CN103789332A - Construction of yeast strain having naringinase producing and recycling functions - Google Patents
Construction of yeast strain having naringinase producing and recycling functions Download PDFInfo
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- CN103789332A CN103789332A CN201410084899.XA CN201410084899A CN103789332A CN 103789332 A CN103789332 A CN 103789332A CN 201410084899 A CN201410084899 A CN 201410084899A CN 103789332 A CN103789332 A CN 103789332A
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Abstract
The invention discloses construction of a yeast strain having naringinase producing and recycling functions and belongs to the field of enzyme engineering. A promoter sequence of saccharomyces cerevisiae TDH3, a secretion signal peptide coding sequence of yeast, a coding sequence of the naringinase, 400 amino acid fragment coding sequences at a clumping factor C end of saccharomyces cerevisiae and a terminator sequence of the saccharomyces cerevisiae TDH3 are compounded in order, restriction enzyme loci are added at two ends of the compounded sequence which is constructed into a pPIC9K plasmid through digestion, and the plasmid is converted into the yeast to obtain the yeast strain having naringinase producing and recycling functions. The recovery rate of naringinase producing reaches 92 percent, and enzyme activity of the naringinase subjected to fermentation reaches 5.0U/g of wet yeast. By means of the construction, producing and synchronous concentrating and recycling of the naringinase can be achieved by one step, energy consumption is reduced, cost is reduced, and therefore, the construction has strong practicability.
Description
Technical field
The invention belongs to enzyme engineering field, be specifically related to a kind of structure of producing and reclaiming the yeast strain of naringinase dual-use function that has.
Background technology
The output of mandarin orange is only low than the U.S. and Brazil, and the output of citrus is arranged in third place in the world.Utilizing the nectar that citrus produces, to have sugar degree high, local flavor delicate fragrance alcohol and etc. feature.And citrus fruit juice has and the feature of being convenient to suitability for industrialized production and processing such as be easy to squeeze, can be used for producing the high value-added products such as fruit wine.But citrus fruit juice usually has dense strong bitter taste, this bitter taste has limited the utilization of fruit juice and the popularization of product greatly.Remove the citrus fruit juice of bitter taste, just can have more wide market and utilize space.
Utilize naringinase the bitter taste in fruit juice can be got rid of effectively.The production of naringinase has solid fermentation and two kinds of methods of liquid fermenting.Although the concentration of solid fermentation enzyme is high, its mass transfer and heat transmission resistance are very large.The solid fermentation that mass transfer and heat transmission resistance are large is unfavorable for large-scale industrial production very much, because its temperature and acidity etc. are not easy to control in industrially scalable.
It is large that liquid fermenting has fermentation volume, easily realize the features such as automatization control, thereby liquid fermenting embodies huge advantage in the production of many products.Particularly, in labor cost rising now, carry forward vigorously under mechanize and automatization background the world, and liquid fermenting has more wide space.The production of naringinase also has more and more tends to liquid fermenting.But the enzyme product concentration that liquid fermenting is produced is low.The naringinase etc. of producing is all free in fermentation liquid, for obtaining the pressed powder of naringinase of high density, need to concentrate fermentation liquid.For purifying concentration of fibre element enzyme often needs to carry out concentration of fibre element enzyme by techniques such as vacuum-evaporation or spraying are dried.In Mierocrystalline cellulose evaporation concentration or spray-dired process, a loss of activity part for naringinase, and also concentration technology has increased substantially the production cost of cellulase.In evaporation or spray-dired process, consume a large amount of energy as electricity or combustion gas etc.Therefore reduce concentrated cost, become the inevitable choice that reduces liquid fermenting production naringinase production cost.
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 yeast strain that produces and reclaim naringinase dual-use function that has.
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:
There is for building the DNA fragmentation of producing and reclaiming the yeast strain of naringinase dual-use function, comprise 400 amino acid fragment encoding sequences, the yeast saccharomyces cerevisiae TDH3 terminator sequence of the yeast saccharomyces cerevisiae TDH3 promoter sequence arranged in order, yeast saccharomyces cerevisiae secretion signal peptide-coding sequence, naringinase encoding sequence, yeast saccharomyces cerevisiae AgF C end; Above-mentioned sequence is respectively as shown in SEQ ID NO.1～5.
