 60
TRPVNVGTLA?PCGGFLIRYS?GDYGRERLQA?MGKVANQGYP?QPGQVLISAN?VPERSRSTGK 120
ACAVGLRPYC?AGTVPTHEEE?SSRDPAFNEL?TTDVSQLNNK?NVPDAGVSRS?VGSRADSTEH 180
KQTVFRQLSR?SLNWPLSFLY?LTRAKLDASC?LLRHPLLAEL?PVISDPVSGT?SALYLTSVRT 240
TIRLGQHSQI?IPEQKWVGIW?DLHTWRASLR?AHGAQRDFLL?RRPPVPRVRI?APGLDPITTA 300
LTSLSGQTQE?EAVGLPASVL?FIIGLDIYLP?NLTGGVEFNA?TRAGEPYNLP?PGAQLKFGGC 360
GLLNDICQKI?QAPLRFITFM?QARGQTKLAF?KGPGGELNLT?KAGPEARNFQ?GCSPAGYAPL 420
AHVANILAWD?L 431
Claims (6)
1, a kind of new phytase is characterized in that this zymoprotein aminoacid sequence is shown in SEQ IDNO.2.
2, the gene of coding phytase as claimed in claim 1, the nucleotide sequence of encoding gene that it is characterized in that this phytase is shown in SEQ ID NO.1.
3, efficiently express the method for gene as claimed in claim 2, comprise the expression of phytase gene in the screening of genetic transformation, recon of structure, the recipient bacterium of a whole set of recombinant expression vector and evaluation and the recon, it is characterized in that in the step of Expressing Recombinant Phytase gene, utilizing the bio-reactor of efficient expression system pichia spp as the Expressing Recombinant Phytase gene.
4, efficiently express the method for gene as claimed in claim 2, comprise the expression of phytase gene in the screening of genetic transformation, recon of structure, the recipient bacterium of a whole set of recombinant expression vector and evaluation and the recon, it is characterized in that described bio-reactor is insect expression system, mould expression system or the plant expression system of baculovirus.
5, comprise the described expression carrier of claim 2.
6, comprise the described expression carrier pPIC9-phyQ of claim 2, it is characterized in that this carrier is to insert the gene constructed of claim 2 by the EcoRI site at expression vector pPIC.
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| CN101935617A (en) * | 2010-07-13 | 2011-01-05 | 湖北大学 | Heat-resisting phytase Pichia pastoris engineering bacterial strain and production method of heat-resisting phytase |
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| CN101368175B (en) * | 2007-08-16 | 2010-12-29 | 中国农业科学院饲料研究所 | Novel phytase, encoding gene, cell and feedstuff additive including the enzyme |
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| CN1126243A (en) * | 1994-04-25 | 1996-07-10 | 弗·哈夫曼-拉罗切有限公司 | Heat tolerant phytases |
| WO1997038096A1 (en) * | 1996-04-05 | 1997-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Novel phytase and gene encoding said phytase |
| WO1997048812A2 (en) * | 1996-06-14 | 1997-12-24 | Her Majesty The Queen In Right Of Canada, Represented By The Department Of Agriculture And Agri-Food Canada | Dna sequences encoding phytases of ruminal microorganisms |
| WO2001090333A2 (en) * | 2000-05-25 | 2001-11-29 | Diversa Corporation | Recombinant bacterial phytases and uses thereof |
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| CN1126243A (en) * | 1994-04-25 | 1996-07-10 | 弗·哈夫曼-拉罗切有限公司 | Heat tolerant phytases |
| WO1997038096A1 (en) * | 1996-04-05 | 1997-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Novel phytase and gene encoding said phytase |
| WO1997048812A2 (en) * | 1996-06-14 | 1997-12-24 | Her Majesty The Queen In Right Of Canada, Represented By The Department Of Agriculture And Agri-Food Canada | Dna sequences encoding phytases of ruminal microorganisms |
| WO2001090333A2 (en) * | 2000-05-25 | 2001-11-29 | Diversa Corporation | Recombinant bacterial phytases and uses thereof |
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| 植酸酶的基因工程 陈艳 等，黑龙江畜牧兽医，第8期 2003 * |
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| CN101935617A (en) * | 2010-07-13 | 2011-01-05 | 湖北大学 | Heat-resisting phytase Pichia pastoris engineering bacterial strain and production method of heat-resisting phytase |
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| CN1775948A (en) | 2006-05-24 |
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