CN114058637A - Efficient expression method of aspergillus niger glucose oxidase gene - Google Patents
Efficient expression method of aspergillus niger glucose oxidase gene Download PDFInfo
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Abstract
The invention relates to the field of genetic engineering, in particular to a coding gene of glucose oxidase and a genetic engineering expression strain thereof. The invention provides a high-efficiency Pichia pastoris expression method of an Aspergillus niger glucose oxidase gene. In order to improve the correct folding of the recombinase, a protein disulfide isomerase pdi gene is introduced by constructing a tandem vector in an engineering bacterium to help protein folding and improve the expression activity of the recombinase. The invention also provides a fermentation medium composition and a process for producing glucose oxidase by using the pichia pastoris.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a high-efficiency expression method of an Aspergillus niger glucose oxidase gene.
Background
Glucose Oxidase (GOD, EC 1.1.3.4) is an important industrial enzyme, and under aerobic conditions, GOD can specifically oxidize Glucose to gluconic acid and hydrogen peroxide. GOD has wide applications in food, sensors, medical fields and in the animal husbandry industry. In food, GOD as an antioxidant and a green antibacterial agent can effectively inhibit the growth of aerobic bacteria and prolong the shelf life of the food; in the medical field, GOD has been widely used for oral care and whitening, and also for establishing H2O2Pathological studies were performed in the oxidative stress model. Another important application of GOD is enzyme biosensor of glucose, which is characterized by that GOD is fixed on the electrode, and the electron transfer medium is used to raise the electron transfer between the redox active centre of enzyme and electrode surface, and the consumption of reactant or product produced in the enzymatic reaction can be converted into electric signal, and recorded.
GOD also has wide application in animal husbandry. The research shows that the addition of GOD in the feed can reduce the pH value of the digestive tract, improve the activity of digestive enzyme and the digestibility of nutrient substances, and improve the morphological structure of the intestinal tract. GOD is also capable of consuming oxygenGas creates an intestinal environment conducive to the survival of beneficial anaerobes, and the produced H2O2Can inhibit the propagation of pathogenic bacteria in intestinal tract such as Escherichia coli and salmonella, and maintain intestinal microecosystem balance. GOD can also relieve oxidative stress, keep livestock and poultry healthy, and improve reproductive performance of female livestock and poultry, thereby improving the production performance of livestock and poultry and improving the quality of livestock products.
GOD is produced by various animals, plants and microorganisms, and is mainly produced by aspergillus and penicillium at present. At present, GOD can be industrially produced, but due to the particularity of the feed industry (such as high-temperature granulation), the development of GOD with good heat resistance has great significance.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a high-efficiency expression method of an Aspergillus niger glucose oxidase gene, which comprises the following steps in sequence:
(1) taking Aspergillus niger DY-2015 slant strain, inoculating sterilized PDA culture medium for culture, collecting mycelia, placing in liquid nitrogen for grinding, taking fine powder, adding a buffer solution, dissolving, centrifuging, taking supernatant, extracting, adding NaAc into the supernatant, adding absolute ethyl alcohol, mixing uniformly, placing on ice, centrifuging, washing precipitates twice with ice-cold ethyl alcohol, placing in a clean bench for drying, dissolving with 20 mu L of sterilized water, and determining the DNA concentration by using Nanodrop 2000;
(2) design of primers, F: 5'-ATGCAGACTCTCCTTGTGAG-3', respectively; r: 5'-TCACTGCATGGAAGCATAATG-3', carrying out PCR amplification by taking the Aspergillus niger DY-2015 genome DNA extracted in the step 1 as a template;
(3) according to the preference of pichia pastoris codons, optimizing a glucose oxidase gene DY-god of aspergillus niger DY-2015, replacing low-frequency codons with high-frequency codons, adjusting the codon adaptation index to be more than 0.8, removing restriction enzyme cutting sites such as EcoRI, BamHI, BglII, Xba I, Sac I and the like from a sequence for convenient subsequent genetic engineering operation, modifying a possible early termination sequence, naming the optimized gene as ts-god, connecting the optimized gene with multiple cloning sites EcoRI and Xba I of a pichia pastoris expression vector pPICzaA, transforming a connecting product into escherichia coli top10 competent cells, and screening an LB plate containing an antibiotic zeocin to obtain a recombinant expression vector pPIC-god;
(4) downloading a pdi sequence of a protein disulfide isomerase gene from NCBI, carrying out total synthesis after online software optimization design, connecting the pdi sequence to EcoRI and Not I enzyme cutting sites of a pGAPzA expression vector, wherein the constructed vector is named as pGAP-pdi, the pGAP-pdi plasmid is subjected to double enzyme cutting by BglII and BamHI, a large fragment is recovered, is connected with a pPIC-god vector subjected to single enzyme cutting by BamHI, an escherichia coli top10 competent cell is transformed, and a recombinant plasmid is obtained by screening of an antibiotic zeocin, and the tandem expression vector is named as pPIC-god-pdi;