Preferably, described for building the sequence with the DNA fragmentation of producing and reclaiming the yeast strain of naringinase dual-use function as shown in SEQ ID NO.6.
Be the eukaryon expression plasmid that comprises above-mentioned DNA fragmentation for building the plasmid that there is production and reclaim the yeast strain of naringinase dual-use function.Described eukaryon expression plasmid is preferably pPIC9K plasmid.
Described prepares by the method that comprises following steps for building the plasmid that has production and reclaim the yeast strain of naringinase dual-use function: 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 pPIC9K plasmid, restriction enzyme is preferably Aat II and Not I.
There is a yeast strain that produces and reclaim naringinase dual-use function, contain above-mentioned plasmid.Described yeast strain is preferably yeast strain SMD1168.
Described has production and reclaims the construction process of the yeast strain of naringinase dual-use function, 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:
Yeast strain of the present invention can, in producing naringinase, be adsorbed on bacterial strain self surface naringinase, by simple filtration, just can reach the object of concentrated naringinase.
The present invention has simplified the concentration technology of naringinase greatly, has reduced energy consumption, has significantly reduced cost, and therefore this invention has stronger practicality.
The present invention expresses naringinase gene in yeast, and has realized that naringinase is produced and a step of synchronous concentration and recovery completes, and prior art does not have relevant report, have higher novelty.
The rate of recovery that yeast strain of the present invention produces naringinase reaches 92%, and after fermentation, the enzyme work of naringinase reaches the wet yeast of 5.0 U/g.
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.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
Embodiment 1
(1) extraction of pPIC9K plasmid
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 bacterium liquid 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 the pre-cold soln III of 0.15 mL (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.
(2) preparation of bacillus coli DH 5 alpha competent cell
1) bacillus coli DH 5 alpha 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) mL overnight culture in transferase 10 .2～1 is to 20 mL LB(or other nutritious substratum are housed) 100 mL shaking flasks.
5) thermal agitation is cultivated 6 hours at 37 ℃.
6) monitoring OD600 value (cultivate and measure once per half an hour after 1 hour).
7) in the time that OD600 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 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) 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.
(3) pPIC9K-Naringinase grain builds
1) with restriction enzyme A at II, Not I digested plasmid pPIC9K respectively.Restriction enzyme A at II, the each 1.5 μ L of Not I (TAkaRA), the pPIC9K plasmid solution 6 μ L of extraction, 10 × K Buffer, 1 μ L joins in 100 μ L EP pipes, and in 30 ℃ of water-baths, enzyme is cut 60 min.Reclaim by agarose gel electrophoresis the plasmid pPIC9K that enzyme is cut again, reclaiming band is 9.25 kb left and right.
2) sequence is synthetic
Synthetic two ends have the sequence of Aat II and Not I recognition sequence, sequence comprises that CCGACGTC-yeast saccharomyces cerevisiae TDH3 opens 400 amino acid fragments volume code order row – yeast saccharomyces cerevisiae TDH3 termination subsequence – GCGGCCGCGG of subsequence – yeast saccharomyces cerevisiae secretion signal peptide-coding sequence – naringinase volume code order row – yeast saccharomyces cerevisiae AgF C end, and each fragment and full length fragment are as shown in SEQ ID NO.1～6.
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) above-mentioned connection product electricity is transformed into bacillus coli DH 5 alpha, it is as follows that electricity transforms concrete grammar:
4.1) thaw on ice bacillus coli DH 5 alpha competent cell add 1～10 μ L connect product, place on ice approximately 5 minutes.
4.2) shift and connect product/cell mixture to cooled 2mm electroporation container.
4.3) add live conversion instrument, be ready to 300 μ L LB or 2 × YT.
4.4) electroporation container is carried out to pulse (200 ohm, 25 μ F, 2.5 kilovolts), testing time constant, should be more than 3.
