CN103820443A - Production and construction of yeast strain with cellulose incision enzyme and glucosidase double activity protein - Google Patents
Production and construction of yeast strain with cellulose incision enzyme and glucosidase double activity protein Download PDFInfo
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- CN103820443A CN103820443A CN201410084775.1A CN201410084775A CN103820443A CN 103820443 A CN103820443 A CN 103820443A CN 201410084775 A CN201410084775 A CN 201410084775A CN 103820443 A CN103820443 A CN 103820443A
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Abstract
The invention discloses production and construction of a yeast strain with a cellulose incision enzyme and glucosidase double activity protein, and belongs to the field of enzyme engineering. A brewer's yeast TDH3 promoter sequence, a brewer's yeast secretion signal peptide coding sequence, a sequence with cellulose incision enzyme activity and glucosidase activity and a brewer's yeast TDH3 terminator sequence are synthesized in sequence, added with restrictive incision enzyme sites at two ends, and constructed into a pPIC9K plasmid through enzyme digestion, and then the plasmid is transformed into yeast so as to obtain the yeast strain with cellulose incision enzyme and glucosidase double activity protein. The production and construction realizes the expression of the cellulose incision enzyme and glucosidase double activity protein in yeast, and has relatively strong practicability. The activity of the cellulose incision enzyme and the glucosidase in the yeast strain production can reach 1.2 U/mL and 0.3 U/mL respectively.
Description
Technical field
The invention belongs to enzyme engineering field, be specifically related to a kind of production and have yeast strain and the structure thereof of Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen.
Background technology
Mierocrystalline cellulose is renewable resources the abundantest on the earth, and the Mierocrystalline cellulose producing because of photosynthesis every year on the earth reaches 10,000,000,000 tons of left and right, utilizes cellulose raw to produce bioenergy, is alleviating energy crisis, realizes the key of human kind sustainable development.
Carbohydrate-glucose that the key of cellulose utilization is is fermentability cellulose degradation.Cellulosic degraded needs the acting in conjunction of excision enzyme, restriction endonuclease, Polyglucosidase.Wherein the Mierocrystalline cellulose of restriction endonuclease degraded long segment becomes dimer, is the committed step of cellulose degradation.There is green, mild condition, feature that transformation efficiency is high with cellulose degraded Mierocrystalline cellulose, but the higher production cost of cellulase becomes the cellulosic bottleneck of cellulose degraded.
Reducing effective means of cellulase cost is the utilising efficiency that improves enzyme.Due to the synergy of three kinds of enzymes of cellulosic degraded needs, the albumen with two kinds of functions or several functions can improve the utilising efficiency of cellulase significantly, thereby reduces the cost of cellulase industrialized utilization.The stability of cellulase is better simultaneously, and the degraded cellulose of cellulase energy longer time, therefore has larger using value.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming that overcomes prior art, with not enough, provides a kind of production to have the yeast strain of Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen.
Another object of the present invention is to be provided for building the DNA fragmentation of above-mentioned yeast strain.
A further object of the present invention is to be provided for building the plasmid of above-mentioned yeast strain.
The present invention also aims to provide the construction process of above-mentioned yeast strain.
Object of the present invention is achieved through the following technical solutions:
For building the DNA fragmentation of producing the yeast strain with Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen, comprise yeast saccharomyces cerevisiae TDH3 promoter sequence, yeast saccharomyces cerevisiae secretion signal peptide-coding sequence, the sequence with Mierocrystalline cellulose restriction endonuclease and glucosidase activity, the yeast saccharomyces cerevisiae TDH3 terminator sequence of arranging in order; Above-mentioned sequence is respectively as shown in SEQ ID NO.1～4.
Preferably, described for the sequence that builds the DNA fragmentation of producing the yeast strain with Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen as shown in SEQ ID NO.5.
The plasmid of producing the yeast strain with Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen for building is the eukaryon expression plasmid that comprises above-mentioned DNA fragmentation.Described eukaryon expression plasmid is preferably pPIC9K plasmid.
The described plasmid of producing the yeast strain with Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen for building preferably prepares by the method that comprises following steps: the sequence that synthetic two ends restricted property restriction endonuclease recognition site contains above-mentioned DNA fragmentation, is connected on eukaryon expression plasmid by restriction enzyme.When described eukaryon expression plasmid is pPIC9K plasmid, restriction enzyme is preferably Aat II and Not I.
Production has a yeast strain for Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen, contains above-mentioned plasmid.Described yeast strain is preferably yeast strain SMD1168.
Described production has the construction process of the yeast strain of Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen, comprises the steps: to prepare yeast Electroporation-competent cells, and above-mentioned plasmid is transformed in yeast and obtained by electricity.
