CN101638642B - Cyclized phytase with improved heat stability and proteinase resistance - Google Patents

Cyclized phytase with improved heat stability and proteinase resistance Download PDF

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CN101638642B
CN101638642B CN2008101175968A CN200810117596A CN101638642B CN 101638642 B CN101638642 B CN 101638642B CN 2008101175968 A CN2008101175968 A CN 2008101175968A CN 200810117596 A CN200810117596 A CN 200810117596A CN 101638642 B CN101638642 B CN 101638642B
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cyclisation
gene
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sequence
enzyme
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CN101638642A (en
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平淑珍
赵仲麟
林敏�
陆伟
陈明
张维
燕永亮
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MIANYANG HABIO BIOENGINEERING CO.,LTD.
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Biotechnology Research Institute of CAAS
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Abstract

The invention relates to cyclase with improved heat stability and proteinase resistance, and a method for cyclizing the cyclase to improve properties of the cyclase. The method cyclizes protein by trans-splicing natural inteins; and the cyclized protein has better heat stability and proteinase resistance. The invention also provides a recombinant bacillus coli cell containing phytase genes.

Description

Improved the cyclisation Sumizyme PHY of thermostability and protease resistant
Technical field
The present invention relates to a kind of cyclase that has improved thermostability and protease resistant, the invention still further relates to proteolytic enzyme is carried out cyclisation to improve the method for its performance.
Background technology
There are many deficiencies in natural protein, especially zymoprotein aspect stable, such as poor heat resistance, are prone to by protease hydrolysis etc., have limited their application in medicine and industrial production.
For example, Sumizyme PHY (E.C.3.1.3.8) be a kind of can be the enzyme of inositol and phosphoric acid with hydrolysis of phytic acid.Can phytic acid and phytate be hydrolyzed into inositol and phosphoric acid salt after adding Sumizyme PHY in the feed, supply animal to absorb, thereby improve the utilization ratio and the mineralization of skeleton degree of phosphorus, can practice thrift phosphor resource, reduce feed cost.But Sumizyme PHY is to thermally labile, and the granulation link of Sumizyme PHY production process, temperature is 75-90 ℃, and will continue several minutes, the activity of enzyme can significantly be lost in this process, influences its application aborning.Therefore the stability that improves Sumizyme PHY becomes urgent problem.
The albumen cyclisation is a kind of new technology, and the albumen of cyclisation has a lot of benefits than the albumen of line style, for example can increase proteic stability, reduces the resistance that albumen is sheared lytic enzyme, strengthens the flexibility of proteic activity and minimizing protein conformation etc.
The application project intein comes cyclisation protein, promptly uses bacterial expression system production cyclisation albumen, and this method has been opened up the approach that improves protein stability.This cyclization method realizes that through the protein splice reaction protein sequence from an inside of amyloid protein precursor release is called intein.In the montage process of inner protein sequence, intein is by in the embedding amyloid protein precursor, the shearing of catalysis self, and then couple together the protein sequence of both sides.The protein fusion expression of purpose in two portions of intein, is formed a sandwich structure.When intein was released, the C of target protein, N end place formed a peptide bond, make the target protein cyclisation, and proteinic stability will improve.
Summary of the invention
General purpose of the present invention is a kind of new zymoprotein cyclization reaction of exploitation, and the cyclase with thermostability and protease resistant is provided.
Another object of the present invention provides and in bacterial expression system, efficiently expresses the method for producing cyclase.
Technical solution
The invention provides a kind of enzyme of cyclisation, it is characterized in that, preceding 5 amino acid of its peptide sequence have SEQ IDNO:1 sequence, and last 5 amino acid have SEQ ID NO:2 sequence; And the N of zymoprotein, C two ends are linked.
The N of the zymoprotein of said cyclisation, C two ends are linked, and are through the cyclization method of intein mediation, link with N, the C two ends of natural peptide bond with zymoprotein.
