CN104480139A - Method for constructing cellulase high-yielding strain by expressing doubleactive protein of celluloseexonucleaseand endonuclease - Google Patents
Method for constructing cellulase high-yielding strain by expressing doubleactive protein of celluloseexonucleaseand endonuclease Download PDFInfo
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- CN104480139A CN104480139A CN201410824320.9A CN201410824320A CN104480139A CN 104480139 A CN104480139 A CN 104480139A CN 201410824320 A CN201410824320 A CN 201410824320A CN 104480139 A CN104480139 A CN 104480139A
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Abstract
The invention discloses amethod for constructing a cellulase high-yielding strain by expressing doubleactive protein of celluloseexonucleaseand endonuclease, and belongs to the field of enzyme engineering. According to the method, coding genes provided with doubleactive protein of celluloseexonucleaseand endonucleaseare integrated to a position of an aspergillusniger heat shock protein gene with adoption of homologous recombination and genetic engineering technologies. Specifically, a homologous recombinant fragment whose nucleotide sequence is represented by SEQ ID NO.1 is synthesized, is digested by Sal I and connected with pWM1 plasmid,DH 5 alpha is transformed, and homologous recombinant plasmid is obtained; the homologous recombinant plasmid is transformed to agrobacterium tumefaciens, and a cellulase high-yielding aspergillusniger strain is obtained through agrobacterium tumefaciens mediated transformation. Expression of interest protein is increased remarkably by increasing the temperature through heat shockexpression, so that the activities of the celluloseexonucleaseand endonuclease are improved remarkably, and the enzyme activity of celluloseis improved.
Description
Technical field
The invention belongs to enzyme engineering field, be specifically related to a kind of method expressing Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated albumen structure High-Cellulase-Yielding Aspergillus niger strain.
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, and utilizing cellulose raw to produce bioenergy, is alleviating energy crisis, the key realizing 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, dextran glycosides enzyme.Wherein, the crystalline structure of excision enzyme degraded cellulose, be the cellulosic rate-limiting step of cellulose degraded, 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 Mierocrystalline cellulose becomes the cellulosic bottleneck of cellulose degraded.
Obtain the bacterial strain of High Cellulase Production, the production cost reducing cellulase becomes the only way of cellulase industrialized utilization.The production bacterial strain of cellulase mainly contains aspergillus niger.Aspergillus niger, due to its higher security, is considered to production of cellulose enzyme one of bacterial strain having application prospect most.The cellulase that aspergillus niger is produced is made up of Mierocrystalline cellulose restriction endonuclease, Polyglucosidase and excision enzyme.Wherein excision enzyme destroys cellulose crystals structure, and crystal fibre element is degraded to the Mierocrystalline cellulose fragment of solubility, this process is the rate-limiting step of cellulose degradation.Cellulosic efficient degradation needs a large amount of Mierocrystalline cellulose excision enzymes.But, aspergillus niger produce cellulase in, the activity of the specific activity excision enzyme of Polyglucosidase and the activity of restriction endonuclease much higher.The Mierocrystalline cellulose 5 prime excision enzyme activity produced due to aspergillus niger and the deficiency of endonuclease activity, the cellulase causing aspergillus niger to produce is very low to the degradation efficiency of natural cellulose.Therefore increase the activity of Mierocrystalline cellulose excision enzyme and restriction endonuclease, become the important measures improving aspergillus niger cellulose degraded efficiency.
The method of existing increase aspergillus niger cellulase has and the cellulase mixing of high 5 prime excision enzyme activity or Aspergillus niger strain and Trichodermareesei mixed fungus fermentation.With mixing of high 5 prime excision enzyme activity cellulase, inherently add this one technique of mixing, add degraded cost.And the cellulase cost of high 5 prime excision enzyme activity is also higher.Mixed fungus fermentation produces toxin due to Trichodermareesei, greatly limit the use range of cellulase.
The method of the structure aspergillus niger cellulase high-yield reported is that the gene that imports has the gene co expression of cellulase with aspergillus niger, but the enzyme of expressing only has a kind of activity, can not increase the activity of restriction endonuclease and excision enzyme simultaneously.Existing genetic engineering bacterium does not control the single expression increasing considerably a certain enzyme by condition (temperature, acidity etc.) simultaneously, limited to the change of cellulase systems composition.
