CN1475572A

CN1475572A - High expressed high temperature resistant phytase gene in methanol yeast

Info

Publication number: CN1475572A
Application number: CNA021365318A
Authority: CN
Inventors: 姚泉洪; 彭日荷; 熊爱生; 吴伟; 刘承训
Original assignee: YONGYE AGRICULTURAL SCIENCE BIOLOGICAL ENGINEERING Co Ltd SHANGHAI
Current assignee: Xinghu Biotech Co., Ltd., Zhaoqing City, Guangdong Prov.
Priority date: 2002-08-16
Filing date: 2002-08-16
Publication date: 2004-02-18
Anticipated expiration: 2022-08-16
Also published as: CN100354421C

Abstract

A refractory phytase gene with high expression in methanol yeast is prepared through PCR amplification and recombining its DNA, that is, configuring mutant phytase gene liabrary on prokaryotic expression plasmid, screening refractory phytase gene, changing its preferably codon to make it be expressed in yeast, and electric shock transfer to integrate it in Pichia yeast for high expression.

Description

The high temperature resistant phytase gene of high expression level in methanol yeast

Technical field

The invention belongs to the microbiological genetic engineering field, more specifically saying so changes phytase gene by gene engineering method, makes expressed protein can at high temperature keep long-time activity.

Technical background

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, the utilization ratio of phosphorus is very low, cause the waste of a large amount of phosphorus source, unserviceable phosphorus is excluded external, causes environment phosphorus to pollute (Cromwell 1991, and biotechnology is made progress in Application in Food Industry), in addition 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 in food and feed, add inorganic phosphorus (Common 1989, Nature Journal).

Phytase can become hydrolysis of phytic acid inositol and phosphoric acid, in feed, add the utilization ratio that phytase not only can improve phosphorus in the plant feed, (Nelson 1968 to reduce the phosphorus environmental pollution of discharging in the ight soil, the herding science), and can be by reducing the anti-oxidant action of phytate, increase the receptivity (Wyss 1999, applied environment microorganism science) of animal to protein and some metal ions.

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.From the mid-80, the scheme that comprehensive solution inorganic phosphorus pollutes just is devoted to seek in Europe.Its direct reason derives from the concern of parliament of former EEC countries to environment protection, in the environmental protection guide (CouncilDirective91/676/EEC) that they implement, 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.Phytase also is present functional study product the most clearly in all feed enzyme preparation products.Use the benefit of phytase maximum to be to reduce the usage quantity of inorganic phosphorus such as secondary calcium phosphate in the feed, amplitude is 50%-70% at least.In China, a lot of culture zones can be reduced the consumption of secondary calcium phosphate more than 70%, especially in the breeding layer chicken district of assorted dregs of rice large usage quantity, and can the replacing whole secondary calcium phosphate.Use the feed of phytase, to the corresponding minimizing of environment excretory phosphorus more than 30%.According to the most conservative estimation, since beginning to introduce phytase, the aquaculture of China few to environmental emission 7200 tons of phosphorus.Amount to 42000 tons of secondary calcium phosphates.Add the pollution and the expense of phosphorate rock mining, and the carrying cost of saving.The comprehensive benefit that phytase is created runs far deeper than more than 9,200 ten thousand yuan that estimate aspect breed and feed.

In fact, the biggest beneficiary of phytase is the mankind itself.In the environment of depending on for existence, phytase can be eliminated phosphorus easily from the source of polluting and pollute, 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.Can improve proteinic digestive utilization ratio in the feed.Use phytase, strengthened the usage ratio of unconventional raw material resources, provide convenience for saving protein feed.Use phytase can directly reduce the raw materials cost of feed, thereby improve the price competitiveness of product.Therefore in China, 1 ton of complete feed of every production can increase the net income of 5-10 unit at least.The Preblend that every production is 1 ton 5% then can additional income more than hundred yuan.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.

Phytase extensively is present in plant, animal and the microorganism.Many microorganisms can both produce phytase, especially in Aspergillus.Nineteen sixty-eight Shien etc. investigates discovery to 2000 bacterial strains from 68 soil samples, have 21 strains can produce phytase in 22 used strain melanomyces.The phytase of first separated purifying derives from Aspergillus terreusNO.9A-1, and its optimal pH is 4.5, and optimal reactive temperature is 70 ℃, and this enzyme all can the stable maintenance activity in pH1.2～9.0.After this, from tens kinds of microorganisms, separate successively and obtain phytase, the phytase phyA that wherein derives from A.ficcum NR-RL3135 (A.niger var.awamori) has the high enzyme activity under acidic conditions, be considered to the feeding phytic acid plum of at present tool application prospect, the research of its zymologic property is also comparatively deep.Phytase phyA is a kind of glycosylated protein, and molecular weight is 85KD.Its 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 is had restraining effect, and wherein preceding two kinds are the noncompetitive inhibition, and back two kinds is competitive inhibition.There are inhibiting inhibitor such as L (+)-tartrate that it is not but had restraining effect to acid phosphatase.