The present invention has the following advantages and effect with respect to prior art tool:
The present invention has built an albumen that has Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated.Simultaneously, the present invention has realized high efficient expression the albumen with Mierocrystalline cellulose restriction endonuclease and glucosidase activity in yeast, the activity of Mierocrystalline cellulose restriction endonuclease and Polyglucosidase reaches 1.2 U/mL and 0.3 U/mL, has further reduced the industrialized utilization cost of cellulase.
Embodiment
Below in conjunction with embodiment, the present invention is done to further detailed description, but embodiments of the present invention are not limited to this.
Embodiment 1
A, intestinal bacteria pPIC9K plasmid extraction
(1) connect 1% Bacillus coli cells containing pPIC9K plasmid in 2 mL LB substratum.
(2) 37 ℃ of shaking culture 12 h.
(3) get 1.5 mL thalline and manage in EP, with centrifugal 3 min of 4000 rpm, abandon supernatant liquor.
(4) adding 0.l mL solution I (1% glucose, 50 mM EDTA pH 8.0,25 mM Tris-HCl pH 8.0) fully mixes.
(5) add 0.2 mL solution II (0.2 mM NaOH, 1% SDS), upset mixes gently, is placed in ice bath 5 min.
(6) add 0.15 mL precooling solution III (5 mol/L KAc, pH4.8), upset mixes gently, ice bath 5 min.
(7), with centrifugal 20 min of 10000 rpm, get supernatant liquor in another new EP pipe.
(8) add isopyknic primary isoamyl alcohol, mix rear 10 min of leaving standstill.
(9) again with centrifugal 20 min of 10000 rpm, abandon supernatant.
(10) wash once with 70% ethanol 0.5 mL, drain all liquid.
(11) after precipitation is dry, be dissolved in 0.05 mL TE damping fluid.
B, pPIC9K-END plasmid construction
(1) with restriction enzyme A at II, Not I digested plasmid pPIC9K respectively.Restriction enzyme A at II, Not I (TAkaRA) 3 μ L, the pPIC9K plasmid solution 6 μ L of extraction, 10 × K Buffer joins in 100 μ L EP pipes, and in 30 ℃ of water-baths, enzyme is cut 60 min.
(2) sequence is synthetic
Synthetic two ends have sequence C CGACGTCGG-yeast saccharomyces cerevisiae TDH3 promotor-yeast saccharomyces cerevisiae secretion signal peptide-coding sequence of Aat II and Not I recognition sequence-have sequence-yeast saccharomyces cerevisiae TDH3 terminator sequence-TTGCGGCCGCAACC of Mierocrystalline cellulose restriction endonuclease and glucosidase activity, and each fragment and full length fragment sequence are as shown in SEQ ID NO.1～5.
(3) connect
Synthetic base sequence 20 μ L, Aat II, Not I (TAkaRA) 12 μ L, 10 × K Buffer joins in 400 μ L EP pipes, in 30 ℃ of water-baths, enzyme is cut 80 min, after enzyme is cut, gets sequence enzyme and cuts liquid 15 μ L, the pPIC9K plasmid 5 μ L that enzyme is cut, T4 DNA ligase (TAkaRa) 5 μ L, 10 × buffer, 5 μ L, ddH
2o 10 μ L, add in 100 μ L EP pipes, and 16 ℃ of connections are spent the night.
(4) be transformed into intestinal bacteria, extract plasmid according to A, obtain plasmid pPIC9K-END.
C, Electroporation-competent cells preparation
(1) E.coli DH5 α is placed on LB or other nutritious substratum to incubated overnight at 37 ℃.
(2) centrifugal bottle (250-500 mL) that high-temperature sterilization is large is used in order to second day shaking flask.
(3) prepare several bottles of aqua sterilisas (approximately 1.5 liters of total amounts), be stored in refrigeration chamber and use in order to second day resuspension cell.
(4) transferase 10 .2-1 mL overnight culture is to 20 ml LB(or other nutritious substratum are housed) 100 mL shaking flasks.
At (5) 37 ℃, thermal agitation is cultivated 6 hours.
(6) monitoring O.D.600 value (cultivate and measure once per half an hour after 1 hour).
(7) in the time that O.D.600 value reaches 0.5-1.0, from shaking table, take out shaking flask, be placed in cooled on ice 15 minutes.
(8) cell under 4 ℃ of 5000 g centrifugal 15 minutes, abandons supernatant liquor.
(9) the frozen water resuspension cell of use sterilizing.First use vortex instrument or pipette resuspension cell (several milliliters) in a small amount of volume, be then diluted with water to 2/3 volume of centrifuge tube.
(10) shine step repeated centrifugation above, careful abandoning supernatant.
(11) the photograph frozen water resuspension cell of sterilizing for step above.
(12) centrifugal, abandon supernatant liquor.
(13) with 10% glycerine resuspension cell after 20 mL sterilizings, ice-cold.
