CN1597961A

CN1597961A - Production for phytase with high living rate high temp. resisting by pichia

Info

Publication number: CN1597961A
Application number: CN 03151019
Authority: CN
Inventors: 姚泉洪; 彭日荷; 熊爱生; 吴伟; 刘承训
Original assignee: XINGHU BIOTECH CO Ltd ZHAOQING CITY GUANGDONG PROV
Current assignee: XINGHU BIOTECH CO Ltd ZHAOQING CITY GUANGDONG PROV
Priority date: 2003-09-17
Filing date: 2003-09-17
Publication date: 2005-03-23
Anticipated expiration: 2023-09-17
Also published as: CN1302112C

Abstract

The invention discloses utilization of pichia pastoris to produce high-specific activity high temperature- resistant phytase, adopts many isogenous phytase genes, using DNA enzyme to make partial enzyme mediation, recovering all the DNA degraded fragments, in vitro rearranging DNA molecules, making enzyme cutting on all the rearranged DNA molecules, then composing them in an expression carrier, electrically shocking them in Escherichia coli, screening mutant phytase gene by phytase expression and activity, and finally obtaining high-specific activity high temperature-resistant phytase gene Phy XH, which is constructed into phytase gene expression carrier pPhy XH, electrically shocking and transferring in pichia pastoris, and selecting recombinant high-phytase expression pichia pastoris.

Description

Utilize pichia spp to produce height ratio high temperature resistant phytase alive

Technical field

The invention belongs to the microbiological genetic engineering field, specifically utilize a plurality of homology phytase gene fragments to carry out vitro recombination, directed screening obtains the phytase gene than height alive and withstand high temperatures, and this gene can efficiently express in pichia spp.

Background technology

Phytase extensively is present in plant, animal and the microorganism.There is very big-difference in the phytase that different species produce in nature than work, optimal reaction pH, thermotolerance etc.Many microorganisms can both produce the phytase that height ratio is lived.Nineteen sixty-eight Shien etc. investigates discovery to 2000 bacterial strains from 68 soil samples, have 21 strains can produce phytase in all 22 strain melanomyces.The phytase of first separated purifying derives from aspergillus Aspergillus terreus NO.9A-1, and its optimal pH is 4.5, and optimal reactive temperature is 70 ℃, and pH is in 1.2～9.0 scopes, and this enzyme all energy stable maintenance is necessarily active.After this, from tens kinds of microorganisms, separate successively and obtain phytase, the phytase phyA that wherein derives from A.ficcum NR-RL3135 (A.nigervar.awamori) has the high enzyme activity under acidic conditions, be considered to the feeding phytic acid plum of at present tool application prospect.Phytase phyA is a kind of glycosylated protein, and molecular weight is 85KD.The enzyme reaction optimal pH is 2.5 and 5.5, and optimal reactive temperature is 55 ℃.Under 37 ℃, the condition of pH2.5, be that the Km value of substrate is 50mmol with the phytic acid, Ca ²⁺, Fe ²⁺Enzymic activity there is not influence, Mn ²⁺, Co ²⁺Activation is arranged, can make enzymic activity improve 30% and 13% respectively.Cu ²⁺, Zn ²⁺, Fe ²⁺, Cu ⁺Enzymic activity there is restraining effect, wherein Cu ²⁺, Zn ²⁺For noncompetitive suppresses, Fe ²⁺, Cu ⁺Be competitive inhibition.There are inhibiting inhibitor such as L (+)-tartrate that it is not but had restraining effect to acid phosphatase.

Cereal class, pulse family class and oil crops are people's food and the main raw material in the animal-feed.Though contain a large amount of phosphorus in these raw materials, wherein 50%-70% is that form with phytate phosphorus (inositol hexaphosphate) exists (Salunkhe 1982, the food research progress).Monogastric animal lacks and decomposes the necessary enzyme of phytate phosphorus, and the utilization ratio of phosphorus is very low.Phytase can be hydrolyzed into phytate phosphorus inositol and phosphoric acid, adds the utilization ratio that phytase can improve phosphorus in the plant feed in feed.

The immediate cause of exploitation phytase derives from the phosphorus pollution that phytic acid brings.On the one hand, the phosphorus of chelating can't absorb in the phytic acid, then is on the other hand to add a large amount of inorganic phosphorus in feed and food, causes waste of phosphorus source and environmental pollution (Cromwell 1991, and biotechnology is made progress in Application in Food Industry).Along with the development of aquaculture, the excretion of inorganic phosphorus rises significantly, and phosphorus pollutes and spreads all over river, mountains and rivers and Plain cultivated land, directly threatens mankind itself's existence.From the mid-80, Europe just is devoted to seek in the scheme (CouncilDirective91/676/EEC) of comprehensive solution inorganic phosphorus pollution, highlights the phosphorus excretion of restriction aquaculture.Phytase is in the additive of all uses at present, has the zymin of direct, the most remarkable environmental protection social benefit.Because phosphorus is the fundamental element in the growth of animal process, in order to remedy the consumption of phosphorus in the metabolism, usually need add inorganic phosphorus (Common1989, Nature Journal) in food and feed.The phosphorus that phytase closes huge legendary turtle in the phytic acid discharges, thereby has reduced the usage quantity of inorganic phosphorus such as secondary calcium phosphate in the feed, and the reduction amplitude reaches 50%-70%.In China, a lot of culture zones can be reduced the consumption of secondary calcium phosphate more than 70%, the breeding layer chicken district of assorted dregs of rice large usage quantity, even can the replacing whole secondary calcium phosphate.Use the feed of phytase, to the corresponding minimizing of environment excretory phosphorus more than 30% (Nelson 1968, the herding science).Use phytase in reducing feed in the inorganic phosphorus addition, alleviate significantly even stopped fluorine and other heavy metal poisoning problem that secondary calcium phosphate causes fully.Use the phytase cost of every year aspect feed can reduce more than 9,200 ten thousand yuan.In the environment of depending on for existence, phytase is eliminated phosphorus easily from the source of polluting and is polluted, and the later stage of using than the routine method of administering is economical and effective more.Can expect that phytase will produce huge promoter action to the Sustainable development of whole industry with remarkable economic efficiency and social benefit.

