CN109468240A - A kind of recombinant yeast pichia pastoris and its construction method for expressing asparagine aminopeptidase - Google Patents
A kind of recombinant yeast pichia pastoris and its construction method for expressing asparagine aminopeptidase Download PDFInfo
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Abstract
The invention discloses a kind of recombinant yeast pichia pastoris and its construction method for expressing asparagine aminopeptidase, belong to bioengineering and genetic engineering field.The present invention clones first obtains aspergillus oryzae winter amide aminopeptidase gene, and construct recombinant expression carrier pPIC9K/AAP, then it is integrated on Pichia chromosome after being linearized, obtain recombinant yeast pichia pastoris GS115-pPIC9K/AAP, with the recombinant bacterial strain energy high efficient expression asparagine aminopeptidase, after shaking flask tentatively optimizes, the enzyme activity in fermented liquid supernatant is 33.5U/mL, is laid a good foundation for industrialized production asparagine aminopeptidase.
Description
Technical field
The present invention relates to a kind of recombinant yeast pichia pastoris and its construction method for expressing asparagine aminopeptidase, belong to biological work
Journey and gene engineering technology field.
Background technique
Asparagine aminopeptidase (AAP) is that one kind can identify in protein and polypeptide N-terminal and cut aspartic acid or paddy
The exopeptidase of histidine residue, is applied in hydrolyzate of soybean protein, can make the aspartic acid or glutamic acid of peptide chain end
In free state, the degree of hydrolysis of hydrolyzate is improved.Aspartic acid and glutamic acid are Fresh ear fields simultaneously, and water can be effectively relieved
The bitter taste for solving liquid plays the role of pressing down bitter fresh adding, in food hydrolyzate of soybean protein it is de- it is bitter there is significant application value, one
Directly attract attention.
Aspergillus oryzae is a kind of fermentation industry bacterium for being widely used in production, but since the protease species that it is generated are various, point
From purification steps troublesome, it is difficult meet the needs of market.
Pichia yeast expression system is widely used in expressing foreign protein at present, and the speed of growth is fast, and expression quantity is high, outside
Source gene can carry out albumen folding and posttranslational modification on stable integration to yeast chromosomal, make the albumen tool of expression
It is active, while the protein secretion of expression being isolated and purified to extracellular.
At present the research of asparagine aminopeptidase is reported it is few, therefore it provides a kind of asparagine aminopeptidase for preparing
Method has important value for its further application industrially.
Summary of the invention
The first purpose of the invention is to provide a kind of recombinant yeast pichia pastoris, express amino acid sequence such as SEQ ID
Asparagine aminopeptidase shown in NO.1.
In one embodiment of the invention, the recombinant yeast pichia pastoris contains the coding as shown in SEQ ID NO.2
The gene of the asparagine aminopeptidase.
In one embodiment of the invention, the host of the recombinant yeast pichia pastoris includes Pichia pastoris GS115；Recombination
The expression vector of Pichia pastoris includes pPIC9K.
A second object of the present invention is to provide the construction methods of above-mentioned recombinant yeast pichia pastoris, mainly comprise the steps that
(1) design primer PCR amplification asparagine aminopeptidase gene；
(2) PCR product is connected on expression vector, obtains recombinant plasmid；
(3) recombinant plasmid transformed is expressed into Pichia pastoris.
In one embodiment of the invention, the conversion includes electroporated method.
Third object of the present invention is to provide application of the above-mentioned recombinant yeast pichia pastoris in the production of asparagine aminopeptidase.
In one embodiment of the invention, the application mainly comprises the steps that
(1) the recombinant yeast pichia pastoris single colonie activated on picking YPD plate, which is inoculated in BMGY culture medium, cultivates；
(2) by medium centrifugal obtained in (1), precipitating is collected, precipitating is dissolved in again in BMMY culture medium and is cultivated, simultaneously
Carry out inducing expression；
(3) supernatant, the as extracellular crude enzyme liquid of asparagine aminopeptidase is collected by centrifugation in the fermentation liquid of acquisition.