(14) according to step is centrifugal above, careful abandoning supernatant (precipitation may be very loose).
(15) be 2-3 mL with 10% glycerine resuspension cell to final volume.
(16) pack cell into Eppendorf tube by 150 μ L equal portions, in-80 ℃ of preservations.
D, electricity transform
(1) Electroporation-competent cells that thaws on ice adds 1-10 μ L pPIC9K-END plasmid, cultivates on ice approximately 5 minutes.
(2) transfer DNA/cell mixture is to cooled 2 mm electroporation containers.
(3) add live conversion instrument, be ready to 300 μ L LB or 2 × YT.
(4) electroporation container is carried out to pulse (200 ohm, 25 μ Fd, 2.5 kilovolts) (testing time constant, should more than 3).
(5) add immediately the LB of 300 μ L or 2 × YT to electroporation container.
At (6) 37 ℃, culturing cell 40 minutes to 1 hour is with recovery.
(7) transitional cell is selected to cultivate on substratum to containing ammonia benzyl (100 μ g/mL).
D, plasmid transformed yeast bacterial strain
The preparation of yeast Electroporation-competent cells
(1) choose a ring yeast (SMD1168) and be inoculated in 5 mL YEPD substratum, 30 ℃, 250-300 rpm overnight incubation obtain first order seed.
(2) get 1 mL first order seed and be inoculated in respectively in two bottle of 50 mL YEPD substratum, 30 ℃, 250-300 rpm are cultivated the about 1.3-1.5 of about 16-18 h(OD600).
(3), in 4 ℃ of centrifugal collection thalline, with after 25 mL ice precooling sterilized water washings once, cell is resuspended with 10 mL ice precooling sterilized waters, can change less centrifuge tube into.
(4) add 10 × TE damping fluid of 1 mL pH 7.5, rock evenly, then add 1 mL 10 × LiAc, rotation shakes up, and shakes gently 45 min in 30 ℃.
(5) add again 0.4 mL 1 mol/L DTT, and rotation shake simultaneously, 15 min shaken gently in 30 ℃.
(6) in 4 ℃ centrifugal, abandon supernatant (inhaling with rifle), then with 25 mL ice precooling sterilized waters washings.
1 mol/L sorbyl alcohol washing of (7) 2.5 mL ice precoolings, centrifugal collection thalline, abandons supernatant (inhaling with rifle);
(8) every effective 100 μ L 1 mol/L sorbyl alcohols dissolve, in point tubulature (80 μ L pipe), in-70 ℃ of Refrigerator stores.
Electricity transforms
(1) in yeast competent cell, add approximately 5～10 μ g(volumes to be less than 10 μ L) plasmid pPIC9K-END, even with rifle pressure-vaccum, be transferred in the electric revolving cup of precooling, leave standstill 5min.
(2) dry electric revolving cup, electric shock, shock parameters: 1.5 KV, 25 μ F, 200 ohm.
(3) add immediately 1 mol/L sorbyl alcohol of 1 mL ice precooling, be transferred in centrifuge tube, leave standstill 1 h in 30 ℃.
(4) centrifugal, abandon supernatant, add after 1 mL YEPD, cultivate 2 h in 30 ℃, 200 rpm.
(5) after centrifugal thalline, absorb 550 μ L supernatant liquors, then get 150 μ L and be coated with 100 μ g/mL ammonia benzyl YEPD plates and be cultured to and grow transformant in 30 ℃.
E, yeast transformant fermentation
The transformant of picking is 30 ℃ of cultivation 24 h on YEPD substratum, be inoculated in fermention medium (yeast extract 10 g/L with 10% inoculum size (v/v), peptone 20 g/L, 50 mM citrate buffer solutions, wheat bran 200 g/L, Walocel MT 20.000PV 20 g/L, 1 L tap water), fermentation is fermented in 500 mL shaking flasks, and liquid amount is 20%(v/v).In fermention medium, cultivate 48 h.
F, restriction endonuclease and glucosidase activity are measured
The mash filter paper filtering of fermentation, the centrifugal 15min of filtrate 4000 r/m of filtration, discards precipitation, and supernatant liquor is the liquid that contains Mierocrystalline cellulose restriction endonuclease and Polyglucosidase recombinant protein.
Restriction endonuclease enzyme activity determination: take Walocel MT 20.000PV as substrate, carry out enzyme liberating reaction at 50 ℃.Walocel MT 20.000PV citrate buffer solution (0.05 M, pH 5.0) 10 mL that contain 20 g/L in 25 mL test tubes, add centrifugal rear supernatant liquor 2.0 mL, and in water-bath, 50 ℃ of insulation 30 min, then boil 5 min with boiling water.Enzyme work is defined as: per minute discharges the needed enzyme amount of 1 μ mol reducing sugar.The enzyme liquid that when mensuration, supernatant liquor boils 5 min in boiling water is for contrasting.Reducing sugar adopts DNS method to measure.