Up-to-date result of study has also been found the potential nutritive value of phytase.Phytic acid by with the chelating of metal ion, reduce the absorption of monogastric animal to nutrition, phytase can improve animal to the digesting and assimilating of phosphorus and calcium, Jongbloed in set up in 1993 one about phytase vigor and feed in phosphatic equivalent relation formula.Interpolation 500IU phytase is equivalent to 1g and comes from the phosphorus of mono-calcium phosphate or the phosphorus that 1.1g comes from dicalcium phosphate dihydrate in phosphatic feed; Report such as Ofier etc. (1992), Mroz (1994), phytase can improve the ileal digestibility of pig fowl to amino acid and nitrogen.Phytase increases the receptivity (Wyss1999, applied environment microorganism science) of animal to protein and some metal ions by reducing the anti-oxidant action of phytate, improves proteinic digestive utilization ratio in the feed.Use phytase, strengthened the usage ratio of unconventional raw material resources, can save protein feed in a large number.Use phytase can directly reduce the raw materials cost of feed, thereby improve the price competitiveness of product.

Produce the phytase of upward employing at present all from aspergillus niger (Aspergillus niger).Phytase in the aspergillus niger (PhyA) has than the advantages such as height, Stability Analysis of Structures of living.In the phytase early stage of development, all adopt the original strain fermentative production, yet the original strain expression amount is low, production cost is higher, can obtain the bacterial strain that yield of enzyme improves by chemomorphosis or physical mutagenesis method, yet mutagenesis causes the loss of other function of bacterial strain easily, in order to obtain goal gene high expression level and the impregnable bacterial strain of other function, need the long-term observation screening, required time is very long, and in addition, mutagenic strain is degenerated easily.Utilize molecule family evolvement method, existing gene is optimized combination, the poly-a lot of functions that can obtain to encode are in the new protein gene of one.

Summary of the invention

The present invention adopts dna molecular vitro recombination method, and the different a plurality of phytase gene fragments hybridization in origin source form the phytase genes storehouse, utilize colibacillus expression plasmid, seek the phytase gene of a kind of height ratio work and withstand high temperatures from the phytase gene storehouse.Another purpose provides the method for yeast expression high-specific-activity phytase.

The phytase gene that utilizes among the present invention contains four different phytase genes, one is the high temperature resistant phytase gene that obtains by the gene recombination screening, it two is by gene recombination screening high specific activity phytase gene, it three is from the phytase gene in the Fructus Fici aspergillus, it four is the intestinal bacteria phytase gene, and they all are made up of yeast preference password by chemosynthesis.

In order to improve the specific activity of phytase, at first with synthetic Fructus Fici phytase gene of PCR and intestinal bacteria phytase gene.Each phytase gene designs and synthesizes 20 Oligonucleolide primers, length is the 70-90 base, connect between the primer by the 20-30bp overlap, the Tm value adds reaction system for 60-66 with all primers, carry out pcr amplification, carry out 35 circulations altogether, synthetic Fructus Fici phytase gene and the intestinal bacteria phytase gene of forming by yeast preference password.The present invention utilizes continuous extension PCR to synthesize the Fructus Fici aspergillus niger phytase gene, and primer length is 70-90bp, is connected by the 20-30bp overlap between 0 Oligonucleolide primers of Synthetic 2, primer and primer, and the Tm value is 60-66.All primers are added reaction system, and middle primer amount is 10-20ng, and the primer amount of both sides is 100-200ng, and the pcr amplification condition is 94 ℃, 30s; 65 ℃, 30s; 72 ℃, 2min.Carry out 35 circulations altogether.

The present invention utilizes continuous extension PCR to synthesize the intestinal bacteria phytase gene.Primer length is 70-90bp, is connected by the 20-30bp overlap between 0 Oligonucleolide primers of Synthetic 2, primer and primer, and the Tm value is 60-66.All primers are added reaction system, and middle primer amount is 10-20ng, and the primer amount of both sides is 100-200ng.The PCR reaction system is 100 μ L.The pcr amplification condition is 94 ℃, 30s; 65 ℃, 30s; 72 ℃, 2min.Carry out 35 circulations altogether.

The present invention adopts that gene is synthetic, gene cluster molecule vitro recombination screening height ratio is lived high temperature resistant phytase gene prepares height ratio high temperature resistant phytase alive by steps such as vector construction, conversions of yeast electric shock, high reactivity bacterial strain screening, high density fermentations.

The present invention is a template with high temperature resistant phytase gene, and FphyZ1, FphyF1 are the high temperature resistant phytase gene of primer amplification, FphyZ1:(5 '-TTGGATCCTCCAAATCCTGCGACACCGTTGACT TG-3 '); FphyF1:(5 '-CGAGCTCTTAGGAGAAGCATTCACCCCAGTTACCA C-3 '). with the high specific activity phytase gene is template, phyiZ1, phyiF1 is the primer amplification high specific activity phytase gene, phyiZ1:(5 '-TTGGATCCTTGGCTGTCCCAGCTTCCAGAAAC-3 '); PhyiF1:(5 '-CGAGCTCTTAAGCGAAGCATTCAGCCCAGTCAC-3 '). with the Fructus Fici aspergillus niger phytase gene is template, phyiZ1, phyiF1 is its phytase gene of primer amplification, phynZ1:(5 '-TTGCCTATTCCTGCTCAAAACACTTC-3 '); PhynF1:(5 '-GAGCTCATTATTCGGAAGGAACGAAACCGCAC-3 '). with the high specific activity phytase gene is template, phyiZ1, phyiF1 is the high temperature resistant phytase gene of primer amplification, EphyZ1:(5 '-ATGAAAGCGATCTTAATCCCATTTTTATC-3 '); EphyF 1:(5 '-CTCATTACAAACTGCACGCCGGTATGCGTGC-3 ').