In an instance of the present invention, the neutral phytase gene PhyC of subtilis (Bacillus subtilis) is carried out cyclisation, the gene of this cyclisation Sumizyme PHY of encoding has the nucleotide sequence shown in SEQ ID NO:3.Through experiment confirm, the thermostability and the resistance towards proteases of the Sumizyme PHY after the cyclisation all increase.
The present invention also provides a kind of method of cyclase protein; Comprise the structure of a whole set of expression vector, the screening of reorganization word and the authentication method of cyclisation molecule; Preceding 5 amino acid are had SEQ ID NO:1 sequence, and last 5 amino acid have the cyclisation of encoding sox in intestinal bacteria of SEQ ID NO:2 sequence expresses.
The method of said cyclase protein is to utilize the commentaries on classics montage characteristic of natural collection born of the same parents cyanobacteria (Synechocystis species PCC6803) Ssp DnaE intein, respectively at vivo and vitro to the protein cyclisation.
In an instance of the present invention; Said zymoprotein is a Sumizyme PHY albumen; Cyclization method is that the cyclisation of gene in intestinal bacteria of nucleotide sequence shown in the SEQ ID NO:3 expressed; Through the commentaries on classics splicing cyclisation neutral phytase PhyC of intein, the gene of said Ssp DnaE intein has the nucleotide sequence shown in SEQ ID NO:4.Concrete grammar is following:
1. the cyclisation vector construction is by ordinary method clone (New York:Cold Spring Harbor LaboratoryPress, 1989).PCR from the dnaE gene of collection born of the same parents cyanobacterias (Synechocystis species PCC6803), the N end that obtains Ssp DnaE intein is respectively held 2 parts with C, after be connected into the pMXB10 carrier, formation expression vector pEB1.
2. gene clone PCR from the genome of subtilis (Bacillus subtilis) obtains a neutral phytase gene phyC, is cloned among the above-mentioned expression vector pEB1.Be transformed into intestinal bacteria, obtain recon.
3. proteic expression and purifying add IPTG abduction delivering PhyC Sumizyme PHY, through the annular Sumizyme PHY that generates in the nickel post affinity purification body; Obtain external annular Sumizyme PHY through the Regitex FA affinity column.Verify proteic cyclisation situation.
Sumizyme PHY with method improvement of the present invention has better thermostability.Present method is that the production of body inner ring type Sumizyme PHY is laid a good foundation.
Description of drawings
Fig. 1 is the physical map of intestinal bacteria recombinant vectors pPEB;
Fig. 2 is the physical map of intestinal bacteria recombinant vectors pEB1;
Fig. 3 representes under 40～75 ℃, and cyclisation Sumizyme PHY cPhyC and wild Sumizyme PHY PhyC be in each temperature insulation after 1 hour, the cyclisation Sumizyme PHY that draws and the thermostability of wild Sumizyme PHY;
The optimal reactive temperature of Fig. 4 cyclisation Sumizyme PHY;
Fig. 5 cyclisation Sumizyme PHY is to the resistance of exopeptidase.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be understood that this embodiment only is used to illustrate method of the present invention, and do not limit the scope of used zymoprotein.
Experiment condition
Bacterial strain and carrier coli strain E.coli BL21 (DE3) are available from Novagen company, and carrier is by this research and establishment and preservation.
Enzyme and test kit restriction enzyme, ligase enzyme, Taq enzyme are the NEB Company products.It is a day root biochemical corp product that genome extracts test kit.Zymoplasm is the Merk Company products.
Biochemical reagents DNA synthetic agent is the Milipore Company products.Primer synthesizes the Cyclone of ABI company dna synthesizer.IPTG, X-Gal, SDS and Sumizyme PHY sodium are the Sigma Company products.TEMED, ammonium persulphate, acrylic amide and methylene diacrylamide are the Promega Company products.The NTA resin is the Qiagen Company products.The Regitex FA resin is the NEB Company products.