Glucosidase activity is measured: take saligenin as substrate, carry out enzyme liberating reaction at 50 ℃.In 25 mL test tubes, add saligenin citrate buffer solution (0.05 M, pH 5.0) 10 mL that contain 20 g/L, add centrifugal rear supernatant liquor 2.0 mL, in water-bath, 50 ℃ of insulation 30 min, then boil 5 min with boiling water.Enzyme work is defined as: per minute discharges the needed enzyme amount of 1 μ mol reducing sugar.The enzyme liquid that when mensuration, supernatant liquor boils 5 min in boiling water is for contrasting.Reducing sugar adopts DNS method to measure.
The activity of the Mierocrystalline cellulose restriction endonuclease of measuring from supernatant liquor is 1.2 U/mL, and the activity of Polyglucosidase is 0.3 U/mL.Result shows that the recombinant protein building has the activity of Mierocrystalline cellulose restriction endonuclease and Polyglucosidase, and this albumen successful expression in yeast.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
SEQUENCE LISTING
<110> Hubei University Of Technology
<120> produces yeast strain and the structure thereof with Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen
<130> 1
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 711
<212> DNA
<213> Saccharomyces cerevisiae
<400> 1
agtttatcat tatcaatact agtttatcat tatcaatact cgccatttca aagaatacgt 60
aaataattaa tagtagtgat tttcctaact ttatttagtc aaaaaattag ccttttaatt 120
ctgctgtaac ccgtacatgc ccaaaatagg gggcgggtta cacagaatat ataacatcgt 180
aggtgtctgg gtgaacagtt tattcctggc atccactaaa tataatggag cccgcttttt 240
aagctggcat ccagaaaaaa aaagaatccc agcaccaaaa tattgttttc ttcaccaacc 300
atcagttcat aggtccattc tcttagcgca actacagaga acaggggcac aaacaggcaa 360
aaaacgggca caacctcaat ggagtgatgc aacctgcctg gagtaaatga tgacacaagg 420
caattgaccc acgcatgtat ctatctcatt ttcttacacc ttctattacc ttctgctctc 480
tctgatttgg aaaaagctga aaaaaaaggt tgaaaccagt tccctgaaat tattccccta 540
cttgactaat aagtatataa agacggtagg tattgattgt aattctgtaa atctatttct 600
taaacttctt aaattctact tttatagtta gtcttttttt tagttttaaa acaccagaac 660
ttagtttcga cggatttagt tttaaaacac cagaacttag tttcgacgga t 711
<210> 2
<211> 253
<212> DNA
<213> Saccharomyces cerevisiae
<400> 2
ggatccaaac gatgagattt ccttcaattt ttactgcagt tttattcgca gcatcctccg 60
cattagctgc tccagtcaac actacaacag aagatgaaac ggcacaaatt ccggctgaag 120
ctgtcatcgg ttactcagat ttagaagggg atttcgatgt tgctgttttg ccattttcca 180
acagcacaaa taacgggtta ttgtttataa atactactat tgccagcatt gctgctaaag 240
aagaaggggt atc 253
<210> 3
<211> 3287
<212> DNA
<213> Artificial Sequence
<220>
<223> has the sequence of Mierocrystalline cellulose restriction endonuclease and dextran glycosides enzymic activity
<400> 3
atggtgagtt ttaaagcagg tataaattta ggcggatgga tatcacaata tcaagttttc 60
agcaaagagc atttcgatac attcattacg gagaaggaca ttgaaactat tgcagaagca 120
gggtttgacc atgtcagact gccttttgat tatccaatta tcgagtctga tgacaatgtg 180
ggagaatata aagaagatgg gctttcttat attgaccggt gccttgagtg gtgtaaaaaa 240
tacaatttgg ggcttgtgtt ggatatgcat cacgctcccg ggtaccgctt tcaagatttt 300
aagacaagca ccttgtttga agatccgaac cagcaaaaga gatttgttga catatggaga 360
tttttagcca agcgttacat aaatgaacgg gaacatattg cctttgaact gttaaatgaa 420
gttgttgagc ctgacagtac ccgctggaac aagttgatgc ttgagtgtgt aaaagcaatc 480
agggaaattg attccaccag gtggctttac attgggggca ataactataa cagtcctgat 540
