CN103820443B - Produce yeast strain and the structure thereof with Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen - Google Patents
Produce yeast strain and the structure thereof with Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen Download PDFInfo
- Publication number
- CN103820443B CN103820443B CN201410084775.1A CN201410084775A CN103820443B CN 103820443 B CN103820443 B CN 103820443B CN 201410084775 A CN201410084775 A CN 201410084775A CN 103820443 B CN103820443 B CN 103820443B
- Authority
- CN
- China
- Prior art keywords
- yeast
- mierocrystalline cellulose
- restriction endonuclease
- plasmid
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses yeast strain and structure thereof that a kind of production has Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen, belong to enzyme engineering field.By yeast saccharomyces cerevisiae TDH3 promoter sequence, yeast saccharomyces cerevisiae secretion signal peptide-coding sequence, have Mierocrystalline cellulose endo-activity and glucosidase activity sequence, make wine and to synthesize in order with regard to yeast TDH3 terminator sequence, restriction endonuclease sites is added at two ends, cut by enzyme and be building up in pPIC9K plasmid, then will obtain producing the yeast strain with Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen in this Plastid transformation to yeast.Present invention achieves the expression of albumen in yeast of Mierocrystalline cellulose restriction endonuclease and glucosidase activity, there is stronger practicality.The activity of the Mierocrystalline cellulose restriction endonuclease that yeast strain of the present invention produces and Polyglucosidase reaches 1.2U/mL and 0.3U/mL.
Description
Technical field
The invention belongs to enzyme engineering field, be specifically related to yeast strain and structure thereof that a kind of production has 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 that the earth produces because of photosynthesis every year reaches about 10,000,000,000 tons, utilizes cellulose raw to produce bioenergy, is alleviating energy crisis, realizes the key of human kind sustainable development.
The key of cellulose utilization is the carbohydrate-glucose of fermentability cellulose degradation.Cellulosic degraded needs the acting in conjunction of excision enzyme, restriction endonuclease, Polyglucosidase.Wherein the Mierocrystalline cellulose of endonucleases long segment becomes dimer, is the committed step of cellulose degradation.With cellulose degraded Mierocrystalline cellulose, there is green, mild condition, feature that transformation efficiency is high, but the higher production cost of cellulase becomes the cellulosic bottleneck of cellulose degraded.
Reducing cellulase cost effective means is the utilising efficiency improving enzyme.Due to the synergy of cellulosic degraded needs three kinds of enzymes, the albumen with two kinds of functions or several functions can improve the utilising efficiency of cellulase significantly, thus 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 overcoming prior art is 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 the DNA fragmentation being provided for building above-mentioned yeast strain.
Another object of the present invention is the plasmid being provided for building above-mentioned yeast strain.
The present invention also aims to the construction process that above-mentioned yeast strain is provided.
Object of the present invention is achieved through the following technical solutions:
For building the DNA fragmentation produced and there is the yeast strain of Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen, comprise the 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 that arrange in order; Above-mentioned sequence is respectively as shown in SEQIDNO.1 ~ 4.
Preferably, described for building the sequence of producing and there is the DNA fragmentation of the yeast strain of Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen as shown in SEQIDNO.5.
The plasmid for building production with the yeast strain of Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen is the eukaryon expression plasmid comprising above-mentioned DNA fragmentation.Described eukaryon expression plasmid is preferably pPIC9K plasmid.
The described plasmid for building production with the yeast strain of Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen prepares preferably by the method comprising following steps: synthesis two ends restricted property endonuclease recognized site contains the sequence of 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, containing 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, is transformed to turn to enter in yeast by above-mentioned plasmid to obtain by electricity.
The present invention has the following advantages and effect relative to prior art tool:
The present invention constructs the albumen that one has Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated.Meanwhile, the present invention achieves high expression the albumen with Mierocrystalline cellulose restriction endonuclease and glucosidase activity in yeast, and the activity of Mierocrystalline cellulose restriction endonuclease and Polyglucosidase reaches 1.2U/mL and 0.3U/mL, reduce further the industrialized utilization cost of cellulase.
Embodiment
Below in conjunction with embodiment, further detailed description is done to the present invention, but embodiments of the present invention are not limited thereto.
Embodiment 1
A, intestinal bacteria pPIC9K plasmid extraction
(1) connect 1% and contain the Bacillus coli cells of pPIC9K plasmid in 2mLLB substratum.
(2) 37 DEG C of shaking culture 12h.