In one embodiment of the invention, the culture in step (1) is specifically included in 28-32 DEG C, 200-220rpm
Under the conditions of cultivate 18-22h.
In one embodiment of the invention, in one embodiment of the invention, step (2) specifically include by
(1) medium centrifugal obtained in collects precipitating, precipitating is dissolved in again in BMMY culture medium, in 28-32 DEG C, 200-220rpm
Under the conditions of cultivate 90-100h, every for 24 hours into culture solution add volume fraction be 1-1.5% methanol solution.
In one embodiment of the invention, the BMMY medium pH is 6.0-7.0, and liquid amount 12-15% adds
The sorbierite of 6-9g/L is added.
Present invention asparagine aminopeptidase gene of the successful expression from aspergillus oryzae in Pichia pastoris for the first time, by shaking
Enzyme activity in the preliminary optimization post-fermentation liquid supernatant of bottle is up to 33.5U/mL.The method of the present invention is simple and effective, and the recombination of building is finished red
Yeast can high efficient expression asparagine peptase, and obtained recombinant protein include histidine tag, be conducive to later period recombinant protein
Purifying and provide required albumen further to study the characteristic of asparagine aminopeptidase, structure etc..
Detailed description of the invention
Fig. 1: influence of the induction time to restructured Pichia pastoris in expression.
Fig. 2: influence of the methanol concentration to restructured Pichia pastoris in expression.
Fig. 3: the pH influence to restructured Pichia pastoris in expression.
Fig. 4: influence of the inducing temperature to restructured Pichia pastoris in expression.
Fig. 5: influence of the liquid amount to restructured Pichia pastoris in expression.
Fig. 6: influence of the additive amount of sorbierite to restructured Pichia pastoris in expression.
Specific embodiment
(1) used medium:
LB culture medium (g/L): yeast powder 5, peptone 10, sodium chloride 10, the agar powder of addition 20 in solid medium.
YPD culture medium (g/L): yeast powder 10, peptone 20, glucose 20, the agar powder of addition 20 in solid medium.
MD culture medium (g/L): glucose 20, YNB 13.4, biotin 0.4, agar powder 20.
BMGY culture medium (g/L): yeast powder 10, peptone 20, YNB 13.4, biotin 0.4, glycerol 10, every 100mL training
Support the kaliumphosphate buffer that the 1mol/L pH 6.0 of 10mL is added in base.
BMMY culture medium (g/L): yeast powder 10, peptone 20, YNB 13.4, biotin 0.4, glycerol 10, every 100mL training
Support base in add 10mL 1mol/L pH 6.0 kaliumphosphate buffer, the additive amount of methanol as the case may be depending on.
YPD-G418 high copies screening flat board: addition final concentration is respectively 1,2,3, the G418 of 4mg/mL trains in YPD solid
It supports in base.
(2) T4DNA ligase, restriction endonuclease Sac I, EcoR I, Not I are purchased from precious bioengineering (Dalian) and have
Limit company.
(3) measurement of asparagine aminopeptidase enzyme activity:
The Tris-HCl buffer of the fermentation liquid of 20 μ L aminopeptidases containing asparagine and 160 μ L pH 8.0 are mixed, 50 DEG C
2min is preheated, the Asp-pNA solution of 20 μ L 10mmol/L, 50 DEG C of water-bath 10min are added.Add immediately after reaction
The acetic acid for entering 200 μ L 40% terminates reaction, and light absorption value is measured at 405nm.
The definition of enzyme activity: in certain pH, under the conditions of temperature is 50 DEG C, asparagine (Asp-pNA) is hydrolyzed per minute and generates 1
Enzyme amount used in μ g paranitroanilinum (pNA) is defined as 1 enzyme activity unit, is indicated with U.
Enzyme activity calculation formula are as follows: U=(N × 20 × 138.12 × A)/10
In formula, U indicates enzyme activity, U/mL；The extension rate of N expression enzyme solution；20 indicate dilution of the reaction total system to enzyme solution
Multiple；138.12 indicating the relative molecular mass of pNA；A indicates to react the A measured according to enzyme activity405nmValue, look into standard curve and obtain
The concentration of the pNA arrived, mmol/L；10 indicate reaction time, min.