Substratum intestinal bacteria substratum be LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).
The experimental technique of other unreceipted actual conditions among the embodiment; Carry out according to ordinary method; Method like people such as Sambrook; Molecular cloning is by the condition described in (New York:Cold Spring Harbor Laboratory Press, 1989) laboratory manual, or the condition of advising according to manufacturer.
Embodiment 1 cyclisation Sumizyme PHY
1) structure of cyclisation expression vector
The coli expression carrier pEB1 that is used for cyclisation.At first extract the genome of collection born of the same parents cyanobacterias (Synechocystis species).With the genome that is extracted is template, the C terminal sequence of pcr amplification dnaE.Employed primer is synthetic according to 5 ' and 3 ' terminal sequence of the C fragment gene of dnaE; 2 primer sequences are respectively: 5 ' ATGGTTAAAGTTATCGGTC 3 ' and 5 ' GTTAGCTGCGATAGCGCC 3 '; Carry out gel electrophoresis after the amplification, reclaim the fragment of big or small 108bp.In addition, be template with the genome that is extracted, the N terminal sequence of pcr amplification dnaE.Employed primer is synthetic according to 5 ' and 3 ' terminal sequence of the N fragment gene of dnaE; 2 primer sequences are respectively: 5 ' TGCCTGTCTTTTGGTACC 3 ' and 5 ' TTTAATTGTC CCAGCGTC 3 '; Carry out gel electrophoresis after the amplification, reclaim the fragment of big or small 369bp.Afterwards the C fragment gene of dnaE is cut through HindIII and AflII enzyme enzyme, the N fragment gene of dnaE is connected into the pMXB10 carrier after AflII and EcoRI enzyme are cut, and forms pEBl carrier (see figure 2), is the T7 promotor before the C fragment gene of dnaE.
2) clone of phytase gene molecule
Extract subtilis (Bacillus subtilis) genome.With the genome that is extracted is template, carries out pcr amplification, and electrophoresis reclaims the about 1124bp fragment of Sumizyme PHY phyc gene.Employed primer is synthetic according to 5 ' and 3 ' terminal sequence of phyc gene, and 2 primer sequences are respectively:
5 ' CTTAAGCACCATCATCATCATCATAAGCATAAGCTGTCCGATC 3 ' with
5’ATCGATGCTACCACGCGGAACCAGTTTTCC GCTTCTGTC 3’，
Carry out gel electrophoresis after the amplification, reclaim the fragment of big or small 1124bp, SEQ ID NO:3 is the base sequence of phytase phyC, and SEQ ID NO:5 is its aminoacid sequence.The dna molecular that obtains is cut through the AflII enzyme, is connected in 4 ℃ with the pEB1 carrier that obtains in the experiment of cutting through enzyme one and spends the night, and transformed into escherichia coli BL21 (DE3) screens positive recombinant behind the LB substratum coated plate, extracts plasmid and identifies, obtains recon.Recombinant plasmid pPEB collection of illustrative plates is seen Fig. 1.