(3) get 1.5mL thalline to manage in EP, with the centrifugal 3min of 4000rpm, abandon supernatant liquor.
(4) add 0.lmL solution I (1% glucose, 50mMEDTApH8.0,25mMTris-HClpH8.0) fully to mix.
(5) add 0.2mL solution II (0.2mMNaOH, 1%SDS), overturn mixing gently, be placed in ice bath 5min.
(6) 0.15mL cooled solution III(5mol/LKAc, pH4.8 is added), overturn mixing gently, ice bath 5min.
(7) with the centrifugal 20min of 10000rpm, supernatant liquor is got in another new EP pipe.
(8) add isopyknic primary isoamyl alcohol, after mixing, leave standstill 10min.
(9) again with the centrifugal 20min of 10000rpm, supernatant is abandoned.
(10) wash once with 70% ethanol 0.5mL, drain all liquid.
(11), after drying to be precipitated, be dissolved in 0.05mLTE 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 × KBuffer joins in 100 μ LEP pipes, and in 30 DEG C of water-baths, enzyme cuts 60min.
(2) sequent synthesis
Synthesis two ends have the sequence-yeast saccharomyces cerevisiae TDH3 terminator sequence-TTGCGGCCGCAACC of the 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 Mierocrystalline cellulose restriction endonuclease and glucosidase activity, and each fragment and full length fragment sequence are as shown in SEQIDNO.1 ~ 5.
(3) connect
The base sequence 20 μ L of synthesis, Aat II, Not I (TAkaRA) 12 μ L, 10 × KBuffer joins in 400 μ LEP pipes, in 30 DEG C of water-baths, enzyme cuts 80min, after enzyme is cut, gets sequence enzyme and cuts liquid 15 μ L, the pPIC9K plasmid 5 μ L that enzyme is cut, T4DNAligase (TAkaRa) 5 μ L, 10 × buffer5 μ L, ddH
2o10 μ L, add in 100 μ LEP pipes, 16 DEG C of connections are spent the night.
(4) be transformed into intestinal bacteria, extract plasmid according to A, obtain plasmid pPIC9K-END.
Prepared by C, Electroporation-competent cells
(1) E.coliDH5 α is placed on LB or other nutritious substratum, incubated overnight at 37 DEG C.
(2) centrifugal bottle (250-500mL) that high-temperature sterilization is large is used in order to second day shaking flask.
(3) prepare several bottles of aqua sterilisas (total amount about 1.5 liters), be stored in refrigeration chamber and use in order to second day resuspension cell.
(4) transferase 10 .2-1mL overnight culture is to 20mlLB(or other nutritious substratum are housed) 100mL shaking flask.
At (5) 37 DEG C, thermal agitation cultivates 6 hours.
(6) O.D.600 value (cultivate and measure once per half an hour after 1 hour) is monitored.
(7) when 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 DEG C of 5000g centrifugal 15 minutes, abandons supernatant liquor.
(9) with the frozen water resuspension cell of 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) according to previous step repeated centrifugation, careful abandoning supernatant.
(11) according to the frozen water resuspension cell of previous step sterilizing.
(12) centrifugal, abandon supernatant liquor.
(13) with 10% glycerine resuspension cell after 20mL sterilizing, ice-cold.
(14) centrifugal according to previous step, careful abandoning supernatant (precipitation may be very loose).
(15) be 2-3mL with 10% glycerine resuspension cell to final volume.
(16) cell is loaded Eppendorf tube, in-80 DEG C of preservations by 150 μ L equal portions.
D, electricity transform
(1) Electroporation-competent cells that thaws on ice adds 1-10 μ LpPIC9K-END plasmid, incubated on ice about 5 minutes.
(2) in transfer DNA/cell mixture to cooled 2mm electroporation container.
(3) add live conversion instrument, get out 300 μ LLB or 2 × YT.
(4) pulse (200 ohm, 25 μ Fd, 2.5 kilovolts) (testing time constant, should more than 3) is carried out to electroporation container.
(5) LB of 300 μ L or 2 × YT is added immediately in electroporation container.
At (6) 37 DEG C, culturing cell 40 minutes to 1 hour is with recovery.
(7) transitional cell is cultivated to containing on ammonia benzyl (100 μ g/mL) Selective agar medium.
D, Plastid transformation yeast strain
The preparation of yeast Electroporation-competent cells
(1) choosing a ring yeast (SMD1168) is inoculated in 5mLYEPD substratum, 30 DEG C, 250-300rpm overnight incubation obtains first order seed.