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, makes production cost higher, can obtain the bacterial strain that yield of enzyme improves by chemomorphosis, yet required time is very long, and mutagenic strain is degenerated easily, utilizes bio-reactor to efficiently express phytase gene, increase substantially phytase output, overcome the low shortcoming of wild type strain content ^[6,7]Feed institute of the Chinese Academy of Agricultural Sciences and biotechnology research institute were since cooperation in 1996, from black mold, cloned the phytase gene that is adapted at using in the feed, and developed jointly and utilized that bio-reactor is extensive, the technology of low cost production fodder additives phytase.But there is a very big defective in this phytase: can not tolerate higher temperature, enzyme activity unit is lost (Kim 1998, enzyme and microbial technique) in a large number in the course of processing.Therefore, screen the focus that thermally-stabilised phytase becomes present feedstuff industry research from the nature searching or by random mutation.

Isolating phytase can tolerate 90-100 ℃ of high temperature from sootiness aspergillus (Aspergillus fumigatus), and the phytic acid of degrading in the pH of broad scope, therefore has very big market potential (van Loon 1998, applied environment microbiology).Yet in wild type strain, the expression amount of phytase is very low, is difficult to promote on producing.

Summary of the invention

The objective of the invention is to seek and a kind ofly have a preference for the phytase gene that password changes, the coded protein resistance toheat improves, and utilize high expression level in the yeast to prepare the method for this phytase.

The present invention attempts to make gene be adapted at expressing in the yeast by changing the preference password of gene, transforms by electric shock heat-stable phytase gene is incorporated in the pichia spp, realizes efficiently expressing.

For the thermotolerance and the ratio that improve phytase gene are lived, the synthetic phytase gene is carried out the library that DNA reorganizes (DNAShuffling), makes up the sudden change phytase gene on prokaryotic expression plasmid, by the screening of sudden change phytase activity, finally obtained the high temperature resistant phytase gene that height ratio is lived.The ratio work of new phytase Fphy is about 5 times of original Fphy1.The amino acid no of Fphy and Fphy1 is identical, but has 9 amino acid that change has taken place.Comprising: 72V＞I, 86F＞Y, 87A＞T, 194Q＞E, 200A＞E, 236S＞P, 292T＞A, 338V＞I, 396A＞V etc.

The overlapping extension PCR method of the synthetic employing of thermotolerance phytase gene, the PWO archaeal dna polymerase in conjunction with high-fidelity is linked in sequence the primer fragment.Synthetic gene length is 1320 bp.

The nucleotide sequence and the amino acid sequence coded thereof of sudden change heat-stable phytase gene are as follows, and from the prlmary structure of protein analysis, this phytase contains 6 sugar and intensifies the site.

The nucleotide sequence of heat resistance phytase gene of the present invention and the amino acid sequence of coding are as follows: TCCAAATCCTGCGACACCGTTGACTTGGGATACCAATGCTCCCCTGCTACCTCCCA CCTTTGGGGACAATAC S K S C D T V D L G Y Q C S P A T S H L W G Q YTCTCCATTCTTCTCTTTGGAGGACGAACTTTCCGTTTCCTCCAAATTGCCAAAAG ATTGCAGAATCACCTTG S P F F S L E D E L S V S S K L P K D C R I T LGTTCAAGTCTTGTCCAGACACGGTGCTAGATACCCAACCTCCTCCAAATCCAAAA AATACAAAAAACTGATC V Q V L S R H G A R Y P T S S K S K K Y K K LIACTGCTATCCAAGCTAACGCTACCGACTTCAAAGGAAAATACACTTTCTTGAAAAC CTACAACTACACCTTG T A I Q A

A T D F K G K Y TF L K T Y Y T LGGAGCTGACGACTTGACCCCATTCGGAGAACAACAACTTGTCAACTCT GAATCAAATTCTACCAAAGATAC G A D D L T P F G E Q Q L V N S G I K F Y Q R YAAAGCACTTGCTAGATCCGTTGTTCCTTTCATCAGAGCTTCTGGCTCCGACAGAGTTATCGCTTCTGGAGAA K A L A R S V V P F I R A S G S D R V I A S G EAAATTCATCGAAGGATTCCAACAAGCTAAATTGGCTGACCCTGGAGCTACCAACAGAGCTGCTCCTGCTATC K F I E G F Q Q A K L A D P G A T N R A A P A ITCCGTTATCATCCCAGAATCCGAAACCTTCAACAACACCTTGGACCACGGTGTTTGCACCAAATTCGAGGCT S V I I P E S E T F