(4) purifying of asparagine aminopeptidase is recombinated:
Recombinant yeast pichia pastoris fermentation liquid obtained in embodiment 1 is collected, centrifuging and taking supernatant places it in bag filter 4 DEG C
Dialysed overnight.Using AKTA protein purification instrument, isolating and purifying for albumen, column purification item are carried out using His Trip HP crude
Part is as follows: with buffer solution A (0.5mol/L sodium chloride, 0.02mol/L sodium phosphate, 0.02mol/L imidazoles, the pH=of 8 times of column volumes
7.4) pillar is balanced, setting flow velocity is that 1mL/min carries out loading；After end of the sample, with the buffer solution A balance columns of 8 times of column volumes
Son；Simultaneously with buffer solution B (0.5mol/L sodium chloride, 0.02mol/L sodium phosphate, 0.5mol/L imidazoles, the pH=of 0-100%
7.4) linear elution is carried out with the flow velocity of 1mL/min.
Embodiment 1 expresses the building of the recombinant yeast pichia pastoris of asparagine aminopeptidase
To realize expression of the asparagine aminopeptidase gene in Pichia pastoris, steps are as follows for specific implementation method:
(1) amplification of asparagine aminopeptidase gene
According to asparagine aminopeptidase gene order (the NCBI number: XM_ of aspergillus oryzae RIB40 disclosed on NCBI
001818318) pair of primers, is designed, design of primers is as follows:
Upstream primer F:5 '-CCGGAATTC(underscore indicates the I digestion position EcoR to ATGACTTCGAAAATCGCCC-3 '
Point)；Downstream primer R:5 '-ATTTGCGGCCGCTCAGTGGTGGTGGTGGTGGTGGTAACAAAGATTGTCTTTGAC-3 ' (under
Scribing line indicates I restriction enzyme site of Not, and the base of overstriking indicates 6 × His label).
With aspergillus oryzae CGMCC NO.758cDNA template, using PCR, the asparagine aminopeptidase with histidine tag is obtained
Genetic fragment, PCR system are as follows:
PCR system is (50 μ L): 0.3 μ L, 10 × Ex Taq Buffer of Ex Taq enzyme, 5 μ L, dNTP Mix, 4 μ L, upstream
Primers F 0.2 μ L, downstream primer R 0.2 μ L, cDNA 5 μ L, ddH2O 36.3μL.PCR condition are as follows: 95 DEG C of initial denaturation 5min；95
DEG C denaturation 30s, 60 DEG C of annealing 2min, 72 DEG C of extension 30s；25 DEG C of heat preservation 5min；30 circulations.
PCR product is verified with SDS-PAGE, the results showed that is expanded successfully.
(2) building of recombinant expression carrier
The target gene fragment and plasmid pPIC9K I double digestion of EcoR I and Not that amplification is obtained, double digestion system is such as
Under:
Double digestion system (50 μ L) system: 30 μ L of target gene or plasmid, restriction endonuclease each 0.5 μ L, ddH2O
14 μ L, dNTP Mix, 5 μ L.After 37 DEG C of water-baths heat preservations 2h, agarose gel electrophoresis 15min, glue recycles purpose band.
By after double digestion target gene fragment and expression vector segment according to molar ratio be 2:3 ratio T4DNA
Ligase carries out Ligation in vitro, and 16 DEG C of connections overnight, are then converted into E.coli JM109.It extracts bacterium colony PCR and is verified as sun
Property bacterial strain plasmid carry out double digestion verifying, verify correctly carry out sequence verification, sequencing correctly be recombinant expression carrier
pPIC9K/AAP。
(3) building of recombinant yeast pichia pastoris
Recombinant plasmid Sac I is linearized, after purification and recovery, 1500V, 5ms are electroporated to Pichia pastoris GS115 sense
By in state cell, the bacterium solution after conversion is coated on MD plating medium, 30 DEG C are cultivated 2-3 days.It is grown on picking MD plate
Single bacterium drop down onto YPD-G418 gradient resistant panel, 5 lists grown on the YPD plate of 4mg/mL G418 concentration of picking
Bacterium colony carries out shake flask fermentation.