Summary of the invention
The object of the invention is to overcome the shortcoming of prior art and deficiency, a kind of method expressing Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated albumen structure High-Cellulase-Yielding Aspergillus niger strain is provided.The present invention also aims to provide a kind of and build the High-Cellulase-Yielding Aspergillus niger strain obtained based on the method.
Object of the present invention is achieved through the following technical solutions:
Express a method for Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated albumen structure High-Cellulase-Yielding Aspergillus niger strain, for the encoding gene with Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated albumen being incorporated into the position of aspergillus niger heat shock protein gene by homologous recombination and genetic engineering technique.
A kind of High-Cellulase-Yielding Aspergillus niger strain, for being integrated with the Aspergillus niger strain of Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated protein coding gene in the position of heat shock protein gene.
The construction process of described High-Cellulase-Yielding Aspergillus niger strain preferably comprises following steps:
(1) synthesize the homologous recombination fragment of the back segment sequence of the leading portion sequence-Mierocrystalline cellulose excision enzyme of aspergillus niger heat shock protein gene and the encoding sequence-aspergillus niger heat shock protein gene of restriction endonuclease double activated albumen, restriction enzyme digestion sites is contained at these homologous recombination fragment two ends; This recombinant fragment is connected on pWM1 plasmid and obtains homologous recombination plasmid.
(2) with homologous recombination Plastid transformation Agrobacterium.
(3) object bacterial strain is obtained by agrobacterium mediation converted.
Preferably, step (1) is the homologous recombination fragment of synthesizing ribonucleotide sequence as shown in SEQ ID NO.1, uses
sali enzyme is cut; PWM1 plasmid is also used
sali enzyme is cut; Recombinant fragment after being cut by enzyme is connected by DNA ligase with plasmid, and transforms DH5 α and obtain homologous recombination plasmid.
The present invention passes through homologous recombination technique, two sections, the front and back of synthesis aspergillus niger heat shock protein encoding sequence are as homologous recombination sequence, be connected with the albumen coded sequence of excision enzyme having Mierocrystalline cellulose restriction endonuclease, pass through genetic engineering technique, allow the protein integration with Mierocrystalline cellulose restriction endonuclease and 5 prime excision enzyme activity to the position of heat shock protein, construct the bacterial strain that heat shock is expressed, the 6.7U/g of the bacterial strain cellulase of structure is 1.2 times of starting strain 5.6U/g.
Aspergillus niger strain of the present invention can in express the albumen of excision enzyme and endonuclease activity, be the superior strain of cellulase.The maximum advantage of the present invention is by expressing a kind of albumen, the activity of excision enzyme and restriction endonuclease can be increased simultaneously, what have more innovative significance is utilize heat shock to express the expression achieving and increase considerably target protein by improving temperature, thus improving the activity of Mierocrystalline cellulose excision enzyme and restriction endonuclease more significantly, the enzyme increasing cellulase is lived.
Embodiment
Following examples are used for further illustrating content of the present invention, but should not be construed as limitation of the present invention, and without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement all belong to scope of the present invention.If specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
Embodiment 1
The structure of A, homologous recombination plasmid
(1) material: aspergillus niger (
aspergillus niger), intestinal bacteria (
e.coli)dH5 α, Agrobacterium EHA105 and plasmid pWM1 are commercially produced product.Wherein, aspergillus niger is bought from ATCC, strain number ATCC10582; Agrobacterium EHA105 and plasmid pMW1 buys from Biovector company.
(2) synthesis of homologous recombination fragment and enzyme are cut
Synthesize the homologous recombination fragment (nucleotide sequence is as shown in SEQ ID NO.1) be made up of the back segment homologous sequence (SEQ ID NO.4) of leading portion homologous sequence (initiator codon that this sequence is possible is substituted to guarantee to transcribe from the initiator codon of double activated albumen by other base, and sequence is as shown in SEQ ID the NO.2)-Mierocrystalline cellulose excision enzyme of aspergillus niger heat shock protein gene and encoding sequence (SEQ ID the NO.3)-aspergillus niger heat shock protein gene of restriction endonuclease double activated albumen.