The high temperature resistant phytase gene that the present invention obtains with screening, reorganization screening high specific activity phytase gene, Fructus Fici phytase gene and intestinal bacteria phytase gene are the gene that sets out, utilize the DNA enzyme to carry out the part enzymolysis, reclaim all dna degradation fragments, reset at the external dna molecular that carries out, after all are reset dna molecular enzymes and cut, be building up in the expression vector, electric shock is gone into colibacillus, express and screening active ingredients sudden change phytase gene by phytase, finally obtain height ratio and live and resistant to elevated temperatures phytase gene PhyXH.New phytase aminoacid sequence and phyA2 have 97.5% homology.The amino acid no of PhyXH and PhyA2 is identical, but has 12 amino acid that change has taken place.Comprising: 11T＞V, 41K＞Q, 53Q＞K, 91K＞E71S＞T, 112E＞F, 157G＞A, 191L＞V etc.

GAGACTTCTCATTTGTGGGGTCAATACGCTCCATTCTTCTCTTTGGCTAACAAGTCTGCTATCTCTCCAGAT

E T S H L W G Q Y A P F F S L A N Q S A I S P D

GTTCCAGCTGGTTGTAAAGTTACTTTCGCTCAAGTTTTGTCCAGACATGGTGCTAGATACCCAACTGATACT

V P A G C K V T F A Q V L S R H G A R Y P T D T

AAAGGTAAGAAATATTCTGCTTTGATTGAGGAGATTCAACAAAACGCTACTACTTTTGAGGAGAAATACGCT

K G K K Y S A L I E E I Q Q N A T T F E E K Y A

TTTTTGAAAACTTACAACTATTCTTTGGGTGCTGATGATTTGACTCCATTTGGTGAACAAGAATTGGTTAAT

F L K T Y N Y S L G A D D L T P F G E Q E L V N

TCTGGTGTTAAGTTTTACCAAAGATACGAATCTTTGACTAGAAATATTGTTCCATTTATTAGATCCTCTGGT

S G V K F Y Q R Y E S L T R N I V P F I R S S G

TCTTCCAGAGTTATTGCTTCTGGTAATAAATTCATTGAAGCTTATCAATCTACTAAATTGAAAGATCCAAGA

S S R V I A S G N K F I E A Y Q S T K L K D P R

GCCCAACCAGGTCACTCTTCTCCAAAAATTGATGTTGTTATTTCTGAGGCTTCTACTTCTAATAATACTGTG

A Q P G H S S P K I D V V I S E A S T S N N T V

GACCCAGGTACTTGTACTGTTTCTGAAGATAACGAATTGGCTGATGATTTCGAAGCTAATTTTACTGCTACT

D P G T C T V S E D N E L A D D F E A N F T A T

TTTGTTCCATCTATTAGACAATCTTTGGAAAATAACTTGTCTGGTGTTGCTTTGACTGATACTGAAGTTACT

F V P S I R Q S L E N N L S G V A L T D T E V T

TACTTGATGGACTTGTGTTCTTTTGATACTATCTCTACTTCTACTGTTGATACTAAATTGTCTCCATTTTGT

Y L M D L C S F D T I S T S T V D T K L S P F C

GATTTGTTTACTCATGAAAAATGGATTAATTATGATTATTTGCAATCTTTGAACAAGTACTACGGTCATGGT

D L F T H E K W I N Y D Y L Q S L N K Y Y G H G

GCTGGTAATCCATTGGGTCCAACTCAAGGTGTTTGTTACGCTAATGAATTGATTTCCAGATTGACTCATTCT

A G N P L G P T Q G V C Y A N E L I S R L T H S

CCAGTTCATGATTACACTTCTTCTAATCATATTTTGGATTCTTCTCAAGATACTTTTCCATTGAACTCTACT

P V H D Y T S S N H I L D S S Q D T F P L N S T

TTGTACGCTGATTTCTCTCTTAACAATGGTATTATCTCTATTTTGTTTGCTTGGGGTTTGAACAAAGGTACT

L Y A D F S L N N G I I S I L F A W G L N K G T

AAACCATTGTCTTCTACTACTGCTGAAAATATCACTCAAACTGATGGTTTTTCTTCTGCTTGGACTGTTCCA

K P L S S T T A E N I T Q T D G F S S A W T V P

TTCGCTTCCAGAATGTACGTTGAAATGATGCAATGTCAATCTGAACAAGAACCATTGGTTAGAGTTTTGGTT

F A S R M Y V E M M Q C Q S E Q E P L V R V L V

AATGATAGAGTTGTTCCATTGCATGGTTGTCCAGTTGATGCTTTGGGTAGATGTACTAGAGATTCTTTTGTT

N D R V V P L H G C P V D A L G R C T R D S F V

AGAGGTTTGTCTTTTGCTAGATCCGGTGGTGATTGGGCTGAATGCTTTGCTAAGGACGAATTGTAA

R G L S F A R S G G D W A E C F A K D E L -

Go out primer by the two ends sequences Design of screening the height ratio high temperature resistant phytase gene alive that obtains, add XhoI point of contact and signal attitude sequence at the gene 5 ' end, primer is PhyXH1, add Not I point of contact at gene 3 ' end, primer is PhyXH2, behind the amplified fragments clone, XhoI and Not I double digestion, the directed pPIC9 carrier that inserts, be built into the Yeast expression carrier (Fig. 1) of PhyXH, the carrier pPhyXH 9.4Kb electric shock that contains phytase gene expression changes the pichia yeast bacterium over to, and this yeast can not utilize methyl alcohol to make carbon source for growth, only contains the phytase gene of a copy in this yeast.Select phytase high expression level recombination yeast P.Pastoris bacterial classification, carry out high density fermentation, the phytase of expression has higher ratio lives, and tolerates higher temperature.