Embodiment 2: to the fermentation optimization of recombinant yeast pichia pastoris
Single factor test optimization in shaking flask level is carried out to the fermentation process of recombinant yeast pichia pastoris obtained in embodiment 1, often
A experimental group is arranged three in parallel, and results are averaged.Specific optimal conditions are as follows:
(1) induction time is originated
Recombinant yeast pichia pastoris obtained in embodiment 1 is inoculated in BMGY culture medium, respectively thalli growth 14,16,
18, it is forwarded in BMMY culture medium and is induced after 20h, carry out inducing expression with the methanol that volume fraction is 1%, while taking few
It measures sample and measures enzyme activity.As a result as shown in Figure 1, thallus is transferred to BMMY training by when thalli growth 20h from BMGY culture medium
It supports and carries out inducing expression in base, the vigor highest of enzyme is 22.0U/mL, and the vigor of cell is larger at this time while having enough bacterium
Bulk concentration, most beneficial for expression.Therefore, 20h is best starting induction time.
(2) methanol concentration is induced
Recombinant yeast pichia pastoris obtained in embodiment 1 is inoculated in BMGY culture medium, after thalli growth 20h, switching
Into BMMY culture medium, induction table is carried out every the methanol that addition volume fraction for 24 hours is respectively 0.5%, 1%, 1.5%, 2%
It reaches, while taking a small amount of sample measurement enzyme activity.As a result as shown in Fig. 2, the enzyme activity of recombination asparagine aminopeptidase is with methanol concentration
Raising first increases and then decreases.Enzyme activity when methanol additive amount is 0.5% and 1% is lower, it may be possible to because of most of methanol quilt
For thalli growth；When methanol additive amount is 2%, enzyme activity is relatively low, may be too high due to methanol concentration, produces to thallus
Toxic action, so that expression quantity be made to reduce；When methanol additive amount is 1.5%, after inducing 96h, expression quantity highest, about
23.1U/mL。
(3) induction starting pH
Recombinant yeast pichia pastoris obtained in embodiment 1 is inoculated in BMGY culture medium, after thalli growth 20h, by bacterium
Body be forwarded to pH be respectively 5.0,6.0,7.0,8.0 BMMY culture medium in, every for 24 hours add volume fraction 1.5% methanol
Inducing expression is carried out, while taking a small amount of sample measurement enzyme activity.As a result as shown in figure 3, induction starting pH finishes red ferment to recombination
Female expression is affected, and when pH is 5 and 8, whole enzyme activity is lower, and pH is conducive to the expression of Pichia pastoris when being 6 and 7.When rise
When beginning pH is 7.0, after inducing 96h, enzyme activity highest is 24.6U/mL.
(4) inducing temperature
Recombinant yeast pichia pastoris obtained in embodiment 1 is inoculated in BMGY culture medium, after thalli growth 20h, by bacterium
After body is forwarded to the BMMY culture medium that pH is 7.0, respectively under conditions of 23,26,30 DEG C, every adding volume fraction for 24 hours
1.5% methanol carries out inducing expression, while taking a small amount of sample measurement enzyme activity.As a result as shown in figure 4, recombination asparagine ammonia
The enzyme activity of peptase increases with the rising of temperature on the whole, it may be possible to because as the temperature rises, being conducive to Pichia pastoris
Growth, cell density is larger, is conducive to the expression of asparagine aminopeptidase, and when temperature is 30 DEG C, after inducing 96h, enzyme activity reaches
It is 26.3U/mL to highest.
(5) liquid amount
Recombinant yeast pichia pastoris obtained in embodiment 1 is inoculated in BMGY culture medium, after thalli growth 20h, by bacterium
Body be forwarded to the liquid amount that pH is 7.0 be respectively 10%, 15%, after 20%BMMY culture medium, under conditions of 30 DEG C, every
The methanol for adding volume fraction 1.5% for 24 hours carries out inducing expression, while taking a small amount of sample measurement enzyme activity.As a result such as Fig. 5 institute
Show, influence of the liquid amount to enzyme activity is more significant, and when liquid amount is 15%, after inducing 96h, enzyme activity reaches highest, is
28.9U/mL, dissolved oxygen effect at this time is best, meets the demand in Pichia pastoris fermentation process to oxygen.