4.5) add immediately the LB of 300 μ L or 2 × YT to electric revolving cup.
4.6) at 37 ℃ culturing cell 40 minutes to 1 hour with recovery.
4.7) the centrifugal 150 μ L supernatants that discard after recovery, residue 150 μ L resuspended thalline is applied on the solid plate of the LB that contains ammonia benzyl (100 μ g/mL) or 2 × YT substratum, in 37 ℃ of overnight incubation.
5) choose the mono-clonal of growing on solid plate, by cultivating, extract plasmid, by Aat II and FspA I, plasmid is carried out to enzyme and cut evaluation, identify the further order-checking again of correct plasmid, obtain plasmid pPIC9K-Naringinase.
(4) preparation of yeast competent cell
1) choose a ring yeast (SMD1168) and be inoculated in 5 mL YEPD substratum, 30 ℃ of 250～300 rpm overnight incubation obtains first order seed.
2) get 1 mL first order seed and be inoculated in respectively in two bottle of 50 mL YEPD substratum, 30 ℃ of 250～300 rpm cultivates approximately 16～18 h(OD600 approximately 1.3～1.5).
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.
7) 1 mol/L sorbyl alcohol washing of 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, and are sub-packed in (80 μ L/ pipe) in 3 EP pipes, in-70 ℃ of Refrigerator stores.
(5) pPIC9K-Naringinase plasmid electricity transformed yeast
1) in yeast competent cell, add approximately 5～10 μ g(volumes to be less than 10 μ L) pPIC9K-Naringinase plasmid, 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, in 30 ℃ of standing 1h.
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 ℃.
(6) yeast transformant fermentation
The transformant of picking is 30 ℃ of cultivation 24 h in YEPD substratum, are inoculated in fermention medium (yeast extract 10 g/L, peptone 20 g/L with 10% inoculum size (v/v), 50 mM citrate buffer solutions, wheat bran 200 g/L, Walocel MT 20.000PV 20 g/L, 1 L water) in.Fermentation is fermented in 500 mL shaking flasks, and liquid amount is 20%(v/v).In fermention medium, train 48 h.
(7) recovery of naringinase
Centrifugal 10 min of fermented liquid 4000 r/m in whizzer, collect supernatant and (in supernatant, have free naringinase, for the calculating of the naringinase rate of recovery), add distilled water in the ratio of yeast sedimentation weight in wet base and distilled water mass ratio 1:10, with twice of distilled water wash, collect yeast sedimentation, naringinase is adsorbed on yeast surface.
(8) naringinase enzyme activity determination: add respectively 2 mL pH 4.0,0.1 M acetic acid-sodium acetate buffer solution in test tube, the naringin solution of 2 mL mass concentration 400 mg/L, at 40 ℃, be incubated 5 min, add enzyme liquid 1 mL of beforehand dilution, be incubated 10 min, use immediately boiling water bath deactivation 10 min, the amount of high effective liquid chromatography for measuring residue naringin, by can be calculated the reduction of naringin.Chromatographic condition: chromatographic column is Dalian Yi Lite C
18hypersil(250 mm × 4.6 mm, 5 Ixm); Moving phase is methyl alcohol: 5% acetic acid solution (55:45); Flow velocity 1 mL/min; Detect wavelength 283 nm; 30 ℃ of column temperatures.
Enzyme work is defined as: at 40 ℃, under pH value 4.0 conditions, the enzyme amount that per minute decomposes 1 μ g naringin is 1 U.
Live/(enzyme of the enzyme work+yeast sedimentation absorption of supernatant liquor is lived) × 100% of the enzyme of the naringinase rate of recovery (%)=yeast sedimentation absorption.
Reach 92% through the rate of recovery of measuring naringinase, after fermentation, the enzyme work of naringinase reaches the wet yeast of 5.0 U/g.The rate of recovery that naringinase is higher, illustrates in fermentation, and the yeast of structure is the concentrated naringinase of enrichment well, has realized fermentation and a concentrated step and has completed.