Add in this homologous recombination fragment of 100 μ L
sali enzyme liquid 2 μ L(TakaRa buys), 30 DEG C of enzymes cut 16h, and enzyme cuts rear 65 DEG C of water-bath 10min.
(3) extraction of pWM1 plasmid and enzyme are cut
Intestinal bacteria E containing pWM1 plasmid
.colidH5 α is (kantlex of 50 μ g/mL) 37 DEG C of shaking culture 12h in LB.Get 1.5mL thalline to manage in EP, with the centrifugal 3min of 4000rpm, abandon supernatant liquor.Add 0.1mL solution I (1% glucose, 50mM EDTA pH8.0,25mM Tris-HCl pH8.0) fully to mix.Add 0.2mL solution II (0.2mM NaOH, 1% SDS), overturn mixing gently, be placed in ice bath 5min.Add 0.15mL cooled solution III(5mol/L KAc, pH4.8), overturn mixing gently, ice bath 5min.With the centrifugal 20min of 10000rpm, get supernatant liquor in another new EP pipe.Add isopyknic primary isoamyl alcohol, after mixing, leave standstill 10min.Again with the centrifugal 20min of 10000rpm, abandon supernatant.Wash once with 70% ethanol 0.5mL, drain all liquid.After drying to be precipitated, be dissolved in 0.05mL TE damping fluid.
The pWM1 plasmid 50 μ L extracted adds
sali enzyme liquid 2 μ L(TakaRa buys), 30 DEG C of enzymes cut 16h, and enzyme cuts rear 65 DEG C of water-bath 10min.
(4) connect
The homologous recombination fragment 100 μ L that enzyme is cut buys with 5 μ L T4 DNA ligase(TakaRa with the pWM1 plasmid 20 μ L that enzyme is cut) 16 DEG C be connected 24h.
The preparation of B, competent escherichia coli cell
(1) by E
.colidH5 α is placed on LB 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 20mL LB(or other nutritious substratum are housed) 100mL shaking flask.
At (5) 37 DEG C, thermal agitation cultivates 6 hours.
(6) nutrient solution OD is monitored
600value (cultivate and measure once per half an hour after 1 hour).
(7) OD is worked as
600when 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.
C, connection product conversion intestinal bacteria
(1) competent cell that step B of thawing is on ice obtained.
(2) every 100 μ L competent cells add the plasmid (the connection product of step A (4)) that 10 μ L connect, incubated on ice about 5 minutes.
(3) in transfer DNA/cell mixture to cooled 2mm electroporation container.
(4) add live conversion instrument, get out 300 μ L LB substratum.
(5) pulse (200 ohm, 25 μ Fd, 2.5 kilovolts) (testing time constant, should more than 3) is carried out to electroporation container.
(6) LB of 300 μ L is added immediately in electroporation container.
At (7) 37 DEG C, culturing cell 40 minutes to 1 hour is to restore.
(8) transitional cell is cultivated to containing on kantlex (50 μ g/mL) Selective agar medium, and the bacterium colony grown is the transformant containing homologous recombination plasmid (being connected into the pMW1 plasmid of homologous recombination fragment).
The extraction of D, homologous recombination plasmid
The transformant that step C obtains 37 DEG C of shaking culture 12h.Get 1.5mL bacterium liquid in EP pipe, with the centrifugal 3min of 4000rpm, abandon supernatant liquor.Add 0.1mL solution I (1% glucose, 50 mM EDTA pH 8.0,25mM Tris-HCl pH 8.0) fully to mix.Add 0.2mL solution II (0.2mM NaOH, 1% SDS), overturn mixing gently, be placed in ice bath 5min.Add 0.15mL cooled solution III(5mol/L KAc, pH4.8), overturn mixing gently, ice bath 5min.With the centrifugal 20min of 10000rpm, get supernatant liquor in another new EP pipe.Add isopyknic primary isoamyl alcohol, after mixing, leave standstill 10min.Again with the centrifugal 20min of 10000rpm, abandon supernatant.Wash once with 70% ethanol 0.5mL, drain all liquid.After drying to be precipitated, be dissolved in 0.05mL TE damping fluid.