N T L D H G V C T K F E ATCTGAACTTGGAGACGAGGTCGAGGCTAACTTCACCGCTTTGTTCGCTCCTGACA TCAGAGCTAGAGCTGAAS EL G D EV E A N F T A L F A P D I R A R A EAAACACTTGCCTGGTGTCACCTTGACCGACGAAGACGTTGTTTCCTTGATGGATA TGTGCCCCTTCGACACC K H L P G V T L T D E D V V S L M D M C PF D TGTTGCTAGAACCTCCGACGCTTCCCAACTTTCCCCATTCTGCCAACTTTTCACCCACAACGAATGGAAAAAA V A R T S D A S Q L S P F C Q L F T H N E W K KTACAACTACCTGCAGTCCTTGGGAAAATACTACGGATACGGAGCTGGAAACCCATTGGGACCAGCTCAAGGA Y N Y L Q S L G K Y Y G Y G A G N P L G P A Q GATCGGATTCGCCAACGAATTGATCGCTAGATTGACCAGATCCCCAGTTCAAGACCACACCTCCACTAACTCC I G F AN E L I A R L T R S P V Q D H T S T

SACCTTGGTTTCCAACCCAGCTACCTTCCCATTGAACGCTACCATGTACGTTGACT TCTCCCACGACAACTCT T L V S N P A T F P L A T M Y V D F S H D N SATGATCTCCATCTTCTTCGCTTTGGGACTTTACAACGGAACTGAACCATTGTCCA GAACTTCCGTTGAATCC M IS I F F A L G L Y

G T E P L S R T S V E SGCTAAAGAACTTGACGGATACTCCGCTTCCTGGGTTGTTCCATTCGGTGCTAGAG CTTACTTTGAAACTATG A K E L D G Y S A S W V V P F G A R A Y F E T MCAATGCAAATCCGAAAAAGAACCATTGGTTAGAGTCTTGATTAACGACAGAGTTG TTCCATTGCACGGATGC Q C K S E K E P L V R VL I N D R V V P L H G CGACGTTGACAAATTGGGAAGATGCAAATTGAACGACTTCGTTAAAGGATTGTCTT GGGCTAGATCCGGTGGT D V D K L G R C K L N D F V K G L S W A R S G GAACTGGGGTGAATGCTTCTCCTAA N W G E C F S-

Sudden change heat-stable phytase gene is compared with original gene, 352 Nucleotide change, the homology of Nucleotide is 73%, the Nucleotide password all adopts yeast preference password in building-up process, wherein arginine adopts AGA, Methionin adopts AAA, and halfcystine adopts TGC, and aspartic acid adopts AAC.In addition, in order to improve expression amount, synthetic gene has excised the intron sequences in 26 amino acid whose signal coding sequences of 5 ' end and the signal peptide, so that the phytase gene encoder block can correctly be inserted after the promotor and secreting signal peptide that has high expression level, stability and high efficiency is expressed.New phytase gene Fphy compares with former phytase gene Fphy1: FPHY1-TCCAAGTCCTGCGATACGGTAGACCTCGGGTACCAGTGCTCCCCTGCGAC-50

|||||?||||||||?||?||?|||?|?||?|||||?|||||||||||?||FPHY -TCCAAATCCTGCGACACCGTTGACTTGGGATACCAATGCTCCCCTGCTAC?-50FPHY1 -TTCTCATCTATGGGGCCAGTACTCGCCATTCTTTTCGCTCGAGGACGAGC?-100

||?||?||?|||||?||?|||||?||||||||?|| |?||||||||?|FPHY -CTCCCACCTTTGGGGACAATACTCTCCATTCTTCTCTTTGGAGGACGAAC?-100FPHY1 -TGTCCGTGTCGAGTAAGCTTCCCAAGGATTGCCGGATCACCTTGGTACAG?-150

|?|||||?|| || |?||?||?||||||?||||||||||||?||FPHY -TTTCCGTTTCCTCCAAATTGCCAAAAGATTGCAGAATCACCTTGGTTCAA?-150FPHY1 -GTGCTATCGCGCCATGGAGCGCGGTACCCAACCAGCTCCAAGAGCAAAAA?-200

|| |?|| |?||?||?|| |?||||||||| |||||| ||||||FPHY -GTCTTGTCCAGACACGGTGCTAGATACCCAACCTCCTCCAAATCCAAAAA?-200FPHY1 -GTATAAGAAGCTTGTGACGGCGATCCAGGCCAATGCCACCGACTTCAAGG?-250

||?||?||?||?||?||?||?|||||?||?||?||?|||||||||||?|FPHY -ATACAAAAAACTGATCACTGCTATCCAAGCTAACGCTACCGACTTCAAAG?-250FPHY1 -GCAAGTTTGCCTTTTTGAAGACGTACAACTATACTCTGGGTGCGGATGAC?-300