gagcttaaaa accttgcaga tattgatgat gattacatag tttacaattt ccatttttac 600
aatccttttt tctttacgca tcagaaagcc cactggtcgg aaagtgccat ggcgtacaac 660
aggactgtaa aatatccggg acaatatgag ggaattgaag agtttgtgaa aaataatcct 720
aagtacagtt ttatgatgga attgaataac ctgaagctga ataaagagct tttgcgcaaa 780
gatttaaaac cagcaattga gttcagggaa aagaaaaaat gcaaactata ttgcggggag 840
tttggcgtaa ttgccattgc tgacctggag tccaggataa aatggcatga agattatata 900
agtcttctgg aggagtatga tatcggcggc gcggtgtgga actacaaaaa aatggatttt 960
gaaatttata atgaggatag aaaacctgtc tcgcaagaat tggtaaatat actggcgaga 1020
agaaaaactt gaagcggtag atatcaagaa aataataaag cagatgcttt ggaagaaaaa 1080
gcagggttgt gctcgggact ggatttttgg cataccaagc ctgttagaga ctgggcattc 1140
cttcaataat gatgactgac ggacctcatg gactgagaaa gcaggggaag atgcagagat 1200
tgcggacatc aacaacagcg ttccagcaac ctgttttccg tctcagcagg tttggcatgt 1260
tcctgggaca gagaactggt tgagagagta ggtgcagcac tagagaagaa tgtcaggcgg 1320
aaaatgtctc aatactgctt ggaccaggtg caaatataaa ggttcacctt tgtgtggaag 1380
aaattttgaa tattttcccg aagaccctta tctttcgtca gctggcggca agccatataa 1440
aaggagttca aagtcaggga gtgggtgcat gtcttaaaca ttttgccgca aacaaccagg 1500
aacaccggag aatgaccgtt gataccattg tagatgaaag aacgttgagg gaaatatatt 1560
ttgcaagctt tgagaatgct gtaaaaaaag cacggccttg ggtggttatg tgtgcatata 1620
acaagctcaa cggtgaatat tgttcggaga acagatatct tttgacggaa gttttaaaga 1680
atgaatggat gcatgacggc tttgtggtat ccgactgggg tgcggtaaat gacagggtca 1740
gcggcctgga tgcaggtctt gacctggaaa tgcccaccag tcatggtatt acggataaaa 1800
agatagttga agccgtaaaa agcggaaagc tgtctgaaaa tattttaaac agagctgtgg 1860
aaagaatttt gaaagtaatt attatggcac tggaaaacaa aaaagaaaac gcgcagtatg 1920
aacaagatgc tcatcacaga ctggcaaggc aggctgcggc cgaatcgatg gttcttctta 1980
aaaacgagga cgatgtgctt cctttaaaaa agagcggaac catagctttg ataggagctt 2040
ttgtgaaaaa accaagatac cagggttcgg gcagttctca tattaccccg acaagacttg 2100
atgatattta tgaagagata aaaaaggccg gagccgacaa agtaaacctt gtatattcgg 2160
aaggatacag gcttgaaaat gacggtattg atgaggaatt gataaacgaa gctaaaaagg 2220
cggcatcaag ctcggatgtt gcggtagtat ttgcagggct tccggatgaa tatgaatctg 2280
aaggatttga cagaactcac atgagtattc cggaaaatca aaacaggctg atagaagcgg 2340
tggccgaagt ccagagtaat attgttgtgg tattgcttaa cggctcaccg gttgaaatgc 2400
cgtggattga caaggtaaaa tccgtgcttg aagcttatct tggaggccag gcgctgggag 2460
gccgctggcg gatgtgctat tcggtgaagt caatcgtcgg aaaacttgcg gagaccttcc 2520
cggtgaaatt aagccataat ccgtcctatt tgaattttcc cggagaggat gaccgagtgg 2580
agtataaaga agggttgttt gtcggataca gatattatga tacaaaggga attgagccat 2640
tgttcccctt tggtcacgga cttagctata ccaaatttga atacagtgat atatcagtcg 2700
ataaaaaaga tgtttcggac aatagcatca taaatgtcag cgttaaagtc aaaaatgttg 2760
gaaaaatggc aggaaaagaa attgtgcagc tgtatgtaaa agatgtgaaa agcagcgtca 2820
gaagacctga gaaagagctt aaaggatttg aaaaggtctt ccttaatccg ggagaagaaa 2880
agacggttac atttactttg gacaaaaggg cttttgcata ttacaatact cagattaagg 2940
actggcatgt tgaaagcgga gagtttctga tattaatagg aaggtcctcc agggacatag 3000
ttttaaaaga atcagtgaga gtaaattcaa cggtgaagat aagaaaaaga ttcacagtga 3060
attcagcggt tgaagatgta atgtccgatt cttcggctgc ggccgtttta gggcctgtac 3120
taaaagagat aaccgatgca ctgcagattg atatggacaa tgctcatgac atgatggcgg 3180
ccaatataaa gaatatgcct ttgcgctcac ttgtcggtta ctctcaggga aggttaagcg 3240
aagaaatgct ggaggaactg gttgacaaaa taaacaacgt ggaataa 3287
<210> 4
<211> 281
<212> DNA
<213> Saccharomyces cerevisiae
<400> 4