(5) the recombinant plasmid is taken to be linearized and then purified by a PCR product purification kit, and the linearized plasmid is dissolved in sterilized ddH2Checking the purity of linearized plasmid by electrophoresis in O, measuring the concentration by using Nanodrop2000, inoculating a Pichia pastoris x33 single colony in a 10mLYPD culture medium, carrying out shaking culture at 30 ℃ and 250rpm for 12hr, transferring to 50mLYPD, continuously culturing to a logarithmic growth phase, centrifuging at 10,000rpm for 5min, washing with sterilized water and 1M ice-cold sorbitol to prepare a Pichia pastoris x33 competent cell, uniformly mixing linearized DNA and 80 microliter of competent cell, placing in an electric shock cup, carrying out 2000V and 5ms electric shock, immediately adding 1mL of ice-cold 1M sorbitol after electric shock, recovering and culturing at 30 ℃ for 3hr, coating the transformed cell on a zeocin-containing YPDS plate culture medium, and culturing at 30 ℃ for 3-4 d;
(6) reacting glucose with oxygen under the action of GOD by adopting an o-dianisidine spectrophotometry, generating hydrogen peroxide and colorless reduced o-dianisidine under the action of horseradish peroxidase to generate water and red oxidized o-dianisidine, and calculating the activity of glucose oxidase by measuring the color change in the reaction;
(7) selecting a single colony, inoculating a 5mLYPD liquid culture medium, performing oscillation culture at 28 ℃ and 250rpm for 24h, transferring to a 500mLYPD culture medium, performing culture for 12h under the same conditions, inoculating a seed solution into a 30-liter fermentation tank, wherein the fermentation tank contains 10L of a sterilized basic culture medium, performing culture at 30 ℃ and 400rpm, after the basic culture medium is cultured for 24h, the relative dissolved oxygen concentration of the fermentation liquid rises to be close to 100 percent, indicating that a carbon source is exhausted, starting a material supplementing stage, wherein the material supplementing is 50 percent sterilized glycerol, and stopping material supplementing when the weight of wet bacteria reaches more than 300g/L, so that the bacteria are starved for 1-2h to exhaust the carbon source. Then, a methanol induction stage is started, 0.2mL of methanol is fed in per liter of culture medium per min, the feeding is increased to 0.5mL of methanol per min after 3 hours, and 4.35mL of PTM1 trace salt, 0.35g of alpha-ketoglutaric acid and 10mM of arginine are added in per liter of methanol. And adjusting the pH value by ammonia water in the fermentation process to ensure that the pH value is between 5.5 and 6.0. The relative dissolved oxygen is ensured to be more than 20 percent through the rotation speed regulation. Changes in wet bacterial weight and enzyme activity were measured every 12 h.
Preferably, the PDA medium: peeling potato 200g, adding water 800mL, boiling for 30min, filtering, adding glucose 20g into supernatant, diluting to 1L, and autoclaving; LB culture medium: 2% tryptone, 1% yeast extract, 1% NaCl, ph 7.0; low-salt LB medium: 2% tryptone, 1% yeast extract, 0.5% NaCl, pH 7.0; YPD medium: 1% yeast extract, 2% peptone, 2% glucose, solid plate plus 1.5% agar. YPDS medium: 1% yeast extract, 2% peptone, 2% glucose, 1.5% agar, 1M sorbitol.
In any of the above schemes, preferably, the amplification conditions in step (2) are: 30s at 95 ℃, 20s at 58 ℃ and 120s at 72 ℃ for 35 cycles.
In any of the above embodiments, preferably, the aspergillus niger glucose oxidase gene sequence:
in any of the above embodiments, preferably, the aspergillus niger glucose oxidase amino acid sequence:
an Aspergillus niger glucose oxidase optimized gene sequence ts-god:
pdi gene sequence:
pdi-optimized gene sequence Pdi-opt:
the invention provides a high-efficiency Pichia pastoris expression method of an Aspergillus niger glucose oxidase gene, which is constructed by a tandem carrier in engineering bacteria in order to improve the correct folding of a recombinase, introduces a protein disulfide isomerase pdi gene, helps protein folding, and improves the expression activity of the enzyme.
Drawings
FIG. 1 is a flow chart of expression vector construction;
FIG. 2 is a graph showing relative activity of GOD at different temperatures;
FIG. 3 is a graph showing relative activity of GOD at different pH values;
FIG. 4 is a graph of the residual activity of GOD after incubation at 80 ℃ for various periods of time.
Detailed Description
In order that the invention may be further understood, the invention will now be described in detail with reference to specific examples.
1. Test materials and reagents:
1.1 strains and vectors:
the invention clones 1 glucose oxidase gene DY-god from Aspergillus niger DY-2015(Aspergillus niger). Pichia pastoris expression vectors pPICzaA, pGAPzA and strain X-33 were purchased from Invitrogen.
1.2 tool enzymes and other biochemicals:
phusion DNA polymerase was purchased from NEB, restriction enzymes and ligases from TaKaRa, Zeocin from Invitrogen, and other biochemicals were home-made analytical purifiers.