3) program of cyclisation Sumizyme PHY expression and purification
The recon that experiment two is obtained is inoculated in the LB substratum of 20mL, and 37 ℃ of shaking culture are spent the night, and are inoculated in the 1LB substratum by 10% inoculum size; With 37 ℃ to be cultured to OD be 0.6; Add IPTG to final concentration 0.5mM, continue to cultivate the abduction delivering Sumizyme PHY 3 hours.Centrifugal collection thalline, thalline are resuspended in Tris-HCl (pH7.0) damping fluid that 100mL contains 500mM NaCl, the ultrasonic disruption thalline, and 15000rpm removed cell debris in centrifugal 30 minutes.Supernatant carries out purifying through the strain of NTA nickel.With the chromatography column that it is suitable that the NTA resin is packed into, chromatography is washed with the NTA-0Buffer (20mM Tris-HCl pH7.9,0.5M NaCl, 10% glycerine) of 10 times of NTA volumes.The centrifugal back of sample supernatant is added in the NTA chromatography column, and flow rate control is collected penetrating component about 15ml/ hour, be used for the combination situation of SDS/PAGE analysing protein.Chromatography is washed with the NTA-0Buffer of 5 times of NTA volumes, and flow rate control is about 30ml/ hour.Use the NTA-20 (20mM Tris-HCl pH7.9,0.5M NaCl, 10% glycerine, 20mM imidazoles) of 5 times of NTA volumes respectively, NTA-40 (20mM Tris-HCl pH7.9; 0.5M NaCl, 10% glycerine, 40mM imidazoles), NTA-60 (20mM Tris-HCl pH7.9,0.5M NaCl; 10% glycerine, the 60mM imidazoles), NTA-80 (20mM Tris-HCl pH7.9,0.5M NaCl, 10% glycerine; The 80mM imidazoles), NTA-100 (20mM Tris-HCl pH7.9,0.5M NaCl, 10% glycerine; The 100mM imidazoles) buffer solution elution, flow rate control are collected elutriant about 15ml/ hour, every pipe is collected a NTA volume.SDS/PAGE confirms the distribution situation of target protein in elutriant.
4) external cyclisation of Sumizyme PHY and purifying
The recon that experiment two is obtained is inoculated in the LB substratum of 20mL, and 37 ℃ of shaking culture are spent the night, and are inoculated in the 1LB substratum by 10% inoculum size; With 37 ℃ to be cultured to OD be 0.6; Add IPTG to final concentration 0.5mM, continue to cultivate the abduction delivering Sumizyme PHY 3 hours.Centrifugal collection thalline, thalline are resuspended in Tris-HCl (pH7.0) damping fluid that 100mL contains 500mM NaCl, the ultrasonic disruption thalline, and 15000rpm removed cell debris in centrifugal 30 minutes.Supernatant is added the Regitex FA post slowly, and (20mM Tris-HCl pH7.9 0.5MNaCl) thoroughly washes post to use the damping fluid 1 of 20 times of column volumes afterwards.Stopping post stream placed 15 hours for 23 ℃.Collect the target protein wash-out with damping fluid 1 back.Approximately collect the 8-10 pipe, every pipe 5ml.Detect through 280nm UV light absorption value or Bradford Protein Detection method.
Embodiment 2 cyclisation Sumizyme PHY property testings
1) measuring method of Sumizyme PHY zymologic property
Have the collection tube of purpose Sumizyme PHY to use and measure phytase activity, the enzyme activity determination method is following: with supernatant with damping fluid (50mM Tris-HCl, 2mM CaCl2,10% glycerine; PH710) do suitable dilution after, get 015ml enzyme liquid, add sodium phytate (the 100mM Tris-HCl of 2ml 2mM; PH710,012mM CaCl2), 37 ℃ of insulation 30min; Add 215ml 20%TCA (w/v) and stop the enzyme reaction of living, the centrifugal 5min of 5000r/min adds 5ml ferrous sulfate-ammonium molybdate colour developing liquid [(115% ammonium molybdate: 515% sulfuric acid): (217% ferrous sulfate)=4: 1 then; V/v], the centrifugal 5min of 5000r/min, 700mM measures inorganic phosphate content.Contrast makes enzyme deactivation in the enzyme diluent of 015ml, adding 215ml20%TCA earlier, adds the substrate insulation with volume again.Unit of enzyme activity (U) is defined as: under certain condition, it is a unit of enzyme activity that PM discharges the required enzyme amount of 1nmol inorganic phosphorus.