(2) getting 1mL first order seed is inoculated in two bottles of 50mLYEPD substratum respectively, 30 DEG C, 250-300rpm cultivates about 16-18h(OD600 and is about 1.3-1.5).
(3) in 4 DEG C of collected by centrifugation thalline, after the washing once of 25mL ice precooling sterilized water, cell 10mL ice precooling sterilized water is resuspended, can change less centrifuge tube into.
(4) add 10 × TE damping fluid of 1mLpH7.5, rock evenly, then add 1mL10 × LiAc, rotation shakes up, and shakes 45min gently in 30 DEG C.
(5) add 0.4mL1mol/LDTT again, and rotate shake simultaneously, shake 15min gently in 30 DEG C.
(6) centrifugal in 4 DEG C, abandon supernatant (inhaling with rifle), then wash with 25mL ice precooling sterilized water.
(7) the 1mol/L sorbitol washes of 2.5mL ice precooling, collected by centrifugation thalline, abandons supernatant (inhaling with rifle);
(8) often effective 100 μ L1mol/L sorbyl alcohols dissolve, in point tubulature (80 μ L manage), in-70 DEG C of Refrigerator stores.
Electricity transforms
(1) in competent yeast cells, add about 5 ~ 10 μ g(volumes 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) electric revolving cup is dried, electric shock, shock parameters: 1.5KV, 25 μ F, 200 ohm.
(3) add the 1mol/L sorbyl alcohol of 1mL ice precooling immediately, be transferred in centrifuge tube, in 30 DEG C of standing 1h.
(4) centrifugal, abandon supernatant, after adding 1mLYEPD, in 30 DEG C, 200rpm cultivates 2h.
(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 in 30 DEG C and grow transformant.
E, yeast transformant ferment
Transformant 30 DEG C of cultivation 24h on YEPD substratum of picking, fermention medium (yeast extract 10g/L is inoculated in 10% inoculum size (v/v), peptone 20g/L, 50mM citrate buffer solution, wheat bran 200g/L, Walocel MT 20.000PV 20g/L, 1L tap water), fermentation is fermented in 500mL shaking flask, and liquid amount is 20%(v/v).Cultivate 48h in the fermentation medium.
F, restriction endonuclease and glucosidase activity measure
The mash filter paper filtering of fermentation, the centrifugal 15min of filtrate 4000r/m of filtration, discards precipitation, and supernatant liquor is the liquid containing 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 DEG C.Walocel MT 20.000PV citrate buffer solution (0.05M, pH5.0) 10mL containing 20g/L in 25mL test tube, adds centrifuged supernatant 2.0mL, and in water-bath, 50 DEG C of insulation 30min, then boil 5min with boiling water.Enzyme is lived and is defined as: per minute discharges the enzyme amount required for 1 μm of ol reducing sugar.During mensuration, supernatant liquor boils the enzyme liquid of 5min for contrast in boiling water.Reducing sugar adopts DNS method to measure.
Glucosidase activity measures: take saligenin as substrate, carry out enzyme liberating reaction at 50 DEG C.In 25mL test tube, add saligenin citrate buffer solution (0.05M, pH5.0) 10mL containing 20g/L, add centrifuged supernatant 2.0mL, in water-bath, 50 DEG C of insulation 30min, then boil 5min with boiling water.Enzyme is lived and is defined as: per minute discharges the enzyme amount required for 1 μm of ol reducing sugar.During mensuration, supernatant liquor boils the enzyme liquid of 5min for contrast in boiling water.Reducing sugar adopts DNS method to measure.