(6) sorbitol concentration
Recombinant yeast pichia pastoris obtained in embodiment 1 is inoculated in BMGY culture medium, after thalli growth 20h, by bacterium
Body is forwarded to after pH is the BMMY culture medium that 7.0 liquid amounts are 15%, adds 3 respectively into BMMY culture medium, 6,9,12g/L
Sorbierite carries out inducing expression every the methanol for adding volume fraction 1.5% for 24 hours, while taking a small amount of sample under conditions of 30 DEG C
Enzyme activity after product measurement induction 96h.As a result as shown in fig. 6, the sorbierite of suitable concentration can promote recombinant yeast pichia pastoris
Expression, when sorbierite additive amount is 9g/L, enzyme activity reaches highest, is 33.5U/mL.But excessively high sorbierite can inhibit weight instead
The expression of group asparagine aminopeptidase, it may be possible to which sorbierite causes osmotic pressure excessively high, hinders Pichia anomala expression foreign protein.
Comparative example: expression of other aminopeptidases in aspergillus oryzae source in Pichia pastoris
According to aspergillus oryzae RIB40 genomic dna sequence, design primer, with aspergillus oryzae CGMCC NO.758 cDNA template,
PCR amplification obtains the aminopeptidase Y gene (NCBI:XM_001727123.1) in aspergillus oryzae source, remaining condition is the same as embodiment 1.
According to aspergillus oryzae RIB40 genomic dna sequence, design primer, with aspergillus oryzae CGMCC NO.758 cDNA template,
PCR amplification obtains the aminopeptidase C gene (NCBI:XM_001824930.1) in aspergillus oryzae source, remaining condition is the same as embodiment 1.
According to aspergillus oryzae RIB40 genomic dna sequence, design primer, with aspergillus oryzae CGMCC NO.758 cDNA template,
PCR amplification obtains the leucine amino peptidase gene (NCBI:XM_001819493.2) in aspergillus oryzae source, the same embodiment of remaining condition
1。
As a result, it has been found that the aminopeptidase Y in the source aspergillus oryzae CGMCC NO.758, aminopeptidase C, leucine amino peptidase are finishing red ferment
It is all hardly expressed in mother.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of recombinant yeast pichia pastoris and its construction method for expressing asparagine aminopeptidase
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 498
<212> PRT
<213> Aspergillus oryzae
<400> 1
Met Thr Ser Lys Ile Ala Gln Asn Leu Lys Gln Pro Ala Leu Asp Phe
1 5 10 15
Leu Ser Phe Val Asn Ala Ser Pro Thr Pro Phe His Ala Val Gln Ser
20 25 30
Ala Lys Glu Leu Leu Ser Lys Ala Gly Phe Gln Glu Ile Lys Glu Lys
35 40 45
Asp Ser Trp Ser Ser Thr Cys Arg Pro Gly Gly Lys Tyr Tyr Leu Thr
50 55 60
Arg Asn Ser Ser Thr Ile Val Ala Phe Ala Ile Gly Lys Lys Trp Lys
65 70 75 80
Pro Gly Asn Pro Ile Ser Met Ile Gly Ala His Thr Asp Ser Pro Val
85 90 95
Leu Arg Ile Lys Pro Val Ser Asn Lys Arg Gly Glu Gly Phe Val Gln
100 105 110
Val Gly Val Glu Thr Tyr Gly Gly Gly Ile Trp His Thr Trp Phe Asp
115 120 125
Arg Asp Leu Gly Val Ala Gly Arg Ala Met Val Arg Thr Gly Asp Gly
130 135 140