1.3 culture Medium:
PDA culture medium: peeling potato 200g, adding water 800mL, boiling for 30min, filtering, adding glucose 20g into supernatant, diluting to 1L, and autoclaving;
LB culture medium: 2% tryptone, 1% yeast extract, 1% NaCl, ph 7.0;
low-salt LB medium: 2% tryptone, 1% yeast extract, 0.5% NaCl, pH 7.0;
YPD medium: 1% yeast extract, 2% peptone, 2% glucose, solid plate plus 1.5% agar.
YPDS medium: 1% yeast extract, 2% peptone, 2% glucose, 1.5% agar, 1M sorbitol.
Example 1: aspergillus niger DY-2015 genome DNA extraction
Taking Aspergillus niger DY-2015 slant strain, inoculating sterilized PDA culture medium, culturing at 30 deg.C and 250rpm for 48hr, centrifuging at 10,000rpm for 10min, and collecting mycelium. Grinding in liquid nitrogen, collecting 0.5g of fine powder, adding 1mL of TE buffer (10mM Tris-HCl, pH 8.0,1mM EDTA), dissolving, centrifuging at 10,000rpm for 5min, collecting supernatant, and extracting with phenol/chloroform (phenol: chloroform: isoamyl alcohol 25:24:1) twice; 1/10 volume of NaAc (pH5.2) is added into the supernatant, 2 times volume of ice-cold absolute ethyl alcohol is added into the supernatant, the mixture is placed on ice for 20min after being mixed evenly, the mixture is centrifuged at 13,000rpm for 20min, the precipitate is washed twice by 1mL of 75% ice-cold ethyl alcohol, the precipitate is placed in an ultraclean bench for drying, 20 mu L of sterilized water is used for dissolving, and the DNA concentration is determined by using Nanodrop 2000.
Example 2: cloning of Aspergillus niger DY-2015 glucose oxidase gene GOD
Designing a primer according to an Aspergillus niger glucose oxidase gene sequence reported by NCBI, wherein F: 5'-ATGCAGACTCTCCTTGTGAG-3', respectively; r: 5'-TCACTGCATGGAAGCATAATG-3' are provided. PCR amplification was carried out using the genomic DNA of Aspergillus niger DY-2015 extracted in example 1 as a template, and the amplification system is shown in Table 1. The amplification conditions were: 30s at 95 ℃, 20s at 58 ℃ and 120s at 72 ℃ for 35 cycles, detecting the amplification product by using 1% agarose gel electrophoresis after the amplification is finished, and sending the detection product to Beijing Optimalaceae New industry biotechnology Limited for sequencing. The sequence analysis software was DNAMAN 6.0.
TABLE 1 glucose oxidase Gene amplification reaction System
Example 3: GOD gene sequence optimization design
According to the preference of pichia pastoris codons, glucose oxidase gene DY-god of aspergillus niger DY-2015 is optimized, high-frequency codons are used for replacing low-frequency codons through online software optimization design, and the Codon Adaptation Index (CAI value) Index is adjusted to be more than 0.8. For the convenience of subsequent genetic engineering operation, restriction enzyme cutting sites such as EcoRI, BamHI, BglII, Xba I and Sac I are deleted from the sequence, and possible early termination sequences (continuously AT-rich sequences) are modified. The optimized gene was named ts-god. The optimized sequence ts-god was synthesized in the complete gene sequence by the Biotech company of the new industry of Beijing Ongjinke, and was ligated to the multiple cloning sites EcoRI and XbaI of the Pichia pastoris expression vector pPICzaA, and the ligation product was transformed into E.coli top10 competent cells, which were screened on LB plate containing antibiotic zeocin (final concentration 0.25. mu.g/mL) to obtain the recombinant expression vector pPIC-god.
Example 4: synthesis of disulfide isomerase pdi gene and construction of tandem expression vector
The pdi sequence (accession number: M62815.1) of the protein disulfide isomerase gene is downloaded from NCBI, and is subjected to full synthesis after optimized design through online software, and is connected with EcoRI and Not I enzyme cutting sites of a pGAPzA expression vector, and the constructed vector is named as pGAP-pdi. The pGAP-pdi plasmid is subjected to double digestion by BglII and BamHI, a large fragment is recovered, the large fragment is connected with a BamHI single digestion pPIC-god vector, an escherichia coli top10 competent cell is transformed, and the recombinant plasmid is obtained by screening through an antibiotic zeocin (the final concentration is 0.25 mu g/mL). The tandem expression vector was named pPIC-god-pdi.
Example 5: GOD recombinant strain construction
10. mu.g of the recombinant plasmid pPIC-god-pdi was taken, linearized with Sac I (reaction system as shown in Table 2), and then purified with a PCR product purification kit, the linearized plasmid was dissolved in 10. mu.L of sterile ddH2Linearized plasmid purity was checked by electrophoresis in O and concentration was determined using Nanodrop 2000. Inoculating single colony of Pichia pastoris x33 in 10mL YPD medium, shaking at 30 deg.C and 250rpm for 12hr, transferring into 50mL YPD medium, culturing to logarithmic phase, centrifuging at 10,000rpm for 5min, washing with sterilized water and 1M ice-cold sorbitol, and preparing competent cell of Pichia pastoris x 33. The linearized DNA was mixed with 80. mu.L of competent cells, placed in a cuvette, shocked at 2000V for 5ms, after shocking, 1mL of ice-cold 1M sorbitol was added immediately, and after recovery culture at 30 ℃ for 3hr, transformed cells were plated on YPDS plate medium containing zeocin (final concentration 100-.