The activity of the Mierocrystalline cellulose restriction endonuclease measured from supernatant liquor is 1.2U/mL, and the activity of Polyglucosidase is 0.3U/mL.Result shows that the recombinant protein built has the activity of Mierocrystalline cellulose restriction endonuclease and Polyglucosidase, and this albumen successful expression in yeast.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
SEQUENCELISTING
<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>PatentInversion3.5
<210>1
<211>711
<212>DNA
<213>Saccharomycescerevisiae
<400>1
agtttatcattatcaatactagtttatcattatcaatactcgccatttcaaagaatacgt60
aaataattaatagtagtgattttcctaactttatttagtcaaaaaattagccttttaatt120
ctgctgtaacccgtacatgcccaaaatagggggcgggttacacagaatatataacatcgt180
aggtgtctgggtgaacagtttattcctggcatccactaaatataatggagcccgcttttt240
aagctggcatccagaaaaaaaaagaatcccagcaccaaaatattgttttcttcaccaacc300
atcagttcataggtccattctcttagcgcaactacagagaacaggggcacaaacaggcaa360
aaaacgggcacaacctcaatggagtgatgcaacctgcctggagtaaatgatgacacaagg420
caattgacccacgcatgtatctatctcattttcttacaccttctattaccttctgctctc480
tctgatttggaaaaagctgaaaaaaaaggttgaaaccagttccctgaaattattccccta540
cttgactaataagtatataaagacggtaggtattgattgtaattctgtaaatctatttct600
taaacttcttaaattctacttttatagttagtcttttttttagttttaaaacaccagaac660
ttagtttcgacggatttagttttaaaacaccagaacttagtttcgacggat711
<210>2
<211>253
<212>DNA
<213>Saccharomycescerevisiae
<400>2
ggatccaaacgatgagatttccttcaatttttactgcagttttattcgcagcatcctccg60
cattagctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaag120
ctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttgccattttcca180
acagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaag240
aagaaggggtatc253
<210>3
<211>3287
<212>DNA
<213>ArtificialSequence
<220>
<223> has the sequence of Mierocrystalline cellulose restriction endonuclease and dextran glycosides enzymic activity
<400>3
atggtgagttttaaagcaggtataaatttaggcggatggatatcacaatatcaagttttc60
agcaaagagcatttcgatacattcattacggagaaggacattgaaactattgcagaagca120
gggtttgaccatgtcagactgccttttgattatccaattatcgagtctgatgacaatgtg180
ggagaatataaagaagatgggctttcttatattgaccggtgccttgagtggtgtaaaaaa240
tacaatttggggcttgtgttggatatgcatcacgctcccgggtaccgctttcaagatttt300
aagacaagcaccttgtttgaagatccgaaccagcaaaagagatttgttgacatatggaga360
tttttagccaagcgttacataaatgaacgggaacatattgcctttgaactgttaaatgaa420
gttgttgagcctgacagtacccgctggaacaagttgatgcttgagtgtgtaaaagcaatc480
agggaaattgattccaccaggtggctttacattgggggcaataactataacagtcctgat540
gagcttaaaaaccttgcagatattgatgatgattacatagtttacaatttccatttttac600
aatccttttttctttacgcatcagaaagcccactggtcggaaagtgccatggcgtacaac660
aggactgtaaaatatccgggacaatatgagggaattgaagagtttgtgaaaaataatcct720
aagtacagttttatgatggaattgaataacctgaagctgaataaagagcttttgcgcaaa780
gatttaaaaccagcaattgagttcagggaaaagaaaaaatgcaaactatattgcggggag840
tttggcgtaattgccattgctgacctggagtccaggataaaatggcatgaagattatata900
agtcttctggaggagtatgatatcggcggcgcggtgtggaactacaaaaaaatggatttt960
gaaatttataatgaggatagaaaacctgtctcgcaagaattggtaaatatactggcgaga1020
agaaaaacttgaagcggtagatatcaagaaaataataaagcagatgctttggaagaaaaa1080
gcagggttgtgctcgggactggatttttggcataccaagcctgttagagactgggcattc1140
cttcaataatgatgactgacggacctcatggactgagaaagcaggggaagatgcagagat1200
tgcggacatcaacaacagcgttccagcaacctgttttccgtctcagcaggtttggcatgt1260
