CN101638642B - Cyclic phytase with improved thermostability and protease resistance - Google Patents

Abstract

The invention relates to a cyclase with improved thermal stability and protease resistance, and also relates to a method for cyclizing the protease to improve its performance. The invention utilizes the natural intein to transsplice the cyclized protein, and the cyclized protein has better thermostability and protease resistance. The present invention also provides recombinant Escherichia coli cells containing the phytase gene of the present invention.

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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