TABLE 2 linearized cleavage of recombinant expression vectors
| Reagent | Volume (μ L) |
| pPIC-god-pdi | 20(10μg) |
| |
5 |
| 10 |
10 |
| ddH2O | 65 |
| |
100 |
Example 6: recombinant GOD enzyme activity assay
O-dianisidine spectrophotometry was used. Under the action of GOD, glucose reacts with oxygen, and the generated hydrogen peroxide and colorless reduced o-dianisidine react under the action of horseradish peroxidase to generate water and red oxidized o-dianisidine. The activity of glucose oxidase was calculated by measuring the change in color during the reaction.
Example 7: recombinant strain fermentation process
A single colony was picked, inoculated with 5mL of YPD liquid medium, cultured at 28 ℃ under shaking at 250rpm for 24hr, transferred to 500mL of YPD medium, and cultured under the same conditions for 12 hr. Inoculating the seed liquid into a 30L fermentation tank containing 10L sterilized basic culture medium (culture medium per liter contains glycerol 40g, phosphoric acid 20g, and CaSO)4·2H2O 0.93g、Mg2SO44.0 g、(NH4)2SO41.65 g, KOH 3.37g, glutamic acid 1.74g, arginine 1.45g, PTM1 trace salt 4.35mL), 30 degrees C400 rpm culture. After the basal medium is cultured for 24 hours, the relative dissolved oxygen concentration of the fermentation broth rises to be close to 100 percent, which indicates that the carbon source is exhausted, and the feeding stage is started, wherein the feeding is 50 percent of sterilized glycerol (containing 4.35mL of PTM1 trace salt, 1.74g of glutamic acid and 1.45g of arginine per liter). And stopping feeding when the weight of the wet bacteria reaches above 300g/L, and starving the bacteria for 1-2h to exhaust the carbon source. Then, the methanol induction phase was started, with 0.2mL of methanol per minute fed per liter of medium, and increased to 0.5mL of methanol per minute after 3 h. Adding 4.35mL of PTM1 trace salt, 0.35g of alpha-ketoglutaric acid and 10mM of arginine into each liter of methanol. During the fermentation process byAdjusting the pH value with ammonia water to ensure that the pH value is between 5.5 and 6.0. The relative dissolved oxygen is ensured to be more than 20 percent through the rotation speed regulation. Changes in wet bacterial weight and enzyme activity were measured every 12 h.
Example 8: recombinant GOD Property determination
1. Determination of optimal catalytic temperature for GOD:
respectively measuring the activity of the enzyme at 10-70 ℃, calculating the relative activity of the enzyme at different temperatures by taking the highest enzyme activity as 100%, and drawing a relative activity curve of the enzyme at different temperatures, as shown in figure 2, it can be seen from the figure that the catalytic activity of the enzyme is highest when the GOD is at 50 ℃, and the catalytic activity of the enzyme is better between 20-60 ℃.
2. Determination of optimal catalytic pH for GOD:
preparing a buffer solution with the pH value of 1.5-9.0, diluting an enzyme solution and a substrate respectively, determining the activity of the enzyme at 50 ℃, setting the pH value with the highest enzyme activity as 100 percent of relative enzyme activity, comparing the enzyme activities with other pH values, drawing a relative enzyme activity curve, and as shown in figure 3, as can be seen from the figure, the optimum catalytic pH value is 6.0, and the relative catalytic activity of the enzyme is more than 60 percent between the pH value of 3.0-7.0.
3. GOD Heat resistance measurement:
the enzyme solution was treated at 80 ℃ for various times, and then the residual activity of the enzyme was measured at 50 ℃ and pH6.0, and an enzyme thermostability curve was plotted with the untreated enzyme activity as 100%, as shown in FIG. 4.