Description of drawings

Fig. 1 is the Yeast expression carrier figure that is built into PHYXH.

Fig. 2 is the relation of recombinant bacterial strain high density fermentation time and phytase expression amount.

Fig. 3 represents the influence of pH value to the phytase vigor.

Beneficial effect of the present invention

Resetting the phytase gene that obtains by preference password transformation and dna molecular has the following advantages:

1 gene can efficiently express in yeast, and expression amount is higher than 1.8 grams per liters.

2 gene expression products have very high enzyme than living, and the ratio work of new phytase gene PhyXH is higher 50 times than original PHYA2.

3 new phytase PhyXH also can glycosylation in yeast.

4 new phytase PhyXH have higher tolerance to temperature, and optimal reaction pH value is consistent with original gene, is pH2.5-5.5.

Embodiment

Embodiment 1: the chemosynthesis of phytase gene

1.1 utilize continuous extension PCR to synthesize the Fructus Fici aspergillus niger phytase gene.Primer length is 70-90bp, is connected by the 20-30bp overlap between 0 Oligonucleolide primers of Synthetic 2, primer and primer, and the Tm value is 60-66.All primers are added reaction system, and middle primer amount is 10-20ng, and the primer amount of both sides is 100-200ng.The PCR reaction system is 100 μ L.The pcr amplification condition is 94 ℃, 30s; 65 ℃, 30s; 72 ℃, 2min.Carry out 35 circulations altogether.

1.2 utilize continuous extension PCR to synthesize the intestinal bacteria phytase gene.Primer length is 70-90bp, is connected by the 20-30bp overlap between 0 Oligonucleolide primers of Synthetic 2, primer and primer, and the Tm value is 60-66.All primers are added reaction system, and middle primer amount is 10-20ng, and the primer amount of both sides is 100-200ng.The PCR reaction system is 100 μ L.The pcr amplification condition is 94 ℃, 30s; 65 ℃, 30s; 72 ℃, 2min.Carry out 35 circulations altogether.

Embodiment 2: the dna molecular of phytase gene is reset (DNA Shuffling)

2.1 acid phytase gene of pcr amplification and recovery

With high temperature resistant phytase gene is template, and FphyZ1, FphyF1 are the high temperature resistant phytase gene of primer amplification, FphyZ1:(5 '-TTGGATCCTCCAAATCCTGCGACACCGTTGACTTG-3 '); FphyF1:(5 '-CGAGCTCTTAGGAGAAGCATTCACCCCAGTTACCAC-3 ').

With the high specific activity phytase gene is template, and phyiZ1, phyiF1 are the primer amplification high specific activity phytase gene, phyiZ1:(5 '-TTGGATCCTTGGCTGTCCCAGCTTCCAGAAAC-3 '); PhyiF1:(5 '-CGAGCTCTTAAGCGAAGCATTCAGCCCAGTCAC-3 ').

With the Fructus Fici aspergillus niger phytase gene is template, and phyiZ1, phyiF1 are its phytase gene of primer amplification, phynZ1:(5 '-TTGCCTATTCCTGCTCAAAACACTTC-3 '); PhynF1:(5 '-GAGCTCATTATTCGGAAGGAACGAAACCGCAC-3 ').

With the high specific activity phytase gene is template, and phyiZ1, phyiF1 are the high temperature resistant phytase gene of primer amplification, EphyZ1:(5 '-ATGAAAGCGATCTTAATCCCATTTTTATC-3 '); EphyF1:(5 '-CTCATTACAAACTGCACGCCGGTATGCGTGC-3 ').

Reaction conditions is: 94 ℃ of pre-sex change of 10min, 94 ℃ of sex change 30s, 72 ℃ of 1.5min that anneal and extend, totally 30 circulations, 1%Agrose electrophoresis, saturating gene fragment of inhaling bag method recovery 1.4kp.

2.2DNase I degradation of dna and recovery small segment

Reclaim the phytase gene fragment with DNase I damping fluid (50mmol/L Tris-ClpH7.4+1mmol/L MgCl ₂) 100 μ l dissolving; Add 0.1U DNase I, handled 15 minutes for 25 ℃.Handled 10 minutes for 70 ℃.10% acrylamide electrophoresis, the saturating small segment of inhaling bag method recovery 10-50bp.With 10 μ l 10 * no primer PCR damping fluid (Primerless PCR Buffer) (50mmol/LKCl+10mmol/LTris-Cl pH9.0+1%Triton) dissolution precipitation.

2.3 there is not primer PCR (Primerless PCR)

Carry out the Primerless pcr amplification.Reaction system: 5 μ l small segment DNA+4 μ l 2.5mmol/LdNTPs+4.5 μ l 25mmoljm/LMgCl ₂+ Taq2U+ddH ₂O to 50 μ l; Response procedures is: 94 ℃ of 30s, 40 ℃ of 30s, 72 ℃ of 30s, totally 45 circulations), 2%Agrose electrophoresis detection pcr amplification result.2.4 primer PCR (Primer PCR) is arranged

Carry out the PrimerPCR amplified reaction.Reaction system: 5 μ lPrimerless PCR product+phyiZ10.2ng+phyiF1 0.2ng+10 * PCR Buffer 5 μ l+2.5mmol/L dNTPs 4 μ l+Taq2U+ddH ₂O to 50 μ l.Response procedures is: 94 ℃ of 30s, and 70 ℃ of 30s, 72 ℃ of 2.0min, totally 35 circulations, the 1%Agrose electrophoresis detection reclaims the 1.4kb gene fragment.