|?||?||?||?||?|||||?||?||||||||?| ||||?||?||?|||FPHY -GAAAATACACTTTCTTGAAAACCTACAACTACAACTTGGGAGCTGACGAC?-300FPHY1 -CTCACTCCCTTTGGGGAGCAGCAGCTGGTGAACTCGGGCATCAAGTTCTA?-350

|?| ||?||?||?||?||?||?||?||?|||||?||?|||||?|||||FPHY -TTGAACCCATTCGGAGAACAACAACTTGTCAACTCTGGAATCAAATTCTA?-350FPHY1 -CCAGAGGTACAAGGCTCTGGCGCGCAGTGTGGTGCCGTTTATTCGCGCCT?-400

|||?||?|||||?||?||?|| | ||?||?||?||?|| |?||?|FPHY -CCAAAGATACAAAGCACTTGCTAGATCCGTTGTTCCTTTCATCAGAGCTT?-400FPHY1 -CAGGCTCGGACCGGGTTATTGCTTCGGGAGAGAAGTTCATCGAGGGGTTC?-450

|?|||||?|||?|?|||||?|||||?|||||?||?||||||||?||?|||FPHY -CTGGCTCCGACAGAGTTATCGCTTCTGGAGAAAAATTCATCGAAGGATTC?-450FPHY1 -CAGCAGGCGAAGCTGGCTGATCCTGGCGCGACGAACCGCGCCGCTCCGGC?-500

||?||?||?|| |||||||?|||||?||?||?|||?|?||?|||||?||FPHY -CAACAAGCTAAATTGGCTGACCCTGGAGCTACCAACAGAGCTGCTCCTGC?-500FPHY1 -GATTAGTGTGATTATTCCGGAGAGCGAGACGTTCAACAATACGCTGGACC?-550

|| ||?||?||?||?|| |||?||?||||||||?|| ||||||FPHY -TATCTCCGTTATCATCCCAGAATCCGAAACCTTCAACAACACCTTGGACC?-550FPHY1 -ACGGTGTGTGCACGAAGTTTGAGGCGAGTCAGCTGGGAGATGAGGTTGCG?-600

|||||||?|||||?||?||?||||| |||?||?|||||?|||||?|?|FPHY -ACGGTGTTTGCACCAAATTCGAGGCTTCTGAACTTGGAGACGAGGTCGAG?-600FPHY1 -GCCAATTTCACTGCGCTCTTTGCACCCGACATCCGAGCTCGCGCCGAGAA?-650

||?||?|||||?|| |?||?||?||?||||||?|||||?|?||?||?||FPHY -GCTAACTTCACCGCTTTGTTCGCTCCTGACATCAGAGCTAGAGCTGAAAA?-650FPHY1 -GCATCTTCCTGGCGTGACGCTGACAGACGAGGACGTTGTCAGTCTAATGG?-700

|| |?|||||?||?|| ||||?|||||?|||||||| |?||||FPHY -ACACTTGCCTGGTGTCACCTTGACCGACGAAGACGTTGTTTCCTTGATGG?-700FPHY1 -ACATGTGTTCGTTTGATACGGTAGCGCGCACCAGCGACGCAAGTCAGCTG?-750

|?||||| |?||?||?||?||?|| |?||| |||||| ||?||FPHY -ATATGTGCCCCTTCGACACCGTTGCTAGAACCTCCGACGCTTCCCAACTT?-750FPHY1 -TCACCGTTCTGTCAACTCTTCACTCACAATGAGTGGAAGAAGTACAACTA?-800

||?||?|||||?|||||?|||||?|||||?||?|||||?||?||||||||FPHY -TCCCCATTCTGCCAACTTTTCACCCACAACGAATGGAAAAAATACAACTA?-800FPHY1 -CCTTCAGTCCTTGGGCAAGTACTACGGCTACGGCGCAGGCAACCCTCTGG?-850

|||?|||||||||||?||?||||||||?|||||?||?||?||||| |||FPHY -CCTGCAGTCCTTGGGAAAATACTACGGATACGGAGCTGGAAACCCATTGG?-850FPHY1 -GACCGGCTCAGGGGATAGGGTTCACCAACGAGCTGATTGCCCGGTTGACT?-900

||||?|||||?||?||?||?|||?||||||| ||||?|| |?|||||FPHY -GACCAGCTCAAGGAATCGGATTCGCCAACGAATTGATCGCTAGATTGACC?-900FPHY1 -CGTTCGCCAGTGCAGGACCACACCAGCACTAACTCGACTCTAGTCTCCAA?-950

|?||?|||||?||?||||||||| |||||||||?|| |?||?|||||FPHY -AGATCCCCAGTTCAAGACCACACCTCCACTAACTCCACCTTGGTTTCCAA?-950FPHY1 -CCCGGCCACCTTCCCGTTGAACGCTACCATGTACGTCGACTTTTCACACG?-1000