Aspergillus niger glucose oxidase gene sequence:
aspergillus niger glucose oxidase amino acid sequence:
an Aspergillus niger glucose oxidase optimized gene sequence ts-god:
pdi gene sequence:
pdi-optimized gene sequence Pdi-opt:
it will be understood by those skilled in the art that the method for expressing the glucose oxidase gene of Aspergillus niger in high efficiency of the present invention includes any combination of the above-mentioned summary and detailed description of the present invention and the parts shown in the drawings, which is limited by space and not described in any of the schemes of the combination for the sake of brevity. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<120> high-efficiency expression method of Aspergillus niger glucose oxidase gene
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1818
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgcagactc tccttgtgag ctcgcttgtg gtctccctcg ctgcggccct gccacactac 60
atcaggagca atggcattga agccagcctc ctgactgatc ccaaggaagt ctccggccgc 120
acagtcgact acatcatcgc tggtggaggt ctgactggac tcaccaccgc tgcccgtctg 180
acggagaatc ccaacatcag cgtgctcgtc atcgaaagtg gctcctacga gtcggataga 240
ggtcctatca ttgaggacct gaacgcctac ggcgacatct ttggcagcag tgtagaccac 300
gcctacgaga ccgtggagct cgctaccaac aatcaaaccg cgctgatccg ctccggaaat 360
ggtctcggtg gctctactct agtgaatggt ggcacctgga ctcgccccca caaggcacag 420
gttgactctt gggagactgt ctttggaaat gagggctgga actgggacaa tgtggccgcc 480
tactccctcc aggctgagcg tgctcgcgca ccaaatgcca aacagatcgc tgctggccat 540
tacttcaacg catcctgtca tggtttgaat ggtactgtcc atgccggacc ccgtgacacc 600
ggcgatgact attcccccat cgtcaaggct ctcatgagcg ctgtcgaaga ccgaggcgtt 660
cccaccaaga aggacttcgg atgcggtgac cctcatggtg tgtccatgtt ccccaacacc 720
ttgcacgaag accaagttcg ctccgatgcc gctcgcgaat ggctccttcc caactaccaa 780
cgtcccaacc tgcaagtcct gaccggacaa tatgttggta aggtgctcct tagccagaac 840
ggcaccaccc ctcgtgccgt cggcgtggaa ttcggcaccc acaagggcaa cacccacaac 900
gtttacgcta aacacgaggt cctcctggcc gcgggctccg ctgtctctcc cacaatcctg 960
gaatattccg gtatcggaat gaagtccatc ctggagcccc ttggtatcga caccgtcgtt 1020
gacctgcccg tcggcctgaa cctgcaggac cagaccaccg ctaccgtccg cagccgcatc 1080
acctctgctg gtgccggaca gggtcaggcc gcttggttcg ccaccttcaa cgagaccttt 1140
ggtgactatt ccgaaaaggc acacgagctg ctcaacacca agctggagca gtgggccgaa 1200
gaggccgtcg cccgtggcgg attccacaac actaccgcct tgctcatcca gtacgagaac 1260
taccgcgact ggattgtcaa ccacaacgtc gcgtactcgg aactcttcct cgacactgcc 1320
ggagtagcca gcttcgatgt gtgggacctt ctgcccttca cccgaggata cgttcacatc 1380
ctcgacaagg acccctacct tagacacttc gcctacgacc ctcagtactt cctcaacgag 1440
ctggacctgc tcggtcaggc tgccgctact caactggccc gcaacatctc caactccggt 1500
gccatgcaga cctacttcgc tggggagact atccccggtg ataacctcgc gtatgatgcc 1560
gatttgagcg cctgggttga gtacatcccg taccacttcc gtcctaacta ccatggcgtg 1620
ggtacttgct ccatgatgcc gaaggagatg ggcggtgttg ttgataatgc tgcccgtgtg 1680
tatggtgtgc agggactgcg tgtcattgat ggttctattc ctcctacgca aatgtcgtcc 1740
catgtcatga cggtgttcta tgccatggcg ctaaaaattt cggatgctat cttggaagat 1800
tatgcttcca tgcagtga 1818
<210> 2
<211> 425
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
mqtllvsslv vslaaalphy irsngieasl ltdpkevsgr tvdyiiaggg ltglttaarl 60
tenpnisvlv iesgsyesdr gpiiedlnay gdifgssvdh ayetvelatn nqtalirsgn 120
glggstlvng gtwtrphkaq vdswetvfgn egwnwdnvaa yslqaerara pnakqiaagh 180
yfnaschgln gtvhagprdt gddyspivka lmsavedrgv ptkkdfgcgd phgvsmfpnt 240
lhedqvrsda arewllpnyq rpnlqvltgq yvgkvllsqn gttpravgve fgthkgnthn 300
vyakhevlla agsavsptil eysgigmksi leplgidtvv dlpvglnlqd qttatvrsri 360
tsagagqgqa awfatfnetf gdysekahel lntkleqwae eavarggfhn ttalliqyen 420
ldllgqaaat qlarnisnsg amqtyfaget ipgdnlayda dlsawveyip yhfrpnyhgv 280
gtcsmmpkem ggvvdnaarv ygvqglrvid gsipptqmss hvmtvfyama lkisdailed 540
yasmq* 546
<210> 3
<211> 1818
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgcaaacat tgcttgtatc ttcacttgtg gtgtcattag ccgccgcatt accacattac 60
atcaggtcca acggtattga agcttctctt ttaacggatc caaaggaggt ttccggaaga 120
actgtggact acattattgc aggaggcggc cttactggtt tgactaccgc cgcaaggtta 180
actgaaaacc caaatatctc tgtattggtt atagaatctg gttcttatga gtcagataga 240