2.5 phytase screening that high reactivity is high temperature resistant

Reclaim the rearrangement phytase gene fragment of 1.4kb, behind BamH I and SacI double digestion, be built between prokaryotic expression carrier pG251 (CN1338515) promotor and the t1t2 terminator, this carrier has ampicillin resistance gene.

Electric shocking method transformed into escherichia coli bacterial strain DH5 α obtains the mutant expression library, and storage capacity reaches 10 ⁷, carry out the screening of high-specific-activity phytase.Method is as follows: utilize 100ml LB substratum (1% peptone, 0.5% yeast powder, 1% sodium-chlor) culturing bacterium spend the night to bacterial concentration be OD600=0.5-0.8.5000 revolutions per seconds of cultured bacteriums of centrifugal collection utilize the distilled water of sterilization to wash centrifugal thalline twice, and each 25ml washes once with 10% glycerine of sterilization again, utilizes 1ml 10% glycerine suspension thalline at last.With recombinant phytase gene and carrier connector utilization electric shock instrument (Bio-rad Gene pulser) (U.S. BIO-RED company) electric shock transformed into escherichia coli, add 2 μ g in the 50 μ l intestinal bacteria and connect dna fragmentation.The conversion parameter is as follows: 25KV, 200 Ω, 25 μ F.Transform connection dna fragmentation concentration and reach every microlitre 1 microgram.

Get 96 times of 1 μ l bacterium dilutions, equivalent adds 96 hole microbial culture plates (12 * 8), adds the LB nutrient solution of 150 μ l to OD in each hole ₆₀₀=0.4-0.6, taking-up 10 μ l bacterium liquid are transferred in the test tube and are cultivated from the hole of culture plate, place in same test tube with the bacterium in 8 holes of 12 holes of delegation and same row and to cultivate, totally 20 test tubes, every pipe adds the LB nutrient solution 3mL of band penbritin, cultivate after 8-12 hour, centrifugal recovery thalline, ultrasonication cell, centrifuging and taking supernatant 100 μ l, handle after 60 minutes for 100 ℃, add POTASSIUM PHYTATE sodium substrate, 37 ℃ were reacted 30 minutes, added molybdenum blue developer (amine molybdate, ferrous sulfate, the vitriol oil) colour developing.

Get in every row the highest active interface point in active the highest and every row, after 96 times of the dilutions of the bacterium in the 10 μ l culture plate interface point holes, add another piece culture plate and cultivate, every hole 150 μ l LB nutrient solutions, 10 μ l bacterium liquid carry out a new round and screen.After the 3-5 wheel screening, get in the interface point bacterium dilution 10-100 and doubly be coated on and contain on the antibiotic LB solid culture of the ammonia benzyl plate, picking list bacterium colony carries out specific activity, obtains the phytase gene phyXH that height ratio is lived.To screen the acquisition phytase gene and carry out sequencing.

Embodiment 4: yeast expression phytase vector construction

Obtain the two ends sequences Design primer of high temperature resistant height ratio gene alive by screening, add XhoI point of contact and signal attitude cutting sequence at the gene 5 ' end, primer is: PHY1Z (5 '-AACTCGAGAAAAGAGAACCTCCGGATT GGCTGTCCCAGCTTCCAGAAACCAGTCC-3 '), and add Not I point of contact at gene 3 ' end: primer is: PHY1F (5 '-AACGCGGCCGCTTAAGCGAAGCATTCAGCCCAGTCACCACCGGTAC-3 ').Behind the amplified fragments clone, Xho I and Not I double digestion, orientation is inserted pPIC9 carrier (Invitrogen company product), is built into the Yeast expression carrier pPphyXH (Fig. 1) of PhyXH.

Embodiment 5: the screening of phytase high expression level recombination yeast

With activatory yeast strain Pichia Pastoris G315 30 ℃ of cultivation 18hr in 500ml YPD, to OD ₆₀₀=1.7, the centrifugal collection thalline of 5000r/min successively uses 500, the aseptic washing thalline of 250ml precooling, and the centrifugal supernatant of going is with the 1mol/L sorbyl alcohol suspension thalline of 20ml precooling.Centrifugal back thalline suspends with the sorbyl alcohol of 0.5ml precooling again, is used to the competence that shocks by electricity.

A large amount of extracting Yeast expression carrier pPphyXH, the BglII enzyme cuts back to close small segment, and get 2 μ g linearizing fragments and add 50 μ L competent cells, ice bath 5min, with Bio-Red GenePulser electric shock instrument electric shock, parameter is 2.5Kv, 25 μ F.The 1mol/L sorbyl alcohol that adds the 1.0ml precooling after electric shock finishes immediately, get 200 μ L and coat solid selection culture medium flat plate (18.6% sorbyl alcohol, 2% glucose, 1.34%YNB, 0.005% L-glutamic acid, 0.005% methionine(Met), 0.005% Methionin, 0.005% leucine, 0.005% Isoleucine, 2% agarose), 30 ℃ of cultivations occur until transformant.With toothpick MM (1.34%YNB is arrived in the corresponding dibbling of transformant; 0.00004% vitamin H; 0.5% methyl alcohol; 1.5% agarose) and MD (1.34%YNB; 0.00004% vitamin H, 2% glucose, 1.5% agarose) on the flat board; cultivate 2d for 30 ℃, the positive clone of transformant undesired or that do not grow that on MM, grows growing normal on the MD.

Recombination yeast is inoculated in 20ml BMGY (1% yeast extract, 2% peptone, the 1.34%YNB0.000004% vitamin H, 1% glycerine), 30 ℃ of cultivations, centrifugal collection thalline adds 20ml inducing culture BMMY (with the glycerine among the 0.5% methyl alcohol replacement BMGY), and 30 ℃ are continued inducing culture 36hr down.

Be the MM substratum of sole carbon source with methyl alcohol and be that the MD substratum of carbon source carries out correspondence and cultivates with glucose, further screen 168 of recombinant conversion of site-directed integration, called after P.pastoris PHYXH1,2,3 ... 168.