gggggtaccg ggcccggccg caaattaaag ccttcgagcg tcccaaaacc ttctcaagca 60
aggttttcag tataatgtta catgcgtaca cgcgtctgta cagaaaaaaa agaaaaattt 120
gaaatataaa taacgttctt aatactaaca taactataaa aaaataaata gggacctaga 180
cttcaggttg tctaactcct tccttttcgg ttagagcgga tgtgggggga gggcgtgaat 240
gtaagcgtga cataactaat tacatgatgc ggccctttaa a 281
<210> 5
<211> 4532
<212> DNA
<213> Artificial Sequence
<220>
<223> is for building the DNA that produces the yeast strain with Mierocrystalline cellulose restriction endonuclease and dextran glycosides enzyme double activated albumen
The sequence of fragment
<400> 5
agtttatcat tatcaatact agtttatcat tatcaatact cgccatttca aagaatacgt 60
aaataattaa tagtagtgat tttcctaact ttatttagtc aaaaaattag ccttttaatt 120
ctgctgtaac ccgtacatgc ccaaaatagg gggcgggtta cacagaatat ataacatcgt 180
aggtgtctgg gtgaacagtt tattcctggc atccactaaa tataatggag cccgcttttt 240
aagctggcat ccagaaaaaa aaagaatccc agcaccaaaa tattgttttc ttcaccaacc 300
atcagttcat aggtccattc tcttagcgca actacagaga acaggggcac aaacaggcaa 360
aaaacgggca caacctcaat ggagtgatgc aacctgcctg gagtaaatga tgacacaagg 420
caattgaccc acgcatgtat ctatctcatt ttcttacacc ttctattacc ttctgctctc 480
tctgatttgg aaaaagctga aaaaaaaggt tgaaaccagt tccctgaaat tattccccta 540
cttgactaat aagtatataa agacggtagg tattgattgt aattctgtaa atctatttct 600
taaacttctt aaattctact tttatagtta gtcttttttt tagttttaaa acaccagaac 660
ttagtttcga cggatttagt tttaaaacac cagaacttag tttcgacgga tggatccaaa 720
cgatgagatt tccttcaatt tttactgcag ttttattcgc agcatcctcc gcattagctg 780
ctccagtcaa cactacaaca gaagatgaaa cggcacaaat tccggctgaa gctgtcatcg 840
gttactcaga tttagaaggg gatttcgatg ttgctgtttt gccattttcc aacagcacaa 900
ataacgggtt attgtttata aatactacta ttgccagcat tgctgctaaa gaagaagggg 960
tatcatggtg agttttaaag caggtataaa tttaggcgga tggatatcac aatatcaagt 1020
tttcagcaaa gagcatttcg atacattcat tacggagaag gacattgaaa ctattgcaga 1080
agcagggttt gaccatgtca gactgccttt tgattatcca attatcgagt ctgatgacaa 1140
tgtgggagaa tataaagaag atgggctttc ttatattgac cggtgccttg agtggtgtaa 1200
aaaatacaat ttggggcttg tgttggatat gcatcacgct cccgggtacc gctttcaaga 1260
ttttaagaca agcaccttgt ttgaagatcc gaaccagcaa aagagatttg ttgacatatg 1320
gagattttta gccaagcgtt acataaatga acgggaacat attgcctttg aactgttaaa 1380
tgaagttgtt gagcctgaca gtacccgctg gaacaagttg atgcttgagt gtgtaaaagc 1440
aatcagggaa attgattcca ccaggtggct ttacattggg ggcaataact ataacagtcc 1500
tgatgagctt aaaaaccttg cagatattga tgatgattac atagtttaca atttccattt 1560
ttacaatcct tttttcttta cgcatcagaa agcccactgg tcggaaagtg ccatggcgta 1620
caacaggact gtaaaatatc cgggacaata tgagggaatt gaagagtttg tgaaaaataa 1680
tcctaagtac agttttatga tggaattgaa taacctgaag ctgaataaag agcttttgcg 1740
caaagattta aaaccagcaa ttgagttcag ggaaaagaaa aaatgcaaac tatattgcgg 1800
ggagtttggc gtaattgcca ttgctgacct ggagtccagg ataaaatggc atgaagatta 1860
tataagtctt ctggaggagt atgatatcgg cggcgcggtg tggaactaca aaaaaatgga 1920
ttttgaaatt tataatgagg atagaaaacc tgtctcgcaa gaattggtaa atatactggc 1980
gagaagaaaa acttgaagcg gtagatatca agaaaataat aaagcagatg ctttggaaga 2040
aaaagcaggg ttgtgctcgg gactggattt ttggcatacc aagcctgtta gagactgggc 2100
attccttcaa taatgatgac tgacggacct catggactga gaaagcaggg gaagatgcag 2160
agattgcgga catcaacaac agcgttccag caacctgttt tccgtctcag caggtttggc 2220
atgttcctgg gacagagaac tggttgagag agtaggtgca gcactagaga agaatgtcag 2280
gcggaaaatg tctcaatact gcttggacca ggtgcaaata taaaggttca cctttgtgtg 2340