E, homologous recombination Plastid transformation Agrobacterium
The competent preparation of Agrobacterium: the Agrobacterium EHA105 of fluid preservation coats on LB substratum, 28 DEG C of cultivations, by the time after single bacterium colony grows, picking list bacterium colony, be inoculated in the liquid nutrient medium of 5mL, 28 DEG C, 150r/m cultivates 24h, is inoculated in the fresh liquid nutrient medium of 5mL according to the inoculative proportion of 1:10,28 DEG C, 150r/m cultivates 24h.The thalline ice bath 40min cultivated, the centrifugal 5min of 5000r/m, with the 0.02mol/L CaCl of 10mL
2the resuspended thalline of thalline.The centrifugal 5min of 5000r/m again, thalline is suspended in the 0.02mol/L CaCl of 1mL
2solution, is positioned over preservation on ice.
The homologous recombination plasmid 1 μ L getting extraction mixes with the Agrobacterium competence suspension of 40 μ L, gently after shake mixing 5min, and rapid freezing 5min on liquid nitrogen, 37 DEG C of water-bath 5min.Add 1mL LB liquid medium, 28 DEG C shake 4h gently.The centrifugal 5min of thalline 5000r/m, abandoning supernatant, adds LB substratum 50 μ L, concussion Eddy diffusion liquid.Liquid spreading in containing on 50 μ g/mL kantlex LB flat boards, the colony inoculation grown in containing the LB liquid nutrient medium of 50 μ g/mL kantlex 28 DEG C, 150r/m cultivates 24h.
F, agrobacterium mediation converted aspergillus niger
Aspergillus niger is inoculated in PDA medium slant, and cultivate 72h for 37 DEG C, with spore under the distilled water flushing of sterilizing, spore distilled water diluting is to 10
8individual spore/mL.Spore mixes with isopyknic Agrobacterium containing homologous recombination plasmid, adds Syringylethanone to 200 μm ol/L, and 30 DEG C of lucifuges cultivate 24h.Then spore being coated on dull and stereotyped upper (200g potato 1L water boil 30min from the beginning, filtered through gauze, adds 20g glucose, 20g Zulkovsky starch, 20g agar, 200 μm of ol cefotaximes, 200mg hygromycin B), cultivating to growing bacterium colony for 30 DEG C.
The bacterium colony grown is inoculated into (200g potato 1L water boil 30min from the beginning on inclined-plane respectively, filtered through gauze, add 20g glucose, 20g agar), cultivate 72h, with transfering loop scraping spore, be inoculated into (wheat bran: tap water=1:3(W/W) in bran mass for 30 DEG C), each 250mL triangular flask loads 30g bran mass, cultivate 4d for 30 DEG C, be then warmed up to 39 DEG C of insulation 12h, cool to 28 DEG C and cultivate 1d.Measure the cellulose enzyme activity of each transformant one by one, what enzyme work was high is transformant.Screening obtains the bacterial strain that a cellulose enzyme activity reaches 6.7U/g, is 1.2 times of starting strain 5.6U/g.The bacterial strain of screening is checked order further and confirms that the encoding gene of Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated albumen has been incorporated into the position of aspergillus niger heat shock protein gene, show that the present invention expresses Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated albumen structure cellulase superior strain successfully constructs.
By the inoculation of above-mentioned screening to (200g potato 1L water boil 30min from the beginning on inclined-plane, filtered through gauze, add 20g glucose, 20g agar), cultivate 72h for 30 DEG C, with transfering loop scraping spore, be inoculated into (wheat bran: tap water=1:3(W/W) in bran mass), 250mL triangular flask loads 30g bran mass, cultivates 5.5d for 28 DEG C.Measure enzyme to live as 5.9U/g, this explanation does not have heat shock, and it is high that cellulose enzyme activity does not have the enzyme of heat shock to live, and illustrates that heat shock expression strategy makes the enzyme of cellulase live and improves.