ggaccaatca tcgaggatct gaatgcttat ggtgatattt ttggctcctc cgtggatcat 300
gcttacgaga ccgttgaatt ggctaccaac aaccagactg ccctgattag gtctggaaac 360
ggattgggag gctcaacttt ggtaaatggt ggtacgtgga ctagacctca taaagctcag 420
gtagacagtt gggagacagt ttttggcaac gaaggctgga actgggataa tgtcgccgct 480
tattccctgc aagctgaacg agctagagca cctaatgcta agcagatagc tgctggacat 540
tacttcaacg caagttgtca cggacttaat ggaactgtcc atgccggtcc aagggacacg 600
ggagacgatt actctcctat cgtaaaggcc ttgatgtctg ccgtcgagga tagaggtgta 660
ccaactaaga aggactttgg ttgtggtgat ccacatggtg tttcaatgtt cccaaacaca 720
ttgcacgagg atcaagtgcg ttctgatgct gctagggaat ggttgcttcc aaattaccaa 780
aggccaaact tgcaggtgct tactggtcaa tatgtcggta aagttttatt gtcccaaaat 840
ggtacaaccc caagagccgt aggcgttgag tttggcactc ataaaggaaa cactcataac 900
gtctacgcta aacatgaggt cctgttggct gctggctcag ccgtctcacc aactattctg 960
gagtattcag gtattggtat gaagtccatt ttggaaccat tgggtatcga tactgttgtt 1020
gaccttccag tgggactgaa tttacaggac cagacaacag caactgtgag aagtcgtatc 1080
acaagtgctg gagctggtca gggacaggct gcctggtttg ccaccttcaa cgaaaccttc 1140
ggagactatt ctgaaaaagc tcacgagttg ctgaatacca aattggagca atgggctgaa 1200
gaagcagtcg caagaggagg atttcataac acaaccgctc ttttgattca atatgagaac 1260
tatcgtgatt ggatcgttaa tcataatgtg gcttattctg agttattttt ggatacagct 1320
ggtgttgctt cttttgatgt ctgggattta ttgcccttta ccaggggata tgtacatatt 1380
ttggataagg acccatattt aagacatttt gcctatgatc ctcagtactt tttgaacgaa 1440
ctagacttgc tgggccaagc tgcagcaacc cagttagctc gtaatataag taactcaggc 1500
gccatgcaga cttacttcgc aggtgagacg attcctggtg acaacttggc ttatgacgct 1560
gatttatctg cctgggtcga gtacattcca taccatttcc gtccaaatta ccatggagtt 1620
ggaacctgca gtatgatgcc aaaagaaatg ggaggtgttg tagacaacgc tgctagagtc 1680
tacggcgtac agggtctaag agttatcgat ggttctatcc cccctactca aatgtcatcc 1740
catgttatga cagtctttta cgccatggcc ttaaagattt ccgacgctat tcttgaggat 1800
tatgccagta tgcagtaa 1818
<210> 4
<211> 1569
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgaagtttt ctgctggtgc cgtcctgtca tggtcctccc tgctgctcgc ctcctctgtt 60
ttcgcccaac aagaggctgt ggcccctgaa gactccctgt cgttaagttg gccaccgact 120
ctttcaatga atacattcag tcgcacgact tggtggcttg cggagttttt tgctccatgg 180
tgtggccact gtaagaacat ggctcctgaa tacgttaaag ccgccgagac tttagttgag 240
aaaaacatta ccttggccca gatcgactgt actgaaaacc aggatctgtg tatggaacac 300
aacattccag ggttcccaag cttgaagatt ttcaaaaaca gcgatgttaa caactcgatc 360
gattacgagg gacctagaac tgccgaggcc attgtccaat tcatgatcaa gcaaagccaa 420
ccggctgtcg ccgttgttgc tgatctacca gcttaccttg ctaacgagac ttttgtcact 480
ccagttatcg tccaatccgg taagattgac gccgacttca acgccacctt ttactccatg 540
gccaacaaac acttcaacga ctacgacttt gtctccgctg aaaacgcaga ggatgatttc 600
aagctttcta tttacttgcc ctccgccatg gacgagcctg tagtatacaa cggtaagaaa 660
gccgatatcg ctgacgctga tgtttttgaa aaatggttgc aagtggaagc cttgccctac 720
tttggtgaaa tcgacggttc cgttttcgcc caatacgtcg aaagcggttt gcctttgggt 780
tacttgttct acaatgacga ggaagaattg gaagaataca agcctctctt taccgagttg 840
gccaaaaaga acagaggtct aatgaacttt gttagcatcg atgccagaaa attcggcaga 900
cacgccggca acttgaacat gaaggaacaa ttccctctat ttgccatcca cgacatgact 960
gaagacttga agtacggttt gcctcaactc tctgaagagg cgtttgacga attgagcgac 1020
aagatcgtgt tggagtccaa ggctattgaa cctttggtta aggacttctt gaaaggtgat 1080
gcctccccaa tcgtgaagtc ccaagagatc ttcgagaacc aagattcctc tgtcttccaa 1140
ttggtcggta agaaccatga cgaaatcgtc aacgacccaa agaaggacgt tcttgttttg 1200
tactatgccc catggtgtgg tcactgtaag agattggccc caacttacca agaactagct 1260
gatacctacg ccaacgccac atccgacgtt ttgattgcta aactagacca cactgaaaac 1320
gatgtcagag gcgtcgtaat tgaaggttac ccaacaatcg tcttctaccc aggtggtaag 1380
aagtccgaat ctgttgtgta ccaaggttca agatccttgg actctttatt cgacttcatc 1440
aaggaaaacg gtcacttcga cgtcgacggt aaggccttgt acgaagaagc ccaggaaaag 1500
gctgctgagg aagccgatgc tgacgctgaa ttggctgacg aagaagatgc cattcacgat 1560
gaattgtaa 1569
<210> 5
<211> 1569
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgaagtttt ccgcaggtgc cgtattaagt tggagttctc tgctgttggc atcaagtgtc 60
tttgctcaac aggaggcagt tgcacccgaa gactcacttt ctctttcttg gccacctacc 120
ctgtctatga atacttttag tagaactaca tggtggttag ccgagttttt tgctccatgg 180