All positive colonies are inoculated separately in the triangular flask, and 30 ℃ are cultured to OD ₆₀₀=4-5 utilizes methanol induction to cultivate 36hr, and each inducible strain is got 5 μ l supernatant liquors and carried out the SDS-PAGE detection, and other gets 100 times of 10 μ l supernatant liquors dilutions, is that substrate carries out enzyme assay with POTASSIUM PHYTATE sodium.The phytase activity measuring method is taked molybdenum yellow method (BASF Company).Phytase activity unit (U) is defined as: under 37 ℃, it is 1U that per minute decomposition phytate discharges the needed enzyme amount of 1nmol/L inorganic phosphate.In conjunction with electrophoretic band and enzyme activity unit, screen the recon P.pastorisPHYXH of 1 plant height efficient expression phytase gene.

Embodiment 6: the high density fermentation of recombinant bacterial strain

Picking phytase high expression level recombination yeast P.pastoris PHYXH inoculation 200ml YPED substratum, 30 ℃ are cultured to OD ₆₀₀=3.0, change in the B.Braun 5L fermentor tank and carry out high density fermentation, cultivate recombination yeast with YPED, utilizing ammoniacal liquor control pH value is 5.5, and Control for Oxygen Content is cultivated 90hr 20% in the fermenting process, and glycerine exhausts, and adds 0.5% methyl alcohol, 30 ℃ of inducing culture.

Behind 30 ℃ of cultivation 6hr, recombination yeast enters logarithmic phase, and oxygen consumption is accelerated, and must charge into pure oxygen, and Control for Oxygen Content is 20%, and during the fermentation, it is constant in 5.5 to continue to add ammoniacal liquor adjusting pH value, behind the 90hr that ferments, and OD ₆₀₀=110, add 0.5% methanol induction and cultivate, induce the inducing culture liquid of getting 3ul behind the different time and do not have thalline to carry out SDS-PAGE and detect.Result (Fig. 2), along with the increase of induction time, the expression amount of phytase improves constantly, and behind the 60h, it is stable that the expression amount in the supernatant liquor keeps.After recon P.pastoris PHYXH induced 60hr, the phytase expression amount was up to 1.8mg/ml.The phytase molecule amount size of expressing is about 79kD.

The molecular weight size is about 79kD.

Under the condition of 37 ℃ of pH5.5 the nutrient solution that contains expression product is carried out phytase activity and measure, after recombination yeast P.pastoris PHYXH cultivated 60hr, enzyme activity was 2,010,000FTU/ml increases the abduction delivering time, and the expression amount of phytase only slowly increases.

Embodiment 7: the phytase property testing

Substrate is mixed with pH=1.5 respectively; 2.5; 3.5; 4.5; 5.5; 6.5 the unit alive of the enzyme during with pH=5.5 is 100%, measures the relative vigor of phytase under the condition of different pH with the high density fermentation supernatant liquor.The result shows that this phytase all has enzyme activity between pH2.5-6.5, and the pH value is 2.5 and 5.5 o'clock, and phytase activity is the highest, shows two peak values (Fig. 3).

Supernatant liquor is handled different time with 90 ℃, place cooled on ice, add enzyme reaction substrate, 37 ℃ of reaction 30min are reference with the fermented liquid without pyroprocessing, measure the remaining enzymic activity in the supernatant liquor.Handle through 90min, the activity of phytase still keeps 50%.90 ℃ of pyroprocessing 120min still have the part activity.

The present invention's culture medium prescription mentioned above all is weight percentage.

(1) general information:

(i) denomination of invention: utilize pichia spp to produce height ratio high temperature resistant phytase alive

(1) sequence number: 2

(2) information of sequence SEQ ID NO:1:

(i) sequence signature:

(A) length: 1290bp

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linear

(ii) molecule type: DNA

(ii) sequence description: SEQ NO:1

GAGACTTCTCATTTGTGGGGTCAATACGCTCCATTCTTCTCTTTGGCTAACAAGTCTGCTATCTCTCCAGAT

GTTCCAGCTGGTTGTAAAGTTACTTTCGCTCAAGTTTTGTCCAGACATGGTGCTAGATACCCAACTGATACT

AAAGGTAAGAAATATTCTGCTTTGATTGAGGAGATTCAACAAAACGCTACTACTTTTGAGGAGAAATACGCT

TTTTTGAAAACTTACAACTATTCTTTGGGTGCTGATGATTTGACTCCATTTGGTGAACAAGAATTGGTTAAT

TCTGGTGTTAAGTTTTACCAAAGATACGAATCTTTGACTAGAAATATTGTTCCATTTATTAGATCCTCTGGT

TCTTCCAGAGTTATTGCTTCTGGTAATAAATTCATTGAAGCTTATCAATCTACTAAATTGAAAGATCCAAGA

GCCCAACCAGGTCACTCTTCTCCAAAAATTGATGTTGTTATTTCTGAGGCTTCTACTTCTAATAATACTGTG

GACCCAGGTACTTGTACTGTTTCTGAAGATAACGAATTGGCTGATGATTTCGAAGCTAATTTTACTGCTACT

TTTGTTCCATCTATTAGACAATCTTTGGAAAATAACTTGTCTGGTGTTGCTTTGACTGATACTGAAGTTACT

TACTTGATGGACTTGTGTTCTTTTGATACTATCTCTACTTCTACTGTTGATACTAAATTGTCTCCATTTTGT

GATTTGTTTACTCATGAAAAATGGATTAATTATGATTATTTGCAATCTTTGAACAAGTACTACGGTCATGGT

GCTGGTAATCCATTGGGTCCAACTCAAGGTGTTTGTTACGCTAATGAATTGATTTCCAGATTGACTCATTCT

CCAGTTCATGATTACACTTCTTCTAATCATATTTTGGATTCTTCTCAAGATACTTTTCCATTGAACTCTACT

TTGTACGCTGATTTCTCTCTTAACAATGGTATTATCTCTATTTTGTTTGCTTGGGGTTTGAACAAAGGTACT

AAACCATTGTCTTCTACTACTGCTGAAAATATCACTCAAACTGATGGTTTTTCTTCTGCTTGGACTGTTCCA

TTCGCTTCCAGAATGTACGTTGAAATGATGCAATGTCAATCTGAACAAGAACCATTGGTTAGAGTTTTGGTT

AATGATAGAGTTGTTCCATTGCATGGTTGTCCAGTTGATGCTTTGGGTAGATGTACTAGAGATTCTTTTGTT

AGAGGTTTGTCTTTTGCTAGATCCGGTGGTGATTGGGCTGAATGCTTTGCTAAGGACGAATTGTAA

Phytase structure gene integral part all adopts yeast preference password.