The measuring method of cellulose enzyme activity: get substratum 5.0g during fermentation ends, adds 0.05M citrate buffer solution 50mL, and 200r/m lixiviate 30min, filter paper filtering obtains enzyme liquid.Enzyme liberating reaction is carried out at 50 DEG C.The 0.05M citrate buffer solution of 10mL pH 5.0 is added in 25mL test tube, filter paper bar (0.5cm × 2cm) two panels, as substrate, adds fermented liquid centrifuged supernatant 2mL, 50 DEG C of insulation 30min in water-bath, then boil 5min with boiling water, measure the content of reducing sugar by DNS method.Enzyme is lived and is defined as: the per minute enzyme amount discharged required for 1 μm of ol reducing sugar is a Ge Meihuo unit U.
SEQUENCE LISTING
<110> Hubei University Of Technology
<120> mono-kind expresses the method for Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated albumen structure cellulase high-yield
<130> 1
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 2296
<212> DNA
<213> Artificial Sequence
<220>
<223> homologous recombination fragment
<400> 1
ccgtcgactt gtctgccgag actttcgagt tccaggctga gatctctcag ctcctctccc 60
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cgggcaagga tctccgcatc gacctgatcc ccaacgccga ggccaagacc ctcaccatcc 240
gtgataccgg tatcggtatg accaaggctg taagtttttc tttcctatca ctccccgctc 300
tttcttctgc cattgagcgg taccctctta cgattagagt gaattctgac tttgtgggct 360
ttttttagga cctcatcaac aacctcggta ccattgctcg ctctggtact aagcagttca 420
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actactgaag atatgttcaa ggttttaatc gacaaccaat ttaatgtctt ccgtattcca 660
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aagagagttc atgaagttgt tgattatcca tacaagaacg gagcatttgt tatcttaaat 780
cttcatcatg aaacctggaa tcatgccttc tctgaaactc ttgatacagc caaggaaatt 840
ttagaaaaga tctggtctca aattgctgaa gaatttaagg attatgatga acacttaatc 900
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gctggtggta ataatccaaa gcgtcatctt atgattccac catatgctgc tgcttgtaat 1080
gaaaactcat tcaacaactt tatcttccca gaagatgatg ataaggttat tgcttctgtt 1140
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gatgcagctg gtaagagaga tcttgaatgg aacattaatt taatgaagaa gagatttgtt 1260
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gaagttaatg ttgttcatgc tattgaaaaa gaaacagagg aataagtatg cccgagcacc 1560
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tggacagcct caagcagaag aacttcgagg ttctgttcct ggttgacccc attgacgagt 1680
acgccttcac tcagctgaag gagttcgacg gcaagaagct ggtcgacatc accaaggact 1740
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agggtctcgc caagagcctc aagaacatcc tcggcgacaa ggtcgagaag gtcgttgtct 1860
cccacaagct cgttggctct ccttgcgcca tccgtaccgg ccagttcggt tggtccgcta 1920
acatggagcg tatcatgaag gcccaggccc tccgtgacac ctccatgagc tcttacatgt 1980
cctccaagaa gactttcgag atctctccca agtcctctat catcaaggag ctccgcaaga 2040
aggttgaggc cgatggcgag ggtgaccgca ctgtcaagtc catcactcag ctgctcttcg 2100
agacctctct cctggtctcc ggtttcacca ttgaggagcc cgccagcttc gctgagcgca 2160
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The leading portion homologous sequence of <223> aspergillus niger heat shock protein gene
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gtgataccgg tatcggtatg accaaggctg taagtttttc tttcctatca ctccccgctc 300
tttcttctgc cattgagcgg taccctctta cgattagagt gaattctgac tttgtgggct 360
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The encoding sequence of <223> Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated albumen
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gaaacttgct ggggtaatcc aaagactact gaagatatgt tcaaggtttt aatcgacaac 180
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actcttgata cagccaagga aattttagaa aagatctggt ctcaaattgc tgaagaattt 420
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ccagttgaat ggactggtgg tgatcaagaa ggttgggatg ctgttaatgc tatgaatgct 540
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ggtgaaggag ctgttgataa gtttgatgca gctggtaaga gagatcttga atggaacatt 780
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The back segment homologous sequence of <223> aspergillus niger heat shock protein gene
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gtatgcccga gcaccagaag cagatctact acatcaccgg cgagtccatc aaggccgttg 60
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agaaggtcgt tgtctcccac aagctcgttg gctctccttg cgccatccgt accggccagt 360
tcggttggtc cgctaacatg gagcgtatca tgaaggccca ggccctccgt gacacctcca 420
tgagctctta catgtcctcc aagaagactt tcgagatctc tcccaagtcc tctatcatca 480
aggagctccg caagaaggtt gaggccgatg gcgagggtga ccgcactgtc aagtccatca 540
ctcagctgct cttcgagacc tctctcctgg tctccggttt caccattgag gagcccgcca 600
gcttcgctga gcgcatccac aagctcgtct cccttggtct gaacatcgac gaggaggctg 660
agaccaccga ggagaaggct gctgaggagg ctgcccctgc cgccgccgct gccgagagct 720
ccatggagga ggttgactaa ccgtcgactt t 751
Claims (4)
1. expressing a method for Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated albumen structure High-Cellulase-Yielding Aspergillus niger strain, it is characterized in that: for the encoding gene with Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated albumen being incorporated into the position of aspergillus niger heat shock protein gene.