tgtggacatt gcaaaaatat ggctccagag tatgtgaagg cagccgagac actggtcgaa 240
aagaatatta ccttggctca gattgactgc accgaaaacc aagacttgtg tatggaacat 300
aacatacccg gcttcccatc tctgaaaatt ttcaaaaatt ccgacgtcaa caattccatc 360
gactatgaag gtccaagaac cgctgaagcc atcgtgcagt tcatgattaa acaatcccaa 420
cctgcagttg ccgttgttgc tgatttgccc gcatatttgg ccaatgaaac cttcgttacg 480
cctgttatag tgcagtctgg taaaattgat gcagacttta acgctacctt ctattcaatg 540
gccaataaac actttaacga ttatgatttt gtctccgccg aaaacgccga agatgacttc 600
aaattatcta tatatttgcc ttccgctatg gatgagccag tggtttacaa tggcaagaag 660
gccgatatag ctgacgccga tgtcttcgag aaatggcttc aagttgaagc cctaccttat 720
ttcggtgaga tcgatggatc agtttttgct caatatgtgg aatcaggatt accattagga 780
tacctgttct ataatgatga ggaagaactg gaagaataca agccattgtt tacggagtta 840
gccaagaaga accgtggatt aatgaacttt gtgagtatcg atgctagaaa attcggaagg 900
catgctggta atttaaatat gaaggaacaa tttcctttgt tcgctattca cgatatgact 960
gaagatttga aatatggatt accacaactt tctgaagaag ctttcgatga gttaagtgat 1020
aaaatcgttc tggagagtaa ggctatcgaa cctttggtta aagatttctt aaagggtgac 1080
gcttctccta tcgtaaagtc tcaggagatt ttcgaaaacc aagatagttc tgtattccaa 1140
ttagtgggta aaaatcacga tgagatcgtt aatgacccaa aaaaggatgt cttggtgcta 1200
tactatgccc catggtgcgg acattgtaag cgtttagctc ccacctacca agaattggca 1260
gatacatatg ccaacgcaac atcagatgtc ttgattgcca aacttgatca tactgagaac 1320
gatgttaggg gtgtagtcat agaaggatat cctaccatcg ttttctatcc tggtggaaaa 1380
aaatcagaat cagtggttta ccagggttcc agatccctgg attctttatt tgattttatt 1440
aaggaaaacg gacatttcga cgttgatggt aaggcacttt atgaagaggc ccaagaaaag 1500
gcagctgagg aagcagacgc tgatgctgaa ttggctgacg aagaagatgc aattcacgat 1560
gaattgtaa 1569
Claims (5)
1. The efficient expression method of the aspergillus niger glucose oxidase gene is characterized by comprising the following steps in sequence:
(1) taking Aspergillus niger DY-2015 slant strain, inoculating sterilized PDA culture medium for culture, collecting mycelia, placing in liquid nitrogen for grinding, taking fine powder, adding a buffer solution, dissolving, centrifuging, taking supernatant, extracting, adding NaAc into the supernatant, adding absolute ethyl alcohol, mixing uniformly, placing on ice, centrifuging, washing precipitates twice with ice-cold ethyl alcohol, placing in a clean bench for drying, dissolving with 20 mu L of sterilized water, and determining the DNA concentration by using Nanodrop 2000;
(2) design of primers, F: 5'-ATGCAGACTCTCCTTGTGAG-3', respectively; r: 5'-TCACTGCATGGAAGCATAATG-3', carrying out PCR amplification by taking the Aspergillus niger DY-2015 genome DNA extracted in the step 1 as a template;
(3) according to the preference of pichia pastoris codons, optimizing a glucose oxidase gene DY-god of aspergillus niger DY-2015, replacing low-frequency codons with high-frequency codons, adjusting the codon adaptation index to be more than 0.8, removing restriction enzyme cutting sites such as EcoRI, BamHI, BglII, XbaI, Sac I and the like from a sequence for convenient subsequent genetic engineering operation, modifying a possible early termination sequence, naming the optimized gene as ts-god, connecting the optimized gene with multiple cloning sites EcoRI and Xba I of a pichia pastoris expression vector pPICzaA, transforming a connecting product into escherichia coli top10 competent cells, and screening an LB plate containing an antibiotic zeocin to obtain a recombinant expression vector pPIC-god;
(4) downloading a pdi sequence of a protein disulfide isomerase gene from NCBI, optimally designing through online software, then carrying out total synthesis, connecting the pdi sequence to EcoRI and NotI enzyme cutting sites of a pGAPzA expression vector, wherein the constructed vector is named as pGAP-pdi, a pGAP-pdi plasmid is subjected to double enzyme cutting by BglII and BamHI, a large fragment is recovered, the large fragment is connected with a pPIC-god vector subjected to single enzyme cutting by BamHI, an escherichia coli top10 competent cell is transformed, a recombinant plasmid is obtained through screening by an antibiotic zeocin, and a tandem expression vector is named as pPIC-god-pdi;