Ser Ile Val Gln Lys Leu Val Lys Ile Asp Arg Pro Ile Leu Arg Ile
145 150 155 160
Pro Thr Leu Ala Ile His Leu Asp Arg Gln Glu Thr Phe Ala Phe Asn
165 170 175
Lys Glu Thr Gln Leu Phe Pro Ile Ala Gly Leu Val Ala Ala Glu Leu
180 185 190
Asn Arg Thr Ala Asp Ser Thr Ala Thr Gly Glu Lys Thr Ala Ala Asn
195 200 205
Asn Glu Thr Glu Lys Gly Asp Phe Ala Pro Leu Lys Ser Val Thr Glu
210 215 220
Arg His His Pro Tyr Leu Val Glu Leu Ile Ala Ala Glu Ala Gly Val
225 230 235 240
Lys Pro Asp Asp Ile Leu Asp Phe Glu Met Ile Leu Phe Asp Thr Gln
245 250 255
Lys Ser Cys Leu Gly Gly Leu Leu Glu Glu Phe Val Phe Ser Pro Arg
260 265 270
Leu Asp Asn Leu Asn Ser Ser Phe Cys Ala Thr Val Gly Leu Ile Asp
275 280 285
Ser Val Ala Asp Ala Ser Ala Leu Asp Asp Glu Pro Ser Ile Arg Leu
290 295 300
Ile Ala Leu Phe Asp His Glu Glu Ile Gly Ser Arg Thr Ala Gln Gly
305 310 315 320
Ala Asp Ser Asn Val Leu Pro Ala Ile Ile Arg Arg Leu Ser Val Leu
325 330 335
Pro Ser Ser Thr Ser Gly Asn Glu Asp Leu Ala Thr Ala Phe Glu Glu
340 345 350
Thr Leu Ser Thr Ser Phe Leu Leu Ser Ala Asp Met Ala His Ala Val
355 360 365
His Pro Asn Tyr Ala Ala Lys Tyr Glu Asn Asp His Arg Pro Glu Ile
370 375 380
Asn Lys Gly Pro Val Ile Lys Ile Asn Ala Asn Ala Arg Tyr Ala Thr
385 390 395 400
Asn Ser Pro Gly Ile Val Leu Leu Gln Glu Val Ala Arg Lys Ala Ala
405 410 415
Glu Asp Gly Gly Glu Gly Val Pro Leu Gln Leu Phe Val Val Arg Asn
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Asp Ser Ser Cys Gly Ser Thr Ile Gly Pro Met Leu Ser Ala Ala Leu
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Gly Ala Arg Thr Leu Asp Leu Gly Asn Pro Gln Leu Ser Met His Ser
450 455 460
Ile Arg Glu Thr Gly Gly Thr Tyr Asp Val Gly His Ser Ile Arg Leu
465 470 475 480
Phe Thr Ser Phe Phe Lys His Tyr Ser Asn Thr Ser Lys Thr Ile Phe
485 490 495
Val Asp
<210> 2
<211> 1497
<212> DNA
<213> Aspergillus oryzae
<400> 2
atgacttcga aaatcgccca aaatttgaag cagccggctc tggacttctt gtcctttgtc 60
aatgcttccc ccactccctt ccacgctgtc caatcggcaa aggaacttct gtcaaaggct 120
ggcttccagg agatcaagga gaaagattct tggtcctcca cttgtcgtcc cggtggaaag 180
tattacctga cccgtaatag ctcaaccatt gtggctttcg ctatcggcaa gaaatggaag 240
cctggaaacc cgatatctat gatcggtgcc cacacggact ctcccgtgtt gaggatcaag 300
cctgtcagca acaagcgcgg cgaaggcttc gttcaagttg gcgtggagac ctacggtggc 360
ggcatttggc acacctggtt cgaccgtgac ttgggtgtcg caggccgggc tatggtacgg 420
accggtgacg gctccattgt gcagaagttg gtcaagatcg accggccgat tctccgaatc 480
ccgaccttgg ctatccacct tgatcgccag gagacttttg ctttcaataa ggagacccaa 540
ttgttcccta tcgcaggcct tgtcgctgct gagctgaacc gcactgctga ttctactgca 600
actggcgaaa agaccgcggc aaacaacgaa acggagaaag gagactttgc tccactaaaa 660
tcagtaaccg agcgtcatca cccctacttg gtggagctta ttgctgccga agcaggagtt 720
aagccggacg acatcttgga ctttgagatg atcttgttcg acactcagaa gtcttgcctt 780
ggtggcttgc tggaggagtt cgttttctcg ccccgtctgg ataacctgaa cagctcgttc 840
tgtgccactg ttggactaat cgactccgtt gccgatgcgt cggcgctgga cgatgaaccg 900
tccattcgtc tcattgcgtt attcgatcac gaagagatcg gcagccgtac cgcacaggga 960
gctgactcaa atgtgcttcc ggcaattatc cgtcgcctgt ctgttctacc ttcttccaca 1020
tctggcaatg aagacttggc tactgctttc gaggagactt tgtcgacttc attcctcctc 1080