2. a High-Cellulase-Yielding Aspergillus niger strain, is characterized in that: for being integrated with the Aspergillus niger strain of Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated protein coding gene in the position of heat shock protein gene.
3. the construction process of High-Cellulase-Yielding Aspergillus niger strain according to claim 2, is characterized in that comprising following steps:
(1) synthesize the homologous recombination fragment of the back segment sequence of the leading portion sequence-Mierocrystalline cellulose excision enzyme of aspergillus niger heat shock protein gene and the encoding sequence-aspergillus niger heat shock protein gene of restriction endonuclease double activated albumen, restriction enzyme digestion sites is contained at these homologous recombination fragment two ends; This recombinant fragment is connected on pWM1 plasmid and obtains homologous recombination plasmid;
(2) with homologous recombination Plastid transformation Agrobacterium;
(3) object bacterial strain is obtained by agrobacterium mediation converted.
4. construction process according to claim 3, is characterized in that step (1) is for the homologous recombination fragment of synthesizing ribonucleotide sequence as shown in SEQ ID NO.1, uses
sali enzyme is cut; PWM1 plasmid is also used
sali enzyme is cut; Recombinant fragment after being cut by enzyme is connected by DNA ligase with plasmid, and transforms DH5 α and obtain homologous recombination plasmid.
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| CN106497959A (en) * | 2016-10-28 | 2017-03-15 | 湖北工业大学 | The method that cellulose excision enzyme is expressed in lactobacillus under sour environment |
| CN114015677A (en) * | 2021-11-26 | 2022-02-08 | 中农华威生物制药(湖北)有限公司 | Cellulase for promoting release of traditional Chinese medicine feed additive in intestinal tract and production method thereof |
| CN114164227A (en) * | 2021-11-26 | 2022-03-11 | 中农华威生物制药(湖北)有限公司 | Construction method of cellulase high-expression strain adapted to traditional Chinese medicine feed fermentation |
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| CN103820443A (en) * | 2014-03-10 | 2014-05-28 | 湖北工业大学 | Production and construction of yeast strain with cellulose incision enzyme and glucosidase double activity protein |
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Non-Patent Citations (2)
| Title |
|---|
| TT LIU等: "Simulation of PMSM Vector Control System Based on Matlab/Simulink", 《INTERNATIONAL CONFERENCE ON MEASURING TECHNOLOGY AND MECHATRONICS AUTOMATION》 * |
| 倪浩: "黑曲霉内、外切葡聚糖酶基因的克隆与表达", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106497959A (en) * | 2016-10-28 | 2017-03-15 | 湖北工业大学 | The method that cellulose excision enzyme is expressed in lactobacillus under sour environment |
| CN114015677A (en) * | 2021-11-26 | 2022-02-08 | 中农华威生物制药(湖北)有限公司 | Cellulase for promoting release of traditional Chinese medicine feed additive in intestinal tract and production method thereof |
| CN114164227A (en) * | 2021-11-26 | 2022-03-11 | 中农华威生物制药(湖北)有限公司 | Construction method of cellulase high-expression strain adapted to traditional Chinese medicine feed fermentation |
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