|||?||?||||||||?||||||||||||||||||||?|||||?||?||||FPHY -CCCAGCTACCTTCCCATTGAACGCTACCATGTACGTTGACTTCTCCCACG?-1000FPHY1 -ACAACAGCATGGTTTCCATCTTCTTTGCATTGGGCCTGTACAACGGCACT?-1050

||||| |||?|?|||||||||||?||?|||||?||?||||||||?|||FPHY -ACAACTCTATGATCTCCATCTTCTTCGCTTTGGGACTTTACAACGGAACT?-1050FPHY1 -GAACCCTTGTCCCGGACCTCGGTGGAAAGCGCCAAGGAATTGGATGGGTA?-1100

|||||?||||||?|?||?||?||?||| |||?||?|||?|?||?||?||FPHY -GAACCATTGTCCAGAACTTCCGTTGAATCCGCTAAAGAACTTGACGGATA?-1100FPHY1 -TTCTGCATCCTGGGTGGTGCCTTTCGGCGCGCGAGCCTACTTCGAGACGA?-1150

||?||?||||||||?||?||?|||||?|| ||||?|||||?||?||?|FPHY -CTCCGCTTCCTGGGTTGTTCCATTCGGTGCTAGAGCTTACTTTGAAACTA?-1150FPHY1 -TGCAATGCAAGTCGGAAAAGGAGCCTCTTGTTCGCGCTTTGATTAATGAC?-1200

||||||||||?||?|||||?||?|| |||?|?|?| ||||||||?|||FPHY -TGCAATGCAAATCCGAAAAAGAACCATTGGTTAGAGTCTTGATTAACGAC?-1200FPHY1 -CGGGTTGTGCCACTGCATGGCTGCGATGTGGACAAGCTGGGGCGATGCAA?-1250

|?|||||?|||?|| ||?|||||?||?||||| |||| |||||||FPHY -AGAGTTGTTCCATTGTTGGGATGCGACGTTGACAAATTGGGAAGATGCAA?-1250FPHY1 -GCTGAATGACTTTGTCAAGGGATTGAGTTGGGCCAGATCTGGGGGCAACT?-1300

||||?|||||?||?||?|||||| ||||||?|||||?||?||?||||FPHY -ATTGAACGACTTCGTTAAAGGATTGTCTTGGGCTAGATCCGGTGGTAACT?-1300FPHY1 -GGGGAGAGTGCTTTAGTTGA?-1320

||||?||?||||| |?|FPHY -GGGGTGAATGCTTCTCCTAA?-1320

Phytase is as animal feedstuff additive, and the promoter action of growth of animal has been obtained conclusive evidence, but full use not also in fodder industry at present, and major cause is that the production bacterial strain expression amount of phytase is low, is difficult to obtain large-tonnage product.Utilize pichia spp (P.pastoris) Expressing Recombinant Phytase can improve the expression level of phytase greatly, expression amount is brought up to the 1-10 milligram by every milliliter of several micrograms of original strain, in pichia spp, expressed the phyA2 gene that derives from A.niger NRRL3135, can make the expression amount of phytase improve nearly 3000 times.Can carry out the glycosylation modified protein translation post-treatment that waits in this external yeast, improve the biological activity of enzyme.

Beneficial effect of the present invention:

1, phytase of the present invention has obviously high thermotolerance, handles 80 minutes for 90 ℃.The activity of enzyme still has 40%.And common product was handled 30 minutes down at 90 ℃, and enzymic activity destroys 80%.

2, phytase gene of the present invention is more suitable for transcriptional expression in methanol yeast, and two genes are transformed in the yeast simultaneously, and expression amount is 13 times of wild type gene.Through high density fermentation, expressing quantity is every liter of 5.6g.

3, after Tu Bian phytase gene was expressed in yeast, enzymic activity pH value scope was big, pH2.5-6.5.When the pH value was 5.5-6.5, it is the highest that phytase activity reaches.

Description of drawings:

Fig. 1 is the yeast expression phytase carrier that is built into.

After Fig. 2 is the phytase strain fermentation, be subjected to the methanol induction different time, the secreting, expressing of phytase.

Fig. 3 represents under the different PH conditions, the relative vigor of phytase.

Fig. 4 is illustrated in 90 ℃ of high temperature different times and handles down, the relative vigor of phytase.

EmbodimentEmbodiment 1: the chemosynthesis of high temperature resistant phytase gene

1. utilize continuous extension PCR to synthesize high temperature resistant phytase gene.Primer length is 70-90bp, is connected by the 20-30bp overlap between 1 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.