2) thermal stability determination of cyclisation Sumizyme PHY and wild-type Sumizyme PHY relatively
Enzymatic reaction is carried out in being determined under Tris-HCl (pH 7.0) buffer solution system and the differing temps of the optimum temperuture of Sumizyme PHY.Thermal stability determination is that Sumizyme PHY was handled 1 hour under differing temps, under 37 ℃, carries out enzyme activity determination.The enzyme reaction optimum temperuture is measured result (Fig. 4) and is shown that its optimum temperuture is 58 ℃.The thermostability experiment of enzyme shows (Fig. 3), handles after 1 hour for 60 ℃, and residual enzyme work is 65%, 70 ℃ to be handled 1 hour, enzyme basic forfeiture alive.And the wild-type Sumizyme PHY is handled after 1 hour for 60 ℃, and residual enzyme work is 39%, 65 ℃ to be handled 1 hour, and residual enzyme work is 20%, 70 ℃ to be handled 1 hour, enzyme basic forfeiture alive.
3) the exopeptidase resistant determination of cyclisation Sumizyme PHY and wild-type Sumizyme PHY relatively
The exopeptidase resistant determination of cyclisation Sumizyme PHY and wild-type Sumizyme PHY adds carboxypeptidase y at Tris-HCl (pH 7.0) buffer solution system, handles 12 hours for 25 ℃.Measure result (Fig. 5) and show, handle after 12 hours for 25 ℃, the cyclisation Sumizyme PHY is not degraded, and the wild-type Sumizyme PHY receives the exopeptidase effect, and partly degraded has taken place.Degraded has tangible resistance to the Sumizyme PHY of description of test cyclisation to exopeptidase.
The sequence table explanation
SEQ ID NO:1 be the cyclase peptide sequence preceding 5 amino acid
SEQ ID NO:2 is last 5 amino acid of cyclase peptide sequence
SEQ ID NO:3 is the gene order of the neutral phytase gene phyc of subtilis (Bacillus subtilis).
SEQ ID NO:4 is the gene order of dnaE
SEQ ID NO:5 is the phyc amino acid sequence coded of deriving from SEQ ID NO:3.
Sequence table
< 110>Biological Technology institute, Chinese Academy of Agricultural Sciences
< 120>improved the cyclisation Sumizyme PHY of thermostability and protease resistant
<160>5
<170>PatentIn version 3.1
<210>1
<211>5
<212>PRT
< 213>artificial sequence
<400>1
Cys Phe Asn Ile Ser
1 5
<210>2
<211>5
<212>PRT
< 213>artificial sequence
<400>2
Lys Phe Ala Glu Tyr
1 5
<210>3
<211>1194
<212>DNA
< 213>subtilis (Bacillus subtilis)
<400>3
tgctttaaca tatccaccgg tcttaagctt aagcaccatc atcatcatca taagcataag 60
ctgtccgatc cttatcattt taccgtgaat gcggcggcgg aaacggaacc ggttgatacg 120
gcaggtgacg cggctgatga tcctgcgatt tggctggatc ccaagactcc tcagaacagc 180
aaattgatta cgaccaataa aaaatcaggt ttagtcgttt acagccttga tggtaagatg 240
cttcattcct ataataccgg gaagctgaac aatgtcgata tccgttatga ttttccgttg 300
aacggaaaaa aagtcgatat cgcggcagca tccaatcgct ctgaaggaaa aaataccatt 360
gatatttacg ccattgacgg gaaaaacggc acattgcaaa gcattacaga tccagaccat 420
ccgattgcat cggcaattaa tgaggtatac ggttttacct tataccacag tcaaaaaaca 480