tcctgggacagagaactggttgagagagtaggtgcagcactagagaagaatgtcaggcgg1320
aaaatgtctcaatactgcttggaccaggtgcaaatataaaggttcacctttgtgtggaag1380
aaattttgaatattttcccgaagacccttatctttcgtcagctggcggcaagccatataa1440
aaggagttcaaagtcagggagtgggtgcatgtcttaaacattttgccgcaaacaaccagg1500
aacaccggagaatgaccgttgataccattgtagatgaaagaacgttgagggaaatatatt1560
ttgcaagctttgagaatgctgtaaaaaaagcacggccttgggtggttatgtgtgcatata1620
acaagctcaacggtgaatattgttcggagaacagatatcttttgacggaagttttaaaga1680
atgaatggatgcatgacggctttgtggtatccgactggggtgcggtaaatgacagggtca1740
gcggcctggatgcaggtcttgacctggaaatgcccaccagtcatggtattacggataaaa1800
agatagttgaagccgtaaaaagcggaaagctgtctgaaaatattttaaacagagctgtgg1860
aaagaattttgaaagtaattattatggcactggaaaacaaaaaagaaaacgcgcagtatg1920
aacaagatgctcatcacagactggcaaggcaggctgcggccgaatcgatggttcttctta1980
aaaacgaggacgatgtgcttcctttaaaaaagagcggaaccatagctttgataggagctt2040
ttgtgaaaaaaccaagataccagggttcgggcagttctcatattaccccgacaagacttg2100
atgatatttatgaagagataaaaaaggccggagccgacaaagtaaaccttgtatattcgg2160
aaggatacaggcttgaaaatgacggtattgatgaggaattgataaacgaagctaaaaagg2220
cggcatcaagctcggatgttgcggtagtatttgcagggcttccggatgaatatgaatctg2280
aaggatttgacagaactcacatgagtattccggaaaatcaaaacaggctgatagaagcgg2340
tggccgaagtccagagtaatattgttgtggtattgcttaacggctcaccggttgaaatgc2400
cgtggattgacaaggtaaaatccgtgcttgaagcttatcttggaggccaggcgctgggag2460
gccgctggcggatgtgctattcggtgaagtcaatcgtcggaaaacttgcggagaccttcc2520
cggtgaaattaagccataatccgtcctatttgaattttcccggagaggatgaccgagtgg2580
agtataaagaagggttgtttgtcggatacagatattatgatacaaagggaattgagccat2640
tgttcccctttggtcacggacttagctataccaaatttgaatacagtgatatatcagtcg2700
ataaaaaagatgtttcggacaatagcatcataaatgtcagcgttaaagtcaaaaatgttg2760
gaaaaatggcaggaaaagaaattgtgcagctgtatgtaaaagatgtgaaaagcagcgtca2820
gaagacctgagaaagagcttaaaggatttgaaaaggtcttccttaatccgggagaagaaa2880
agacggttacatttactttggacaaaagggcttttgcatattacaatactcagattaagg2940
actggcatgttgaaagcggagagtttctgatattaataggaaggtcctccagggacatag3000
ttttaaaagaatcagtgagagtaaattcaacggtgaagataagaaaaagattcacagtga3060
attcagcggttgaagatgtaatgtccgattcttcggctgcggccgttttagggcctgtac3120
taaaagagataaccgatgcactgcagattgatatggacaatgctcatgacatgatggcgg3180
ccaatataaagaatatgcctttgcgctcacttgtcggttactctcagggaaggttaagcg3240
aagaaatgctggaggaactggttgacaaaataaacaacgtggaataa3287
<210>4
<211>281
<212>DNA
<213>Saccharomycescerevisiae
<400>4
gggggtaccgggcccggccgcaaattaaagccttcgagcgtcccaaaaccttctcaagca60
aggttttcagtataatgttacatgcgtacacgcgtctgtacagaaaaaaaagaaaaattt120
gaaatataaataacgttcttaatactaacataactataaaaaaataaatagggacctaga180
cttcaggttgtctaactccttccttttcggttagagcggatgtggggggagggcgtgaat240
gtaagcgtgacataactaattacatgatgcggccctttaaa281
<210>5
<211>4532
<212>DNA
<213>ArtificialSequence
<220>
<223> is for building the DNA producing and have the yeast strain of Mierocrystalline cellulose restriction endonuclease and dextran glycosides enzyme double activated albumen
The sequence of fragment
<400>5
agtttatcattatcaatactagtttatcattatcaatactcgccatttcaaagaatacgt60
aaataattaatagtagtgattttcctaactttatttagtcaaaaaattagccttttaatt120
ctgctgtaacccgtacatgcccaaaatagggggcgggttacacagaatatataacatcgt180
aggtgtctgggtgaacagtttattcctggcatccactaaatataatggagcccgcttttt240
aagctggcatccagaaaaaaaaagaatcccagcaccaaaatattgttttcttcaccaacc300