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
Mono-kind of <120> has the structure of producing and reclaiming the yeast strain of naringinase dual-use function
<130> 1
<160> 8
<170> PatentIn version 3.5
<210> 1
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<212> DNA
<213> Saccharomyces cerevisiae
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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
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ggatccaaac gatgagattt ccttcaattt ttactgcagt tttattcgca gcatcctccg 60
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ctgtcatcgg ttactcagat ttagaagggg atttcgatgt tgctgttttg ccattttcca 180
acagcacaaa taacgggtta ttgtttataa atactactat tgccagcatt gctgctaaag 240
aagaaggggt atc 253
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atggccttgt caatctccca agtggctttc gaacaccatc gaacggccct cggcattggc 60
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gcgtatgaaa ttgaggtcaa gcgagccggc cacgacgcag acgtcttccg ttcggagtct 180
tccgattcgg tcctggttcc ctggccgagc tctccgctgc aatcgggtga agaggcgacg 240
gtgcgcgtac gctccttcgg atcggacggc cagcacgaca cgccttggtc ggacgctgtc 300
accgtcgagc cgggtctcct cacgccggac gattggcacg atgcggtcgt gattgcctct 360
gaccggccga cggaggtgga cgccacccat cgaccgatcc agtttcgcaa ggaattctcc 420
gtggatgatt cgtacgtctc agcgcggttg tacatcacgg cgctggggct ctacgaggcc 480
cgtatcaacg accagcgcgt tggtgaccat gtcatggcgc ccgggtggca gtcctatcag 540
tatcgccatg aatacaacac atacgacgtg accgatctgc tgaagcaggg gcccaatgcc 600
attggcgtca ctgtgggaga gggatggtac tccgggcgta tcggatacga tggcggaaag 660
cgcaatatct atggcgacac acttgggctg ctctcgctac ttgtcgttac caagtcggat 720
gggagtaagc tctacatccc tagcgacagc agctggaagt ccagcacggg gccgatcatc 780
tcgtcggaga tatatgatgg agaagaatat gactccaggc tggaacagaa gggttggtcc 840
caggtgggct ttaacagcac cggctggctt ggtacgcacg agctgtcgtt ccctaaggag 900
agattggcct cgccagacgg cccccccgtg cgacgcgtgg ccgagcacaa gcttgcgaac 960
gtcttctcta gcgcttccgg aaagacggtt ctcgactttg gacagaacct tgtagggtgg 1020
ctacgcatcc gagtcaaggg ccccaagggt cagactattc gctttgttca tactgaagtg 1080
atggagaatg gagaggtagc cactcgaccc ttacgccagg ccaaagcaac ggaccatttt 1140
accctctcgg gcgaaggagt ccaggagtgg gagccctcgt ttacctatca cggattccgt 1200
tacgtgcagg tcgacggctg gccagcagac acgcctcttg atgaaaactc cgtgaccgcc 1260
atcgtggtgc attcggacat ggaacggacg gggtactttg aatgttccaa cccgctcatc 1320
agcaaacttc acgagaacat cctctggagc atgagaggga acttctttag tatccccacc 1380
gactgccctc agcgagatga gcggcttggt tggaccggcg acattcatgc cttttcccga 1440
acggccaact tcatctatga cacggcgggc ttcttgagag catggctcaa agacgcccgt 1500
tccgagcaat tgaaccactc gtactcactg ccttatgtta ttcccaacat ccacggaaac 1560
ggagagaccc caacctctat ctggggagat gcaattgtcg gagtaccatg gcagctttac 1620
gagagctttg gtgataaggt catgctggaa gagcagtatg ggggcgccaa agactgggtg 1680
gacaagggga ttgtacgcaa cgatgtcggt ctgtgggatc ggtcaacgtt ccaatgggcc 1740
gactggctcg atcccaaggc tccggccgat gacccggggg atgccaccac gaacaagtat 1800