High temperature resistant phytase gene primer is:

phy1：

TCCAAATCCTGCGACACCGTTGACTTGGGATACCAATGCTCCCCTGCTACCTCCCACCTTTGG

GGACAATAC

phy2：

ACCTCCCACCTTTGGGGACAATACTCTCCATTCTTCTCTTTGGAGGACGAACTTTCCGTTTCCTC

CAAATTGCCAAAAGATTGCAGAATC

Phy3：

CAAATTGCCAAAAGATTGCAGAATCACCTTGGTTCAAGTCTTGTCCAGACACGGTGCTAGATA

CCCAACCTCCTCCAAATCCAAAAAATAC

Phy4：

CAACCTCCTCCAAATCCAAAAAATACAAAAAACTGGTCACTGCTATCCAAGCTAACGCTACCG

ACTTCAAAGGAAAATTCGCTTTCTTG

Phy5：GACTTCAAAGGAAAATTCGCTTTCTTGAAAACCTACAACTACACCTTGGGAGCTGACGA

CTTGACCCCATTCGGAGAACAACAACTTG

Phy6：

GACCCCATTCGGAGAACAACAACTTGTCAACTCTGGAATCAAATTCTACCAAAGATACAAAGC

ACTTGCTAGATCCGTTGTTCCTTC

Phy7：

CACTTGCTAGATCCGTTGTTCCTTTCATCAGAGCTTCTGGCTCCGACAGAGTTATCGCTTCTGG

AGAAAAATTCATCGAAGGATTCCAAC

Phy8：

CTGGAGAAAAATTCATCGAAGGATTCCAACAAGCTAAATTGGCTGACCCTGGAGCTACCAACA

GAGCTGCTCCTGCTATCTCCG

Phy9：

CCAACAGAGCTGCTCCTGCTATCTCCGTTATCATCCCAGAATCCGAAACCTTCAACAACACCTT

GGACCACGGTGTTTGCACCAAATTC

Phy10：

GGACCACGGTGTTTGCACCAAATTCGAGGCTTCTCAACTTGGAGACGAGGTCGCTGCTAACTT

CACCGCTTTGTTCGCTCCTGACATC

Phy11：

CAAGGAAACAACGTCTTCGTCGGTCAAGGTGACACCAGGCAAGTGTTTTTCAGCTCTAGCTCT

GATGTCAGGAGCGAACAAAGCGGTG

Phy12：

GCAGAATGGGGAAAGTTGGGAAGCGTCGGAGGTTCTAGCAACGGTGTCGAAGGAGCACATAT

CCATCAAGGAAACAACGTCTTCGTC

Phy13：

CCGTAGTATTTTTCCCAAGGACTGCAGGTAGTTGTATTTTTTCCATTCGTTGTGGGTGAAAAGTTG

GCAGAATGGGGAAAGTTGGGAAG

Phy14：

CAATCTAGCGATCAATTCGTTGGTGAATCCGATTCCTTGAGCTGGTCCCAATGGGTTTCCAGCT

CCGTATCCGTAGTATTTTTCCCAAGGACTG

Phy15：

GTAGCTGGGTTGGAAACCAAGGTGGAGTTAGTGGAGGTGTGGTCTTGAACTGGGGATCTGGTC

AATCTAGCGATCAATTCGTTGGTG

Phy16：

GAAGAAGATGGAGACCATAGAGTTGTCGTGGGAGAAGTCAACGTACTAGGTAGCGTTCAATGG

GAAGGTAGCTGGGTTGGAAACCAAGGTGG

Phy17：

GTTCTTTAGCGGATTCAACGGAAGTTCTGGACAATGGTTCAGTTCCGTTGTAAAGTCCCAAAG

CGAAGAAGATGGAGACCATAGAGTTG

Phy18：

CATTGCATAGTTTCAAAGTAAGCTGTAGCACCGAATGGAACAACCCAGGAAGCGGAGTATCCG

TCAAGTTCTTTAGCGGATTCAACGGAAGTTC

Phy19：

GCATCCGTGCAATGGAACAACTCTGTCGTTAATCAAGGCTCTAACCAATGGTTCTTTTCTTATTT

GCATTGCATAGTTTCAAAGTAAGC

Phy20：

ACCACCGGATCTAGCCCAAGACAATCCTTTAACGAAGTCGTTCAATTTGCATCTTCCCAATTTG

TCAACGTCGCATCCGTGCAATGGAACAACTC

Phy21：

TTAGGAGAAGCATTCACCCCAGTTACCACCGGATCTAGCCCAAGACAATC embodiment 2: the dna molecular of high temperature resistant phytase gene is reset acid phytase gene of (DNA Shuffling) 2.1 pcr amplifications and recovery