atcagttcataggtccattctcttagcgcaactacagagaacaggggcacaaacaggcaa360
aaaacgggcacaacctcaatggagtgatgcaacctgcctggagtaaatgatgacacaagg420
caattgacccacgcatgtatctatctcattttcttacaccttctattaccttctgctctc480
tctgatttggaaaaagctgaaaaaaaaggttgaaaccagttccctgaaattattccccta540
cttgactaataagtatataaagacggtaggtattgattgtaattctgtaaatctatttct600
taaacttcttaaattctacttttatagttagtcttttttttagttttaaaacaccagaac660
ttagtttcgacggatttagttttaaaacaccagaacttagtttcgacggatggatccaaa720
cgatgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagctg780
ctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcg840
gttactcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaa900
ataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaagggg960
tatcatggtgagttttaaagcaggtataaatttaggcggatggatatcacaatatcaagt1020
tttcagcaaagagcatttcgatacattcattacggagaaggacattgaaactattgcaga1080
agcagggtttgaccatgtcagactgccttttgattatccaattatcgagtctgatgacaa1140
tgtgggagaatataaagaagatgggctttcttatattgaccggtgccttgagtggtgtaa1200
aaaatacaatttggggcttgtgttggatatgcatcacgctcccgggtaccgctttcaaga1260
ttttaagacaagcaccttgtttgaagatccgaaccagcaaaagagatttgttgacatatg1320
gagatttttagccaagcgttacataaatgaacgggaacatattgcctttgaactgttaaa1380
tgaagttgttgagcctgacagtacccgctggaacaagttgatgcttgagtgtgtaaaagc1440
aatcagggaaattgattccaccaggtggctttacattgggggcaataactataacagtcc1500
tgatgagcttaaaaaccttgcagatattgatgatgattacatagtttacaatttccattt1560
ttacaatccttttttctttacgcatcagaaagcccactggtcggaaagtgccatggcgta1620
caacaggactgtaaaatatccgggacaatatgagggaattgaagagtttgtgaaaaataa1680
tcctaagtacagttttatgatggaattgaataacctgaagctgaataaagagcttttgcg1740
caaagatttaaaaccagcaattgagttcagggaaaagaaaaaatgcaaactatattgcgg1800
ggagtttggcgtaattgccattgctgacctggagtccaggataaaatggcatgaagatta1860
tataagtcttctggaggagtatgatatcggcggcgcggtgtggaactacaaaaaaatgga1920
ttttgaaatttataatgaggatagaaaacctgtctcgcaagaattggtaaatatactggc1980
gagaagaaaaacttgaagcggtagatatcaagaaaataataaagcagatgctttggaaga2040
aaaagcagggttgtgctcgggactggatttttggcataccaagcctgttagagactgggc2100
attccttcaataatgatgactgacggacctcatggactgagaaagcaggggaagatgcag2160
agattgcggacatcaacaacagcgttccagcaacctgttttccgtctcagcaggtttggc2220
atgttcctgggacagagaactggttgagagagtaggtgcagcactagagaagaatgtcag2280
gcggaaaatgtctcaatactgcttggaccaggtgcaaatataaaggttcacctttgtgtg2340
gaagaaattttgaatattttcccgaagacccttatctttcgtcagctggcggcaagccat2400
ataaaaggagttcaaagtcagggagtgggtgcatgtcttaaacattttgccgcaaacaac2460
caggaacaccggagaatgaccgttgataccattgtagatgaaagaacgttgagggaaata2520
tattttgcaagctttgagaatgctgtaaaaaaagcacggccttgggtggttatgtgtgca2580
tataacaagctcaacggtgaatattgttcggagaacagatatcttttgacggaagtttta2640
aagaatgaatggatgcatgacggctttgtggtatccgactggggtgcggtaaatgacagg2700
gtcagcggcctggatgcaggtcttgacctggaaatgcccaccagtcatggtattacggat2760
aaaaagatagttgaagccgtaaaaagcggaaagctgtctgaaaatattttaaacagagct2820
gtggaaagaattttgaaagtaattattatggcactggaaaacaaaaaagaaaacgcgcag2880
tatgaacaagatgctcatcacagactggcaaggcaggctgcggccgaatcgatggttctt2940
cttaaaaacgaggacgatgtgcttcctttaaaaaagagcggaaccatagctttgatagga3000
gcttttgtgaaaaaaccaagataccagggttcgggcagttctcatattaccccgacaaga3060
cttgatgatatttatgaagagataaaaaaggccggagccgacaaagtaaaccttgtatat3120
tcggaaggatacaggcttgaaaatgacggtattgatgaggaattgataaacgaagctaaa3180
aaggcggcatcaagctcggatgttgcggtagtatttgcagggcttccggatgaatatgaa3240