ctcgtttcgg acgcctacct cctccacagc acggacatgc tcgccaacat ctcgacttcc 1860
ctttccaaag gagaggaggc gtcgaactac accgagtggc acgcgaaact caccaaggaa 1920
ttccagaagg catggattac gtcgaacggg acgatggcca atgaaacgca gacaggcctg 1980
gccctcccgc tgtactttga tctgttcccc agcgctgaac aggcccagtc ggccgccaag 2040
cgcttggtca acatcatcaa gcagaacgac tacaaggtgg gcaccgggtt tgcggggacc 2100
catctcctcg gccatacgtt gtccaagtac ggagaatccg acgcgttcta ttcaatgctg 2160
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caggcgtcta ccaagttctt gactccgtac ggcatggctt cgacgaagtg gtggctggac 2460
gggcaagacc agtcgtgcgg cgggttcgat ttccacctcg tggctgaagt gccgcccaac 2520
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aagggtatcg gattctctgg ttcttatcaa gacgttacca atatggatga aaacactggt 180
aaatgtactc aaaaatcata ttcctttagt ggtaacttgt ctccattaga cgaagaatta 240
tctgttcatt tcagaggacc tttgaaatta ttagaatttg gtgtttacta cccaagcagt 300
aatggtaatt caaagagaca agttgacgaa caagattgta atacaaaaca cgttcatcat 360
aaacacaaga gagcaactga agttgttcaa gtcacacaaa cagttttcgt tgatggtaat 420
ggtaacactg ttacttctca agccctccaa acctctacta ttgaaacaaa ttcagctgct 480
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tatggatctg tttctgccct tgacggtgaa ggcaaagctt atagaaccga tatctcaacc 600
aaatctgctc caacctcaac atctgctcaa ccatcttcat cagaaactgc ctctgctgat 660
ggtgcttgga ccagagacag tcattacact ccaggatcca ctgataactg tgtgttcttg 720
aactatcatg gtggttctgg ttctggtgtt tggtctgcta attttggtaa ctcattatct 780
tatgccaact ccgataattc tggtgggtct tcaactcctg ttgccttggg agaaaccact 840
atcaaatctg gcgaagaatt catcatcttc tctggttcta aatgtggtag tagttccgat 900
tgtggttatt atagagatgg tactgttgct tatcacggtt tcaaaggaac tagcaagatt 960
tttgtttttg aatttgaaat gccaagtgat actaatggta acggttataa ccaagacatg 1020
ccagccgttt ggttattgaa tgctaagatc ccaagaactt tacaatacgg tgaagccact 1080
tgttcttgtt ggaagaccgg ttgtggtgaa ttagatttgt ttgaagtctt aagcagtggt 1140
tccagtaaaa tgatttctca cttgcacgat ggtcaaggtt cttctcaaaa cagtaacaat 1200
taa 1203
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gaaatataaa taacgttctt aatactaaca taactataaa aaaataaata gggacctaga 180
cttcaggttg tctaactcct tccttttcgg ttagagcgga tgtgggggga gggcgtgaat 240
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<210> 6
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<213> Artificial Sequence
<220>
<223> has for building the DNA fragmentation of producing and reclaiming the yeast strain of naringinase dual-use function
<400> 6
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
tatcatggcc ttgtcaatct cccaagtggc tttcgaacac catcgaacgg ccctcggcat 1020
tggcgagacg cagccccggg tctcatggcg gttcgacggc aacgtctccg actgggagca 1080
acgtgcgtat gaaattgagg tcaagcgagc cggccacgac gcagacgtct tccgttcgga 1140
gtcttccgat tcggtcctgg ttccctggcc gagctctccg ctgcaatcgg gtgaagaggc 1200
gacggtgcgc gtacgctcct tcggatcgga cggccagcac gacacgcctt ggtcggacgc 1260
tgtcaccgtc gagccgggtc tcctcacgcc ggacgattgg cacgatgcgg tcgtgattgc 1320