gaagaaattt tgaatatttt cccgaagacc cttatctttc gtcagctggc ggcaagccat 2400
ataaaaggag ttcaaagtca gggagtgggt gcatgtctta aacattttgc cgcaaacaac 2460
caggaacacc ggagaatgac cgttgatacc attgtagatg aaagaacgtt gagggaaata 2520
tattttgcaa gctttgagaa tgctgtaaaa aaagcacggc cttgggtggt tatgtgtgca 2580
tataacaagc tcaacggtga atattgttcg gagaacagat atcttttgac ggaagtttta 2640
aagaatgaat ggatgcatga cggctttgtg gtatccgact ggggtgcggt aaatgacagg 2700
gtcagcggcc tggatgcagg tcttgacctg gaaatgccca ccagtcatgg tattacggat 2760
aaaaagatag ttgaagccgt aaaaagcgga aagctgtctg aaaatatttt aaacagagct 2820
gtggaaagaa ttttgaaagt aattattatg gcactggaaa acaaaaaaga aaacgcgcag 2880
tatgaacaag atgctcatca cagactggca aggcaggctg cggccgaatc gatggttctt 2940
cttaaaaacg aggacgatgt gcttccttta aaaaagagcg gaaccatagc tttgatagga 3000
gcttttgtga aaaaaccaag ataccagggt tcgggcagtt ctcatattac cccgacaaga 3060
cttgatgata tttatgaaga gataaaaaag gccggagccg acaaagtaaa ccttgtatat 3120
tcggaaggat acaggcttga aaatgacggt attgatgagg aattgataaa cgaagctaaa 3180
aaggcggcat caagctcgga tgttgcggta gtatttgcag ggcttccgga tgaatatgaa 3240
tctgaaggat ttgacagaac tcacatgagt attccggaaa atcaaaacag gctgatagaa 3300
gcggtggccg aagtccagag taatattgtt gtggtattgc ttaacggctc accggttgaa 3360
atgccgtgga ttgacaaggt aaaatccgtg cttgaagctt atcttggagg ccaggcgctg 3420
ggaggccgct ggcggatgtg ctattcggtg aagtcaatcg tcggaaaact tgcggagacc 3480
ttcccggtga aattaagcca taatccgtcc tatttgaatt ttcccggaga ggatgaccga 3540
gtggagtata aagaagggtt gtttgtcgga tacagatatt atgatacaaa gggaattgag 3600
ccattgttcc cctttggtca cggacttagc tataccaaat ttgaatacag tgatatatca 3660
gtcgataaaa aagatgtttc ggacaatagc atcataaatg tcagcgttaa agtcaaaaat 3720
gttggaaaaa tggcaggaaa agaaattgtg cagctgtatg taaaagatgt gaaaagcagc 3780
gtcagaagac ctgagaaaga gcttaaagga tttgaaaagg tcttccttaa tccgggagaa 3840
gaaaagacgg ttacatttac tttggacaaa agggcttttg catattacaa tactcagatt 3900
aaggactggc atgttgaaag cggagagttt ctgatattaa taggaaggtc ctccagggac 3960
atagttttaa aagaatcagt gagagtaaat tcaacggtga agataagaaa aagattcaca 4020
gtgaattcag cggttgaaga tgtaatgtcc gattcttcgg ctgcggccgt tttagggcct 4080
gtactaaaag agataaccga tgcactgcag attgatatgg acaatgctca tgacatgatg 4140
gcggccaata taaagaatat gcctttgcgc tcacttgtcg gttactctca gggaaggtta 4200
agcgaagaaa tgctggagga actggttgac aaaataaaca acgtggaata agggggtacc 4260
gggcccggcc gcaaattaaa gccttcgagc gtcccaaaac cttctcaagc aaggttttca 4320
gtataatgtt acatgcgtac acgcgtctgt acagaaaaaa aagaaaaatt tgaaatataa 4380
ataacgttct taatactaac ataactataa aaaaataaat agggacctag acttcaggtt 4440
gtctaactcc ttccttttcg gttagagcgg atgtgggggg agggcgtgaa tgtaagcgtg 4500
acataactaa ttacatgatg cggcccttta aa 4532
<210> 6
<211> 10
<212> DNA
<213> Artificial Sequence
<220>
<223> Aat II recognition sequence
<400> 6
ccgacgtcgg 10
<210> 7
<211> 14
<212> DNA
<213> Artificial Sequence
<220>
<223> Not I recognition sequence
<400> 7
ttgcggccgc aacc 14
Claims (10)
1. for building the DNA fragmentation of producing the yeast strain with Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen, it is characterized in that: comprise yeast saccharomyces cerevisiae TDH3 promoter sequence, yeast saccharomyces cerevisiae secretion signal peptide-coding sequence, the sequence with Mierocrystalline cellulose restriction endonuclease and glucosidase activity, the yeast saccharomyces cerevisiae TDH3 terminator sequence of arranging in order.