With synthetic phytase gene is template, and FphyZ1, FphyF1 are the primer amplification phytase gene, FphyZ1:(5 '-TTGGATCCTCCAAATCCTGCGACACCGTTGACTTG-3 '); FphyF1:(5 '-CGAGCTCTTAGGAGAAGCATTCACCCCAGTTACCAC-3 ') reaction conditions is: 94 ℃ of pre-sex change of 10min, 94 ℃ of sex change 30s anneal and extension 1.5min, 30 circulations for 72 ℃ totally, the 1%Agrose electrophoresis, saturating gene fragment of inhaling bag method recovery 1.4kp.2.2 DNase I degradation of dna and recovery small segment

Reclaim the Fphy gene fragment with DNase I damping fluid (50mmol/L Tris-Cl pH7.4+1mmol/LMgCl 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 l10 * no primer PCR damping fluid (Primerless PCRBuffer) (50mmol/L KCl+10mmol/L Tris-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/L dNTPs+4.5 μ l25mmol/L MgCl 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+FphyZ10.2ng+FphyF1 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 1.4kp gene fragment.2.5 high-activity phytase screening

Reclaim the rearrangement phytase gene fragment of 1.4kb, behind BamH I and Sac I 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.

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 takes out 10 μ l bacterium liquid and transfers in the test tube and cultivate 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 cultivate, totally 20 test tubes, the LB nutrient solution 3mL that every pipe adds the band penbritin cultivates after 8-12 hour, centrifugal recovery thalline, the ultrasonication cell, centrifuging and taking supernatant 100 μ l add POTASSIUM PHYTATE sodium substrate, 37 ℃ were reacted 30 minutes, add 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 Fphy that height ratio is lived.Embodiment 3: yeast expression phytase vector construction

Press the two ends sequences Design primer of synthetic gene, add Xho I point of contact and signal attitude cutting sequence at the gene 5 ' end, primer is: FP1Z (5 '-AACTC GAGAAAAGAGAGGCTGAGGCTTCCAAATCCTGCGAC ACCGTTGACTTG-3 ') adds Not I point of contact at gene 3 ' end: primer is FP1F (5 '-AACGCGGCCGCTTAGGAGAAGCATTCACCCCAGTTAC-3 ').Behind the amplified fragments clone, Xho I and Not I double digestion, orientation is inserted pPIC9 carrier (Invetrogen company), is built into the Yeast expression carrier pPfphy (Fig. 1) of fphy.Embodiment 4: the screening of phytase high expression level recombination yeast

With activatory yeast strain Pichia Pastoris 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 liquor that goes 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 pPfphy, 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%Biotin; 0.5% methyl alcohol; 1.5% agarose) and MD (1.34%YNB; 0.00004%Biotin, 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, 1.34%YNB 0.000004%Biotin, 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 68 of recombinant conversion of site-directed integration, called after P.pastoris FPHY1,2,3.....68.

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.pastoris FPHY34 of 3 plant height efficient expression phytase genes, P.pastoris FPHY41, P.pastoris FPHY57.Embodiment 5: the high density fermentation of recombinant bacterial strain

Picking phytase high expression level recombination yeast P.pastoris FPHY34 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 FPHY34 induced 60hr, the phytase expression amount was up to 5.6mg/ml.The phytase molecule amount size of expressing is about 85kD.

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 FPHY34 cultivated 60hr, enzyme activity was 130, and 000u/ml increases the abduction delivering time, and the expression amount of phytase only slowly increases.Embodiment 6: 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 when the pH value was 5.5-6.5, it is the highest that phytase activity reaches, and does not have considerable change (Fig. 3) toward neutral range skew enzymic activity.

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 80min, the activity of phytase still keeps 40%.90 ℃ of pyroprocessing 120min still have the part activity.(Fig. 4)