ctctgaccgg ccgacggagg tggacgccac ccatcgaccg atccagtttc gcaaggaatt 1380
ctccgtggat gattcgtacg tctcagcgcg gttgtacatc acggcgctgg ggctctacga 1440
ggcccgtatc aacgaccagc gcgttggtga ccatgtcatg gcgcccgggt ggcagtccta 1500
tcagtatcgc catgaataca acacatacga cgtgaccgat ctgctgaagc aggggcccaa 1560
tgccattggc gtcactgtgg gagagggatg gtactccggg cgtatcggat acgatggcgg 1620
aaagcgcaat atctatggcg acacacttgg gctgctctcg ctacttgtcg ttaccaagtc 1680
ggatgggagt aagctctaca tccctagcga cagcagctgg aagtccagca cggggccgat 1740
catctcgtcg gagatatatg atggagaaga atatgactcc aggctggaac agaagggttg 1800
gtcccaggtg ggctttaaca gcaccggctg gcttggtacg cacgagctgt cgttccctaa 1860
ggagagattg gcctcgccag acggcccccc cgtgcgacgc gtggccgagc acaagcttgc 1920
gaacgtcttc tctagcgctt ccggaaagac ggttctcgac tttggacaga accttgtagg 1980
gtggctacgc atccgagtca agggccccaa gggtcagact attcgctttg ttcatactga 2040
agtgatggag aatggagagg tagccactcg acccttacgc caggccaaag caacggacca 2100
ttttaccctc tcgggcgaag gagtccagga gtgggagccc tcgtttacct atcacggatt 2160
ccgttacgtg caggtcgacg gctggccagc agacacgcct cttgatgaaa actccgtgac 2220
cgccatcgtg gtgcattcgg acatggaacg gacggggtac tttgaatgtt ccaacccgct 2280
catcagcaaa cttcacgaga acatcctctg gagcatgaga gggaacttct ttagtatccc 2340
caccgactgc cctcagcgag atgagcggct tggttggacc ggcgacattc atgccttttc 2400
ccgaacggcc aacttcatct atgacacggc gggcttcttg agagcatggc tcaaagacgc 2460
ccgttccgag caattgaacc actcgtactc actgccttat gttattccca acatccacgg 2520
aaacggagag accccaacct ctatctgggg agatgcaatt gtcggagtac catggcagct 2580
ttacgagagc tttggtgata aggtcatgct ggaagagcag tatgggggcg ccaaagactg 2640
ggtggacaag gggattgtac gcaacgatgt cggtctgtgg gatcggtcaa cgttccaatg 2700
ggccgactgg ctcgatccca aggctccggc cgatgacccg ggggatgcca ccacgaacaa 2760
gtatctcgtt tcggacgcct acctcctcca cagcacggac atgctcgcca acatctcgac 2820
ttccctttcc aaaggagagg aggcgtcgaa ctacaccgag tggcacgcga aactcaccaa 2880
ggaattccag aaggcatgga ttacgtcgaa cgggacgatg gccaatgaaa cgcagacagg 2940
cctggccctc ccgctgtact ttgatctgtt ccccagcgct gaacaggccc agtcggccgc 3000
caagcgcttg gtcaacatca tcaagcagaa cgactacaag gtgggcaccg ggtttgcggg 3060
gacccatctc ctcggccata cgttgtccaa gtacggagaa tccgacgcgt tctattcaat 3120
gctgcgacag acagaagtcc cgtcgtggct gtatcaggtg gtgatgaacg gcaccacgac 3180
atgggagcgc tgggacagca tgttgcccaa tgggagcatc aacccgggcc agatgacctc 3240
gttcaaccac tacgctgtcg gctctgtggg gagctggctc catgaggtca tcgggggtct 3300
gtctcctgct gagcccggtt ggaggagaat caatattgag gtagtgcctg gcggcgatct 3360
tcagcaggcg tctaccaagt tcttgactcc gtacggcatg gcttcgacga agtggtggct 3420
ggacgggcaa gaccagtcgt gcggcgggtt cgatttccac ctcgtggctg aagtgccgcc 3480
caacacccgg gccactgtcg ttcttcctgg aaaggggggt gagaaggttg atgttggttc 3540
aggggttcat gaatatcatg ttcggtgtgt gaagctgtag atgaaattta gttctactac 3600
attattagct gtgttagcat cactttcggc cactgtcaat gccggatgtt catttgaagg 3660
tggaaactac tactgttcag aaaccaaaaa agtcgtctac aagggtatcg gattctctgg 3720
ttcttatcaa gacgttacca atatggatga aaacactggt aaatgtactc aaaaatcata 3780