(3) information of sequence SEQ ID NO:2:

(i) sequence signature:

(A) length: 430AA

(B) type: amino acid

(C) topological framework: linear

(ii) molecule type: polypeptide

(iii) sequence description: SEQ NO:2

(benefit is gone into aminoacid sequence)

E T S H L W G Q Y A P F F S L A N Q S A I S P D

V P A G C K V T F A Q V L S R H G A R Y P T D T

K G K K Y S A L I E E I Q Q N A T T F E E K Y A

F L K T Y N Y S L G A D D L T P F G E Q E L V N

S G V K F Y Q R Y E S L T R N I V P F I R S S G

S S R V I A S G N K F I E A Y Q S T K L K D P R

A Q P G H S S P K I D V V I S E A S T S N N T V

D P G T C T V S E D N E L A D D F E A N F T A T

F V P S I R Q S L E N N L S G V A L T D T E V T

Y L M D L C S F D T I S T S T V D T K L S P F C

D L F T H E K W I N Y D Y L Q S L N K Y Y G H G

A G N P L G P T Q G V C Y A N E L I S R L T H S

P V H D Y T S S N H I L D S S Q D T F P L N S T

L Y A D F S L N N G I I S I L F A W G L N K G T

K P L S S T T A E N I T Q T D G F S S A W T V P

F A S R M Y V E M M Q C Q S E Q E P L V R V L V

N D R V V P L H G C P V D A L G R C T R D S F V

R G L S F A R S G G D W A E C F A K D E L -

The phytase aminoacid sequence constitutes the primary structure of high temperature resistant high-specific-activity phytase, and the peptide molecule size is 50kD.There are 12 amino acid differences in this aminoacid sequence and original gene amino acid sequence coded.Contain 6 glycosylation sites between the molecule, generally in yeast, give expression to 79kD glycosylation molecule.Form specific higher structure through glycosylated phytase molecule, contain the combination and the catalytic site of phytic acid.

Claims

1, a kind of have a following phytase gene PhyXH of coding that high temperature resistant and height ratio is lived:

GAGACTTCTCATTTGTGGGGTCAATACGCTCCATTCTTCTCTTTGGCTAACAAGTCTGCTATCTCTCCAGAT

E T S H L W G Q Y A P F F S L A N Q S A I S P D

GTTCCAGCTGGTTGTAAAGTTACTTTCGCTCAAGTTTTGTCCAGACATGGTGCTAGATACCCAACTGATACT

V P A G C K V T F A Q V L S R H G A R Y P T D T

AAAGGTAAGAAATATTCTGCTTTGATTGAGGAGATTCAACAAAACGCTACTACTTTTGAGGAGAAATACGCT

K G K K Y S A L I E E I Q Q N A T T F E E K Y A

TTTTTGAAAACTTACAACTATTCTTTGGGTGCTGATGATTTGACTCCATTTGGTGAACAAGAATTGGTTAAT

F L K T Y N Y S L G A D D L T P F G E Q E L V N

TCTGGTGTTAAGTTTTACCAAAGATACGAATCTTTGACTAGAAATATTGTTCCATTTATTAGATCCTCTGGT

S G V K F Y Q R Y E S L T R N I V P F I R S S G

TCTTCCAGAGTTATTGCTTCTGGTAATAAATTCATTGAAGCTTATCAATCTACTAAATTGAAAGATCCAAGA

S S R V I A S G N K F I E A Y Q S T K L K D P R

GCCCAACCAGGTCACTCTTCTCCAAAAATTGATGTTGTTATTTCTGAGGCITCTACTTCTAATAATACTGTG

A Q P G H S S P K I D V V I S E A S T S N N T V

GACCCAGGTACTTGTACTGTTTCTGAAGATAACGAATTGGCTGATGATTTCGAAGCTAATTTTACTGCTACT

D P G T C T V S E D N E L A D D F E A N F T A T

TTTGTTCCATCTATTAGACAATCTTTGGAAAATAACTTGTCTGGTGTTGCTTTGACTGATACTGAAGTTACT

F V P S I R Q S L E N N L S G V A L T D T E V T

TACTTGATGGACTTGTGTTCTTTTGATACTATCTCTACTTCTACTGTTGATACTAAATTGTCTCCATTTTGT

Y L M D L C S F D T I S T S T V D T K L S P F C

GATTTGTTTACTCATGAAAAATGGATTAATTATGATTATTTGCAATCTTTGAACAAGTACTACGGTCATGGT

D L F T H E K W I N Y D Y L Q S L N K Y Y G H G

GCTGGTAATCCATTGGGTCCAACTCAAGGTGTTTGTTACGCTAATGAATTGATTTCCAGATTGACTCATTCT

A G N P L G P T Q G V C Y A N E L I S R L T H S

CCAGTTCATGATTACACTTCTTCTAATCATATTTTGGATTCTTCTCAAGATACTTTTCCATTGAACTCTACT

P V H D Y T S S N H I L D S S Q D T F P L N S T

TTGTACGCTGATTTCTCTCTTAACAATGGTATTATCTCTATTTTGTTTGCTTGGGGTTTGAACAAAGGTACT

L Y A D F S L N N G I I S I L F A W G L N K G T

AAACCATTGTCTTCTACTACTGCTGAAAATATCACTCAAACTGATGGTTTTTCTTCTGCTTGGACTGTTCCA

K P L S S T T A E N I T Q T D G F S S A W T V P

TTCGCTTCCAGAATGTACGTTGAAATGATGCAATGTCAATCTGAACAAGAACCATTGGTTAGAGTTTTGGTT

F A S R M Y V E M M Q C Q S E Q E P L V R V L V

AATGATAGAGTTGTTCCATTGCATGGTTGTCCAGTTGATGCTTTGGGTAGATGTACTAGAGATTCTTTTGTT

N D R V V P L H G C P V D A L G R C T R D S F V

AGAGGTTTGTCTTTTGCTAGATCCGGTGGTGATTGGGCTGAATGCTTTGCTAAGGACGAATTGTAA

R G L S F A R S G G D W A E C F A K D E L -

Be integrated into the Pichia yeast engineering that yeast obtains to produce phytase by the PhyXH plasmid vector.

2, the Pichia yeast engineering that can produce phytase according to claim 1, amino acid number is with identical from the Phy2A amino acid number of aspergillus niger coding in the phytase gene that it is characterized in that encoding, sport Xie Ansuan in 11 amino acids by the Threonine of Phy2A, 41 amino acids are glutamine by the lysine mutation of Phy2A; 53 amino acids sport Methionin by the Phy2A glutamine; 91 amino acids are L-glutamic acid by the lysine mutation of Phy2A; 71 amino acids are Threonine by the mutant serine of Phy2A; 112 amino acids sport phenylalanine by the L-glutamic acid of Phy2A; 157 amino acids are L-Ala by the glycine mutation of Phy2A; 191 amino acids sport Xie Ansuan by the leucine of Phy2A.

3, the Pichia yeast engineering that can produce phytase according to claim 1 is characterized in that the Yeast expression carrier pPhy XH that makes up, contains the described height ratio of the claim 1 high temperature resistant phytase gene sequence of living.

4, the Pichia yeast engineering that can produce phytase according to claim 1, it is characterized in that pPhy XH plasmid vector contains to be reset with dna molecular family by Fructus Fici phytase gene, intestinal bacteria phytase gene, high temperature resistant phytase gene, high specific activity phytase gene carries out vitro recombination, rearrangement phytase gene fragment is building up in the prokaryotic expression carrier, and electric shock changes coli strain over to and obtains height ratio resistant to elevated temperatures phytase gene alive by screening.

5, the Pichia yeast engineering that can produce phytase according to claim 1 is characterized in that Phy XH gene two ends aligning primer adds Xhol point of contact and signal attitude cutting sequence at gene 5, end, and primer is:

PHY1Z，

(5 '-AACTCGAGAAAAGAGAACCTCCGGATTGGCTGTCCCAGCTTCCAGAAACCAGTCC-3 '), add Not I point of contact at gene 3` end, primer is PHY1F (5 '-AACGCGGCCGCTTAAGCGAAGCATICAGCCCAGTCACCACCGGTAC-3 ').

6, the preparation method that can produce the Pichia yeast engineering of phytase as claimed in claim 1,

A, four of employings have the phytase genes of yeast preference password;

B, be template with above-mentioned four phytase genes, utilize dna molecular family rearrangement technology, synthetic gene is carried out vitro recombination, PhyIZ1, PhyIF1 are the phytase gene that primer amplification is reset, reclaim the phytase gene fragment that 1.4Kb resets, be building up in the prokaryotic expression carrier, electric shocking method changes coli strain over to and obtains the mutant expression library, carry out the high-specific-activity phytase screening, obtain height ratio and live and resistant to elevated temperatures phytase gene Phy XH;

C, adopting screening to obtain the gene height ratio lives and resistant to elevated temperatures phytase gene Phy XH two ends sequences Design primer, add Xhol point of contact and signal attitude cutting sequence at gene 5` end, primer is: PHY1Z, (5 '-AACTCGAGAAAAGAGAACCTCCGGATTGGCTGTCCCAGCTTCCAGAAACCAGTCC-3 '), add Not I point of contact at gene 3` end, primer is PHY1F (5 '-AACGCGGCCGCTTAAGCGAAGCATTCAGCCCAGTCACCACCGGTAC-3 '), behind the amplified fragments clone, Xho I and Not I double digestion, orientation is inserted the pPIC9 carrier, is built into the Yeast expression carrier pPhy XH of PhyXH;

D, the screening of phytase gene high expression level recombination yeast;

E, with the restructuring yeast strains high density fermentation.

7, the preparation method that can produce the Pichia yeast engineering of phytase according to claim 6, after it is characterized in that phytase gene synthetic primer is Phyl Z and Phyl F amplified fragments clone, at XhoI and Not I double digestion, orientation is inserted the pPIC9 carrier, is built into the Yeast expression carrier pPhyXH of Phy XH.

8, the preparation method that can produce the Pichia yeast engineering of phytase according to claim 6, it is characterized in that the screening of high reactivity and high temperature resistant phytase is by reclaiming the rearrangement phytase gene fragment of 1.4kb, through being built into behind Bam HI and the SacI double digestion between the whole son of prokaryotic expression carrier pG251 promotor and t1t2, this carrier has ampicillin resistance gene, electric shocking method transformed into escherichia coli bacterial strain DH5 α obtains the mutant expression library, carries out the screening of high-specific-activity phytase.

9, the preparation method that can produce the Pichia yeast engineering of phytase according to claim 6 is characterized in that containing phytase gene expression carrier pPhy XH electric shock and changes Pichia yeast over to, selects the phytase restructuring yeast strains and carries out high density fermentation.