tctgaaggatttgacagaactcacatgagtattccggaaaatcaaaacaggctgatagaa3300
gcggtggccgaagtccagagtaatattgttgtggtattgcttaacggctcaccggttgaa3360
atgccgtggattgacaaggtaaaatccgtgcttgaagcttatcttggaggccaggcgctg3420
ggaggccgctggcggatgtgctattcggtgaagtcaatcgtcggaaaacttgcggagacc3480
ttcccggtgaaattaagccataatccgtcctatttgaattttcccggagaggatgaccga3540
gtggagtataaagaagggttgtttgtcggatacagatattatgatacaaagggaattgag3600
ccattgttcccctttggtcacggacttagctataccaaatttgaatacagtgatatatca3660
gtcgataaaaaagatgtttcggacaatagcatcataaatgtcagcgttaaagtcaaaaat3720
gttggaaaaatggcaggaaaagaaattgtgcagctgtatgtaaaagatgtgaaaagcagc3780
gtcagaagacctgagaaagagcttaaaggatttgaaaaggtcttccttaatccgggagaa3840
gaaaagacggttacatttactttggacaaaagggcttttgcatattacaatactcagatt3900
aaggactggcatgttgaaagcggagagtttctgatattaataggaaggtcctccagggac3960
atagttttaaaagaatcagtgagagtaaattcaacggtgaagataagaaaaagattcaca4020
gtgaattcagcggttgaagatgtaatgtccgattcttcggctgcggccgttttagggcct4080
gtactaaaagagataaccgatgcactgcagattgatatggacaatgctcatgacatgatg4140
gcggccaatataaagaatatgcctttgcgctcacttgtcggttactctcagggaaggtta4200
agcgaagaaatgctggaggaactggttgacaaaataaacaacgtggaataagggggtacc4260
gggcccggccgcaaattaaagccttcgagcgtcccaaaaccttctcaagcaaggttttca4320
gtataatgttacatgcgtacacgcgtctgtacagaaaaaaaagaaaaatttgaaatataa4380
ataacgttcttaatactaacataactataaaaaaataaatagggacctagacttcaggtt4440
gtctaactccttccttttcggttagagcggatgtggggggagggcgtgaatgtaagcgtg4500
acataactaattacatgatgcggccctttaaa4532
<210>6
<211>10
<212>DNA
<213>ArtificialSequence
<220>
<223>Aat II recognition sequence
<400>6
ccgacgtcgg10
<210>7
<211>14
<212>DNA
<213>ArtificialSequence
<220>
<223>Not I recognition sequence
<400>7
ttgcggccgcaacc14
Claims (9)
1., for building the DNA fragmentation produced and there is the yeast strain of Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen, it is characterized in that: comprise the 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 that arrange in order; Described sequence is respectively as shown in SEQIDNO.1 ~ 4.
2. DNA fragmentation according to claim 1, is characterized in that: the sequence of described DNA fragmentation is as shown in SEQIDNO.5.
3., for building the plasmid produced and there is the yeast strain of Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen, it is characterized in that: for comprising the eukaryon expression plasmid of the DNA fragmentation described in claim 1 or 2.
4. plasmid according to claim 3, is characterized in that: described eukaryon expression plasmid is pPIC9K plasmid.
5. plasmid according to claim 3, it is characterized in that the method by comprising following steps prepares: synthesis two ends restricted property endonuclease recognized site contains the sequence of the DNA fragmentation described in claim 1 or 2, is connected on eukaryon expression plasmid by restriction enzyme.
6. plasmid according to claim 5, is characterized in that: when described eukaryon expression plasmid is pPIC9K plasmid, and restriction enzyme is Aat II and Not I.
7. 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 any one of claim 3-6.
8. yeast strain according to claim 7, is characterized in that: described yeast strain is yeast strain SMD1168.