ttcctttagt ggtaacttgt ctccattaga cgaagaatta tctgttcatt tcagaggacc 3840
tttgaaatta ttagaatttg gtgtttacta cccaagcagt aatggtaatt caaagagaca 3900
agttgacgaa caagattgta atacaaaaca cgttcatcat aaacacaaga gagcaactga 3960
agttgttcaa gtcacacaaa cagttttcgt tgatggtaat ggtaacactg ttacttctca 4020
agccctccaa acctctacta ttgaaacaaa ttcagctgct tcatcacctg ctgctaataa 4080
tgatgccaac tcaggttctg gttctggttc cggttccggt tatggatctg tttctgccct 4140
tgacggtgaa ggcaaagctt atagaaccga tatctcaacc aaatctgctc caacctcaac 4200
atctgctcaa ccatcttcat cagaaactgc ctctgctgat ggtgcttgga ccagagacag 4260
tcattacact ccaggatcca ctgataactg tgtgttcttg aactatcatg gtggttctgg 4320
ttctggtgtt tggtctgcta attttggtaa ctcattatct tatgccaact ccgataattc 4380
tggtgggtct tcaactcctg ttgccttggg agaaaccact atcaaatctg gcgaagaatt 4440
catcatcttc tctggttcta aatgtggtag tagttccgat tgtggttatt atagagatgg 4500
tactgttgct tatcacggtt tcaaaggaac tagcaagatt tttgtttttg aatttgaaat 4560
gccaagtgat actaatggta acggttataa ccaagacatg ccagccgttt ggttattgaa 4620
tgctaagatc ccaagaactt tacaatacgg tgaagccact tgttcttgtt ggaagaccgg 4680
ttgtggtgaa ttagatttgt ttgaagtctt aagcagtggt tccagtaaaa tgatttctca 4740
cttgcacgat ggtcaaggtt cttctcaaaa cagtaacaat taagggggta ccgggcccgg 4800
ccgcaaatta aagccttcga gcgtcccaaa accttctcaa gcaaggtttt cagtataatg 4860
ttacatgcgt acacgcgtct gtacagaaaa aaaagaaaaa tttgaaatat aaataacgtt 4920
cttaatacta acataactat aaaaaaataa atagggacct agacttcagg ttgtctaact 4980
ccttcctttt cggttagagc ggatgtgggg ggagggcgtg aatgtaagcg tgacataact 5040
aattacatga tgcggccctt taaa 5064
<210> 7
<211> 8
<212> DNA
<213> Artificial Sequence
<220>
<223> Aat II recognition sequence
<400> 7
ccgacgtc 8
<210> 8
<211> 10
<212> DNA
<213> Artificial Sequence
<220>
<223> Not I recognition sequence
<400> 8
gcggccgcgg 10
Claims (10)
1. there is for building the DNA fragmentation of producing and reclaiming the yeast strain of naringinase dual-use function, it is characterized in that: 400 the amino acid fragment encoding sequences, the yeast saccharomyces cerevisiae TDH3 terminator sequence that comprise the yeast saccharomyces cerevisiae TDH3 promoter sequence arranged in order, yeast saccharomyces cerevisiae secretion signal peptide-coding sequence, naringinase encoding sequence, yeast saccharomyces cerevisiae AgF C end.
2. DNA fragmentation according to claim 1, is characterized in that: described sequence is respectively as shown in SEQ ID NO.1～5.
3. DNA fragmentation according to claim 1, is characterized in that: the sequence of described DNA fragmentation is as shown in SEQ ID NO.6.
4. there is for building the plasmid of producing and reclaiming the yeast strain of naringinase dual-use function, 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. there is a yeast strain that produces and reclaim naringinase dual-use function, 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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