2. DNA fragmentation according to claim 1, is characterized in that: described sequence is respectively as shown in SEQ ID NO.1～4.
3. DNA fragmentation according to claim 1, is characterized in that: the sequence of described DNA fragmentation is as shown in SEQ ID NO.5.
4. produce the plasmid of yeast strain with Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen for building, it is characterized in that: for comprising the eukaryon expression plasmid of the DNA fragmentation described in claim 1-3 any one.
5. plasmid according to claim 4, is characterized in that: described eukaryon expression plasmid is pPIC9K plasmid.
6. plasmid according to claim 4, it is characterized in that the method by comprising following steps prepares: the sequence that synthetic two ends restricted property restriction endonuclease recognition site contains the DNA fragmentation described in claim 1-3 any one, is connected on eukaryon expression plasmid by restriction enzyme.
7. plasmid according to claim 6, is characterized in that: when described eukaryon expression plasmid is pPIC9K plasmid, restriction enzyme is Aat II and Not I.
8. production has a yeast strain for Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen, it is characterized in that: comprise the plasmid described in claim 4-7 any one.
9. yeast strain according to claim 8, is characterized in that: described yeast strain is yeast strain SMD1168.
10. the construction process of the yeast strain described in claim 8 or 9, is characterized in that comprising the steps: to prepare yeast Electroporation-competent cells, and the plasmid described in claim 4-7 any one is transformed in yeast and obtained by electricity.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480139A (en) * | 2014-12-26 | 2015-04-01 | 湖北工业大学 | Method for constructing cellulase high-yielding strain by expressing doubleactive protein of celluloseexonucleaseand endonuclease |
| CN106191085A (en) * | 2016-07-27 | 2016-12-07 | 湖北工业大学 | Utilize cellulose restriction endonuclease and β glucosidase structuring food prods safe level carrier and screening culture medium |
| CN109182360A (en) * | 2018-10-23 | 2019-01-11 | 怀化学院 | A kind of small molecule cellulose incision enzyme gene and its albumen and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103124783A (en) * | 2010-06-03 | 2013-05-29 | 马斯科马公司 | Yeast expressing saccharolytic enzymes for consolidated bioprocessing using starch and cellulose |
| CN103589719A (en) * | 2013-11-19 | 2014-02-19 | 湖北工业大学 | Construction of yeast strain with dual functions of producing and recycling cellulose excision enzyme |
-
2014
- 2014-03-10 CN CN201410084775.1A patent/CN103820443B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103124783A (en) * | 2010-06-03 | 2013-05-29 | 马斯科马公司 | Yeast expressing saccharolytic enzymes for consolidated bioprocessing using starch and cellulose |
| CN103589719A (en) * | 2013-11-19 | 2014-02-19 | 湖北工业大学 | Construction of yeast strain with dual functions of producing and recycling cellulose excision enzyme |
Non-Patent Citations (1)
| Title |
|---|
| HSIAO-LIN LEE等: "Construction and characterization of different fusion proteins between cellulases and b-glucosidase to improve glucose production and thermostability", 《BIORESOURCE TECHNOLOGY》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480139A (en) * | 2014-12-26 | 2015-04-01 | 湖北工业大学 | Method for constructing cellulase high-yielding strain by expressing doubleactive protein of celluloseexonucleaseand endonuclease |
| CN104480139B (en) * | 2014-12-26 | 2017-12-15 | 湖北工业大学 | A kind of method for expressing cellulose excision enzyme and restriction endonuclease double activated albumen structure cellulase high-yield |
| CN106191085A (en) * | 2016-07-27 | 2016-12-07 | 湖北工业大学 | Utilize cellulose restriction endonuclease and β glucosidase structuring food prods safe level carrier and screening culture medium |
| CN106191085B (en) * | 2016-07-27 | 2019-10-25 | 湖北工业大学 | Utilize cellulose restriction endonuclease and the safe grade carrier of β-glucosyl enzym structuring food prods and screening and culturing medium |
| CN109182360A (en) * | 2018-10-23 | 2019-01-11 | 怀化学院 | A kind of small molecule cellulose incision enzyme gene and its albumen and application |
| CN109182360B (en) * | 2018-10-23 | 2020-06-05 | 怀化学院 | Micromolecular cellulose endonuclease gene and protein and application thereof |
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| CN103820443B (en) | 2016-01-13 |
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