9. the construction process of the yeast strain described in claim 7 or 8, is characterized in that comprising the steps: to prepare yeast Electroporation-competent cells, is transformed to turn to enter in yeast by the plasmid described in any one of claim 3-6 to obtain by electricity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410084775.1A CN103820443B (en) | 2014-03-10 | 2014-03-10 | Produce yeast strain and the structure thereof with Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410084775.1A CN103820443B (en) | 2014-03-10 | 2014-03-10 | Produce yeast strain and the structure thereof with Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103820443A CN103820443A (en) | 2014-05-28 |
| CN103820443B true CN103820443B (en) | 2016-01-13 |
Family
ID=50755733
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410084775.1A Active CN103820443B (en) | 2014-03-10 | 2014-03-10 | Produce yeast strain and the structure thereof with Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103820443B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN109182360B (en) * | 2018-10-23 | 2020-06-05 | 怀化学院 | Micromolecular cellulose endonuclease gene and protein and application thereof |
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 |
|---|
| Construction and characterization of different fusion proteins between cellulases and b-glucosidase to improve glucose production and thermostability;Hsiao-Lin Lee等;《Bioresource Technology》;20101129;第102卷;3973-3975 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103820443A (en) | 2014-05-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103820273B (en) | Production technology of lotus seed spirit | |
| CN103865949B (en) | A kind of method of gene knockout seed selection cellulase high-yield | |
| CN105368882A (en) | Method for producing ethyl alcohol through crop stalks by use of recombinant zymomonas mobilis | |
| CN103820443B (en) | Produce yeast strain and the structure thereof with Mierocrystalline cellulose restriction endonuclease and Polyglucosidase double activated albumen | |
| CN101423855B (en) | Method for preparing polysaccharide by using lucidum strain fermented laminaria leftover | |
| CN103849576B (en) | One strain has the recombinant Saccharomyces cerevisiae bacterial strain of stress tolerance | |
| CN101633896B (en) | Saccharmyces cerevisiae strain for resisting high-concentration acetic acid and application thereof | |
| CN103409333B (en) | Recombinant saccharomyces cerevisiae strain for continuously and efficiently secreting beta-glucosidase and applications thereof | |
| CN103382444A (en) | Gene recombinant saccharomyces cerevisiae capable of degrading crystalline cellulose | |
| CN103589719B (en) | A kind of structure with the yeast strain of production and recycled fiber element excision enzyme dual-use function | |
| CN104862344A (en) | Method for producing cellulosic ethanol by fermenting agricultural and forest biomass waste thick mash | |
| CN101914579A (en) | Method for preparing resveratrol | |
| CN102703543B (en) | Method for preparing bacterial cellulose by tuberous raw materials | |
| CN103589718B (en) | A kind of structure with the yeast strain of production and recycled fiber element dextran glycosides enzyme dual-use function | |
| CN103555720B (en) | A kind of structure with the yeast strain of production and recycled fiber element restriction endonuclease dual-use function | |
| CN101376905B (en) | Method for producing fermentable sugar by red ramie bark fibre enzymolysis | |
| CN102080050B (en) | Trichoderma viride W2 capable of producing thermophilic ethanol-resistant beta-glucosidase and application thereof | |
| CN106396758A (en) | Method for separating and refining salix biological components and controlling desert soil | |
| CN102732437B (en) | Saccharomyces cerevisiae engineering bacterium and its application in production of ethanol | |
| CN105062928A (en) | Zymomonas mobilis resistant to high-concentration acetic acid and high-concentration furfural and application thereof | |
| CN101979535A (en) | Allele knockout method, recombination GAL strain constructed by same and application | |
| CN104480139A (en) | Method for constructing cellulase high-yielding strain by expressing doubleactive protein of celluloseexonucleaseand endonuclease | |
| CN101235390B (en) | Method for producing fuel ethanol by solid fermentation of sugar grass straw | |
| CN104073524A (en) | Method for preparing fuel ethanol by saccharifying and fermenting carbon-rich microalgae solid acid | |
| CN104805170A (en) | Submerged fermentation and extraction process method for ganoderma triterpene acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190802 Address after: 435400 Shifosi Railway Station Industrial Park, Wuxue City, Huanggang City, Hubei Province Patentee after: Wuxue Ganghua Technical Service Center Address before: 430068 Wuhan Province, Wuchang District, South Lake, Lee Ka pier village, No. 1, No. 1, No. Patentee before: Hubei Industry University |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20191105 Address after: 435400 village committee office building, hujialong village, Shifosi Town, Wuxue City, Huanggang City, Hubei Province Patentee after: Wuxue Liangda science and Technology Development Center Address before: 435400 Shifosi Railway Station Industrial Park, Wuxue City, Huanggang City, Hubei Province Patentee before: Wuxue Ganghua Technical Service Center |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20191204 Address after: Room 006, 3rd floor, Lishizhen Avenue Building, Caohe Town, Qichun County, Huanggang City, Hubei Province Patentee after: Zhongnong Huawei Biopharmaceutical (Hubei) Co., Ltd. Address before: 435400 village committee office building, hujialong village, Shifosi Town, Wuxue City, Huanggang City, Hubei Province Patentee before: Wuxue Liangda science and Technology Development Center |
|
| TR01 | Transfer of patent right |