ggaaaatatt acgcgatggt gacagggaaa gagggtgaat ttgaacaata cgaattaaag 540
gcggacaaaa atggatacat atccggcaaa aaggtacggg cgtttaaaat gaattcccag 600
acggaaggga tggcagcaga cgatgaatac ggcaggcttt atatcgcaga agaagatgag 660
gccatttgga agttcagcgc cgagccggac ggcggcagta acggaacagt tatcgaccat 720
gccgacggca ggcatttaac tcctgatatt gaaggattga cgatttacta cgctgctgac 780
gggaaaggct atctgatggc atcaagccag ggaaacagca gctacgccat ttatgacaga 840
aaaggacaga acaaatatgt tgcggatttt cgcataacag atggtcctga aacagacggc 900
acaagcgata cagacggaat tgacgttctg ggtttcggac ttgggcctga atatccgttc 960
ggtatttttg tcgcacagga cggtgaaaat atagatcacg gccaaaaggc caatcaaaat 1020
cttaaaatcg tgccgtggga aagaattgct gatcaaatcg gcttccgccc gctggcaaat 1080
aaacaggttg acccgagaaa actgaccgac agaagcggaa aactggttcg gcgtggtagc 1140
atcgataacc tcgggatcga gggaaggggt acgctcgaga aatttgctga atat 1194
<210>4
<211>477
<212>DNA
< 213>collection born of the same parents cyanobacterias (Synechocystis species PCC6803)
<400>4
atggttaaag ttatcggtcg tagatctctg ggcgtgcagc gcatctttga tatcggtctg 60
ccgcaggacc ataactttct gctagccaac ggcgctatcg cagctaactg cctgtctttt 120
ggtaccgaaa ttttaaccgt tgagtacggc ccattgccca ttggcaaaat tgtgagtgaa 180
gaaattaatt gttctgtgta cagtgttgat ccagaaggga gagtttacac ccaggcgatc 240
gcccaatggc atgaccgggg agagcaggaa gtattggaat atgaattgga agatggttca 300
gtaatccgag ctacctctga ccaccgcttt ttaaccaccg attatcaact gttggcgatc 360
gaagaaattt ttgctaggca actggacttg ttgactttag aaaatattaa gcaaactgaa 420
gaagctcttg acaaccatcg tcttcccttt ccattacttg acgctgggac aattaaa 477
<210>5
<211>398
<212>PRT
< 213>subtilis (Bacillus subtilis)
<400>5
Cys Phe Asn Ile Ser Thr Gly Leu Lys Leu Lys His His His His His
1 5 10 15
His Lys Hi s Lys Leu Ser Asp Pro Tyr His Phe Thr Val Asn Ala Ala
20 25 30
Ala Glu Thr Glu Pro Val Asp Thr Ala Gly Asp Ala Ala Asp Asp Pro
35 40 45
Ala Ile Trp Leu Asp Pro Lys Thr Pro Gln Asn Ser Lys Leu Ile Thr
50 55 60
Thr Asn Lys Lys Ser Gly Leu Val Val Tyr Ser Leu Asp Gly Lys MET
65 70 75 80
Leu His Ser Tyr Asn Thr Gly Lys Leu Asn Asn Val Asp Ile Arg Tyr
85 90 95
Asp Phe Pro Leu Asn Gly Lys Lys Val Asp Ile Ala Ala Ala Ser Asn
100 105 110
Arg Ser Glu Gly Lys Asn Thr Ile Asp Ile Tyr Ala Ile Asp Gly Lys
115 120 125
Asn Gly Thr Leu Gln Ser Ile Thr Asp Pro Asp His Pro Ile Ala Ser
130 135 140
Ala Ile Asn Glu Val Tyr Gly Phe Thr Leu Tyr His Ser Gln Lys Thr
145 150 155 160
Gly Lys Tyr Tyr Ala MET Val Thr Gly Lys Glu Gly Glu Phe Glu Gln
165 170 175
Tyr Glu Leu Lys Ala Asp Lys Asn Gly Tyr Ile Ser Gly Lys Lys Val
180 185 190
Arg Ala Phe Lys MET Asn Ser Gln Thr Glu Gly MET Ala Ala Asp Asp
195 200 205
Glu Tyr Gly Arg Leu Tyr Ile Ala Glu Glu Asp Glu Ala Ile Tr pLys
210 215 220
Phe Ser Ala Glu Pro Asp Gly Gly Ser Asn Gly Thr Val Ile Asp His
225 230 235 240
Ala Asp Gly Arg His Leu Thr Pro Asp Ile Glu Gly Leu Thr Ile Tyr
245 250 255
Tyr Ala Ala Asp Gly Lys Gly Tyr Leu MET Ala Ser Ser Gln Gly Asn
260 265 270
Ser Ser Tyr Ala Ile Tyr Asp Arg Lys Gly Gln Asn Lys Tyr Val Ala