Claims

1, a kind of high temperature resistant phytase gene with following coding: TCCAAATCCTGCGACACCGTTGACTTGGGATACCAATGCTCCCCTGCTACCTCCCA CCTTTGGGGACAATAC S K S C D T V D L G Y Q C S P A T S H L W G Q YTCTCCATTCTTCTCTTTGGAGGACGAACTTTCCGTTTCCTCCAAATTGCCAAAAG ATTGCAGAATCACCTTG S P F F S L E D E L S V S S K L P K D C R I T LGTTCAAGTCTTGTCCAGACACGGTGCTAGATACCCAACCTCCTCCAAATCCAAAA AATACAAAAAACTGATC V Q V L S R H G A R Y P T S S K S K K Y K K LIACTGCTATCCAAGCTAACGCTACCGACTTCAAAGGAAAATACACTTTCTTGAAAAC CTACAACTACACCTTG T A I Q A A T D F K G K Y TF L K T Y Y T LGGAGCTGACGACTTGACCCCATTCGGAGAACAACAACTTGTCAACTCTGGAATCAAATTCTACCAAAGATAC G A D D L T P F G E Q Q L V N S G I K F Y Q R YAAAGCACTTGCTAGATCCGTTGTTCCTTTCATCAGAGCTTCTGGCTCCGACAGAGTTATCGCTTCTGGAGAA K A L A R S V V P F I R A S G S D R V I A S G EAAATTCATCGAAGGATTCCAACAAGCTAAATTGGCTGACCCTGGAGCTACCAACAGAGCTGCTCCTGCTATC K F I E G F Q Q A K L A D P G A T N R A A P A ITCCGTTATCATCCCAGAATCCGAAACCTTCAACAACACCTTGGACCACGGTGTTTGCACCAAATTCGAGGCT S V I I P E S E T F N T L D H G V C T K F E ATCTGAACTTGGAGACGAGGTCGAGGCTAACTTCACCGCTTTGTTCGCTCCTGACA TCAGAGCTAGAGCTGAAS EL G D E V EA N F T A L F A P D I R A R A EAAACACTTGCCTGGTGTCACCTTGACCGACGAAGACGTTGTTTCCTTGATGGATA TGTGCCCCTTCGACACC K H L P G V T L T D E D V V S L M D M C PF D TGTTGCTAGAACCTCCGACGCTTCCCAACTTTCCCCATTCTGCCAACTTTTCACCCACAACGAATGGAAAAAA V A R T S D A S Q L S P F C Q L F T H N E W K KTACAACTACCTGCAGTCCTTGGGAAAATACTACGGATACGGAGCTGGAAACCCATTGGGACCAGCTCAAGGA Y N Y L Q S L G K Y Y G Y G A G N P L G P A Q GATCGGATTCGCCAACGAATTGATCGCTAGATTGACCAGATCCCCAGTTCAAGACCACACCTCCACTAACTCC I G F AN E L I A R L T R S P V Q D H T S T SACCTTGGTTTCCAACCCAGCTACCTTCCCATTGAACGCTACCATGTACGTTGACT TCTCCCACGACAACTCT T L V S N P A T F P L A T M Y V D F S H D N SATGATCTCCATCTTCTTCGCTTTGGGACTTTACAACGGAACTGAACCATTGTCCA GAACTTCCGTTGAATCC M IS I F F A L G L Y G T E P L S R T S V E SGCTAAAGAACTTGACGGATACTCCGCTTCCTGGGTTGTTCCATTCGGTGCTAGAG CTTACTTTGAAACTATG A K E L D G Y S A S W V V P F G A R A Y F E T MCAATGCAAATCCGAAAAAGAACCATTGGTTAGAGTCTTGATTAACGACAGAGTTG TTCCATTGCACGGATGC Q C K S E K E P L V R VL I N D R V V P L H G CGACGTTGACAAATTGGGAAGATGCAAATTGAACGACTTCGTTAAAGGATTGTCTT GGGCTAGATCCGGTGGT D V D K L G R C K L N D F V K G L S W A R S G GAACTGGGGTGAATGCTTCTCCTAA N W G E C F S-

2, according to the described high temperature resistant phytase gene of claim 1, it is characterized in that this phytase contains 6 sugar and intensifies the site, mrna length is 1320bp, and nucleotide homology is 73%, and the Nucleotide password all is a yeast preference password.

3, high temperature resistant according to claim 1 phytase gene preparation method is following steps:

A, the synthetic high temperature resistant phytase gene of the continuous extension PCR of employing;

B, the high temperature resistant phytase gene of above synthetic are template, utilize dna molecular rearrangement technology that synthetic gene is carried out vitro recombination, the phytase gene that FphyZ 1, FphyF 1 reset for primer amplification, reclaim the phytase gene fragment that 1.4Kb resets, be building up in the prokaryotic expression carrier, electric shocking method changes coli strain DH5 α over to, obtains the mutant expression library and carries out the high-specific-activity phytase screening;

C, press the two ends sequences Design primer of synthetic gene, add Xho I point of contact and signal attitude cutting sequence at the gene 5 ' end, primer is: FP1Z (5 '-AACTCGAGAAAAGAGAGGCTGAGGCTTCCAA ATCCTGCGACACCGTTGAC TTG-3 '), and add Not I point of contact at gene 3 ' end: primer is: FP1F (5 '-AACGCGGCCGCTTAGGAGAAGCATTCACCCCAGTTAC-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 pPfphy of high temperature resistant phytase gene.

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

E, with recombination yeast bacteria strain high density fermentation.

4,, it is characterized in that the Yeast expression carrier pPfphy that makes up contains the described high temperature resistant phytase gene sequence of claim 1 according to the described preparation method of claim 3 with the high temperature resistant phytase gene of yeast expression.

5, the preparation method with the high temperature resistant phytase gene of expression according to claim 3, it is characterized in that synthetic phytase gene is by behind the external molecular transposition, adopting primer is FphyZ 1 and FphyF 1, amplified fragments, BamHI and SacI double digestion are directed to be inserted between promotor and the t1+2 terminator, is built into phytase gene prokaryotic expression carrier storehouse.