(5) the recombinant plasmid is taken to be linearized and then purified by a PCR product purification kit, and the linearized plasmid is dissolved in sterilized ddH2Checking the purity of linearized plasmid by electrophoresis in O, measuring the concentration by using Nanodrop2000, inoculating Pichia pastoris x33 single colony in 10mL YPD culture medium, shaking and culturing at 30 ℃ and 250rpm for 12hr, transferring to 50mL YPD, continuing culturing to logarithmic growth phase, centrifuging at 10,000rpm for 5min, washing with sterilized water and 1M ice-cold sorbitol to prepare Pichia pastoris x33 competent cells, mixing linearized DNA with 80 μ L competent cells, placing in an electric shock cup, shocking at 2000V and 5ms, immediately adding 1mL ice-cold 1M sorbitol, recovering and culturing at 30 ℃ for 3hr, spreading the transformed cells on Zeocin-containing YPDS plate culture medium, and culturing at 30 ℃Culturing for 3-4 days;
(6) reacting glucose with oxygen under the action of GOD by adopting an o-dianisidine spectrophotometry, generating hydrogen peroxide and colorless reduced o-dianisidine under the action of horseradish peroxidase to generate water and red oxidized o-dianisidine, and calculating the activity of glucose oxidase by measuring the color change in the reaction;
(7) selecting a single colony, inoculating 5mL of YPD liquid culture medium, carrying out shaking culture at 28 ℃ and 250rpm for 24h, transferring to 500mL of YPD culture medium, carrying out culture for 12h under the same conditions, inoculating seed liquid into a 30-liter fermentation tank, wherein the fermentation tank contains 10L of sterilized basic culture medium, carrying out culture at 30 ℃ and 400rpm, after the basic culture medium is cultured for 24h, the relative dissolved oxygen concentration of the fermentation liquid rises to be close to 100 percent, indicating that the carbon source is exhausted, starting a material supplementing stage, wherein the material supplementing is 50 percent sterilized glycerol, and stopping material supplementing when the weight of wet bacteria reaches more than 300g/L, so that the bacteria are starved for 1-2h to exhaust the carbon source. Then, a methanol induction stage is started, 0.2mL of methanol is fed in per liter of culture medium per min, the feeding is increased to 0.5mL of methanol per min after 3 hours, and 4.35mL of PTM1 trace salt, 0.35g of alpha-ketoglutaric acid and 10mM of arginine are added in per liter of methanol. And adjusting the pH value by ammonia water in the fermentation process to ensure that the pH value is between 5.5 and 6.0. The relative dissolved oxygen is ensured to be more than 20 percent through the rotation speed regulation. Changes in wet bacterial weight and enzyme activity were measured every 12 h.
2. The efficient expression method of aspergillus niger glucose oxidase gene according to claim 1, characterized in that the PDA culture medium: peeling potato 200g, adding water 800mL, boiling for 30min, filtering, adding glucose 20g into supernatant, diluting to 1L, and autoclaving; LB culture medium: 2% tryptone, 1% yeast extract, 1% NaCl, ph 7.0; low-salt LB medium: 2% tryptone, 1% yeast extract, 0.5% NaCl, pH 7.0; YPD medium: 1% yeast extract, 2% peptone, 2% glucose, solid plate plus 1.5% agar. YPDS medium: 1% yeast extract, 2% peptone, 2% glucose, 1.5% agar, 1M sorbitol.
3. The method for efficiently expressing the aspergillus niger glucose oxidase gene according to claim 1, wherein the amplification conditions of the step (2) are as follows: 30s at 95 ℃, 20s at 58 ℃ and 120s at 72 ℃ for 35 cycles.
4. The efficient expression method of Aspergillus niger glucose oxidase gene according to claim 1, characterized in that the Aspergillus niger glucose oxidase gene sequence:
5. the efficient expression method of the aspergillus niger glucose oxidase gene according to claim 1, characterized in that the amino acid sequence of the aspergillus niger glucose oxidase is as follows:
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