275 280 285
Asp Phe Arg Ile Thr Asp Gly Pro Glu Thr Asp Gly Thr Ser Asp Thr
290 295 300
Asp Gly Ile Asp Val Leu Gly Phe Gly Leu Gly Pro Glu Tyr Pro Phe
305 310 315 320
Gly Ile Phe Val Ala Gln Asp Gly Glu Asn Ile Asp His Gly Gln Lys
325 330 335
Ala Asn Gln Asn Leu Lys Ile Val Pro Trp Glu Arg Ile Ala Asp Gln
340 345 350
Ile Gly Phe Arg Pro Leu Ala Asn Lys Gln Val Asp Pro Arg Lys Leu
355 360 365
Thr Asp Arg Ser Gly Lys Leu Val Arg Arg Gly Ser Ile Asp Asn Leu
370 375 380
Gly Ile Glu Gly Arg Gly Thr Leu Glu Lys Phe Ala Glu Tyr
385 390 395

Claims (6)

1. the Sumizyme PHY of a cyclisation, preceding 5 amino acid of its peptide sequence have SEQ ID NO:1 sequence, and last 5 amino acid have SEQ ID NO:2 sequence; And the N of zymoprotein, C two ends are linked; It is characterized in that encoding the nucleotide sequence of gene of this enzyme shown in SEQ ID NO:3.
2. it is the cyclization method through the intein mediation that the enzyme of the said cyclisation of claim 1, the N of said zymoprotein, C two ends are linked, and links with N, the C two ends of natural peptide bond with zymoprotein.
3. recombinant Bacillus coli cells contains the gene of nucleotide sequence shown in the SEQ ID NO:3.
4. the method for a cyclase protein comprises the structure of expression vector, the screening of recon and the authentication method of cyclisation molecule, it is characterized in that the cyclisation of gene in intestinal bacteria of the said polypeptide of coding claim 1 expressed.
5. the method for the described cyclase protein of claim 4 is a commentaries on classics montage characteristic of utilizing natural collection born of the same parents cyanobacteria (Synechocystis species) PCC6803Ssp DnaE intein, respectively at vivo and vitro to the protein cyclisation.
6. the method for claim 4 or 5 described cyclase proteins, the gene that the cyclisation in intestinal bacteria is expressed is the gene of sequence shown in the SEQ ID NO:3.
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MX2018003389A (en) 2015-09-18 2019-09-18 Agrivida Inc Engineered phytases and methods of using the same.

Citations (1)

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Publication number Priority date Publication date Assignee Title
CN1354788A (en) * 1998-12-18 2002-06-19 宾州研究基金会 Cyclic peptides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1354788A (en) * 1998-12-18 2002-06-19 宾州研究基金会 Cyclic peptides

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Akira Kanno 等.Cyclic luciferase for Real-Time Sensing of Caspase-3 Activities in Living Mammals.《Angewandte Chemie》.2007,第119卷(第40期),图b. *
刘梅英 等.从后处理工艺提高植酸酶热稳定性的研究进展.《饲料工业》.2007,第28卷(第11期),2-4. *
李素丽 等.蛋白质内含肽及其生物学意义.《生物技术通讯》.2005,第16卷(第5期),552-555. *
梁如冰.大肠杆菌条件性基因表达调控系统的构建与应用.《中国博士学位论文(基础科学辑)》.2007,(第4期),全文. *
郝岗平，杨清.蛋白质内含肽的研究进展.《生命科学研究》.2002,第6卷(第4期),136-139. *

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