tctgcggaca tggctcatgc tgtccaccct aactacgctg ctaagtacga gaatgatcac 1140
cgaccggaga tcaacaaggg tcctgtgatc aagatcaacg ccaatgctcg ctacgcgacg 1200
aactcccctg gcattgtcct acttcaggag gttgcacgca aggcagcaga agacggtgga 1260
gaaggcgttc ctctccaact cttcgtcgtt cgcaacgact ccagctgcgg aagcacaatt 1320
ggtcccatgt tgtccgctgc gcttggtgcc cgcacgctgg acttgggtaa cccacagcta 1380
agcatgcaca gtatccggga gactggtggt acatatgatg ttgggcattc tattcggttg 1440
ttcactagct tcttcaagca ttactccaac acgtcaaaga caatctttgt tgactga 1497
Claims (10)
1. a kind of recombinant yeast pichia pastoris, which is characterized in that express amino acid sequence asparagine ammonia as shown in SEQ ID NO.1
Peptase.
2. recombinant yeast pichia pastoris as described in claim 1, which is characterized in that containing encoding institute as shown in SEQ ID NO.2
State the gene of asparagine aminopeptidase.
3. recombinant yeast pichia pastoris as claimed in claim 1 or 2, which is characterized in that its host includes Pichia pastoris GS115；Its
Expression vector includes pPIC9K.
4. the construction method of any recombinant yeast pichia pastoris of claim 1-3, mainly comprises the steps that
(1) design primer PCR amplification asparagine aminopeptidase gene；
(2) PCR product is connected on expression vector, obtains recombinant plasmid；
(3) recombinant plasmid transformed is expressed into Pichia pastoris.
5. construction method as claimed in claim 4, which is characterized in that the conversion includes electroporated method.
6. application of any recombinant yeast pichia pastoris of claim 1-3 in the production of asparagine aminopeptidase.
7. application as claimed in claim 6, which is characterized in that the application mainly comprises the steps that
(1) the recombinant yeast pichia pastoris single colonie activated on picking YPD plate, which is inoculated in BMGY culture medium, cultivates；
(2) by medium centrifugal obtained in (1), precipitating is collected, precipitating is dissolved in again in BMMY culture medium and is cultivated, is carried out simultaneously
Inducing expression；
(3) supernatant, the as extracellular crude enzyme liquid of asparagine aminopeptidase is collected by centrifugation in the fermentation liquid of acquisition.
8. the use as claimed in claim 7, which is characterized in that the culture in step (1) is specifically included in 28-32 DEG C, 200-
18-22h is cultivated under the conditions of 220rpm.
9. application as claimed in claim 7 or 8, which is characterized in that step (2) is specifically included culture solution obtained in (1)
Precipitating is collected in centrifugation, and precipitating is dissolved in again in BMMY culture medium, cultivates 90-100h under the conditions of 28-32 DEG C, 200-220rpm,
Volume fraction is added into culture solution as the methanol solution of 1-1.5% every for 24 hours.
10. application as claimed in claim 9, which is characterized in that the BMMY medium pH is 6.0-7.0, liquid amount 12-
15%, it is added to the sorbierite of 6-9g/L.
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