CN105420252A - Glucose oxidase gene GOD, protein coded by GOD, pichia pastoris transformed by GOD and preparation method of pichia pastoris - Google Patents
Glucose oxidase gene GOD, protein coded by GOD, pichia pastoris transformed by GOD and preparation method of pichia pastoris Download PDFInfo
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- CN105420252A CN105420252A CN201510893562.8A CN201510893562A CN105420252A CN 105420252 A CN105420252 A CN 105420252A CN 201510893562 A CN201510893562 A CN 201510893562A CN 105420252 A CN105420252 A CN 105420252A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
Abstract
The invention belongs to the preparation of a new gene, and in particular discloses a glucose oxidase gene GOD, protein coded by the GOD and pichia pastoris transformed by the GOD. The glucose oxidase gene GOD is prepared by taking 5'-ATGAAGTCCACTATTATCACCTCCA and 3'-CTAGGCACTTTTGGCATAGTCTTCA as specific primers and penicillium notatum as a template through polymerase chain reaction (PCR) amplification. According to the invention, the problem of an existing recombinant glucose oxidase gene in heterologous expression is solved; the total-length GOD gene and a shuttle expression vector constructed by the gene are obtained and the gene and the shuttle expression vector thereof are further transformed to the pichia pastoris, and upon screening and identification, a strain, which is higher in secretion expression of the glucose oxidase than an original strain, is obtained. The invention can greatly save cost and expense for further industrialized production and can improve the economic benefits of the glucose oxidase.
Description
Technical field
The invention belongs to the preparation of new gene, refer to the pichia pastoris phaff Pichiapastoris of a kind of glucose oxidase gene GOD, the albumen utilizing it to encode and conversion especially.
Background technology
Glucose oxidase (EC1.1.3.4) is that a kind of catalysis β-D-Glucose that can be single-minded is oxidized to δ-maltonic acid, and utilizes molecular oxygen to produce the aerobic dehydrogenase of hydrogen peroxide as acceptor.Glucose oxidase is
countryallow one of zymin used, to human non-toxic, be used as without pair, there is effects such as removing glucose, dehydrogenation, sterilization, be widely used in the industries such as food, weaving, chemical industry, feed, medicine.
Glucose oxidase is distributed widely in animals and plants and microbe, current is aspergillus niger (Abperrillusniger) and some mould (Penicilliumnotatum) for studying and produce the main bacterial strain of glucose oxidase, this is because mould enzymatic productivity is strong, is easy to mass-producing and generates; But in aspergillus niger and Penicillium notatum fermentative production GOD process, the existence of the assorted enzyme in a large number such as catalase, cellulase and amylase brings suitable difficulty to purifying.The demand of market to glucose oxidase increased year by year in recent years.But China's glucose oxidase technique level is still in the primary stage of researchdevelopment, the problems such as existence yields poorly, enzyme work is low, it is loaded down with trivial details to purify, Enzyme activity assay method complicated operation, make domestic glucose oxidase still based on import.And tradition improves with optimization of fermentation conditions the method that glucose oxidase output and enzyme live, also exist that tolerance range is low, experiment number is many, the cycle is long, fast and effeciently can not improve the technological deficiency of the output of glucose oxidase and enzyme work.
Restructuring glucose oxidase gene carries out heterogenous expression and can effectively address these problems, especially Pichia anomala expression foreign protein has that expression amount is high, good stability, toxigenic capacity are low and the advantage such as the easily separated purifying of product, be suitable for large volume high-density to continuously ferment, there is alcohol oxidase (Alcoholoxidase that is strong and easily control, AOX) advantage such as promotor, strictly can control the expression of foreign gene.
Produce bacterial strain by genetic engineering means transformation glucose oxidase, obtain production and the extracting method of grape carbohydrate oxidase precise and high efficiency, improve yield of enzyme, the suitability for industrialized production for the later stage provides guiding basis, improves its economic benefit.
Summary of the invention
An object of the present invention is to provide a kind of glucose oxidase gene GOD.
Two of object of the present invention is the preparation method providing a kind of glucose oxidase gene GOD.
Three of object of the present invention is to provide a kind of albumen utilizing glucose oxidase gene GOD to encode.
Four of object of the present invention is to provide a kind of method utilizing glucose oxidase gene GOD transforming Pichia pastoris Pichiapastoris.
Overall technology design of the present invention is:
Glucose oxidase gene GOD, its gene order is SEQIDNo.1.
The preparation method of glucose oxidase gene GOD is that with a mould DNA for template, employing PCR amplification method obtains with 5 ' end ATGAAGTCCACTATTATCACCTCCA and 3 ' end CTAGGCACTTTTGGCATAGTCTTCA for special primer.
Utilize the albumen that glucose oxidase gene GOD encodes, its sequence is SEQIDNo.2.
Utilize the pichia pastoris phaff Pichiapastoris that glucose oxidase gene GOD transforms, its
preservationbe numbered CGMCCNo.11626.
Pichia pastoris phaff Pichiapastoris applicant in the present invention submits Chinese microorganism strain on November 6th, 2015
preservationmanagement
the councilcommon micro-organisms center
preservation,
preservationbe numbered CGMCCNo.11626, should
preservationthe address of mechanism is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, should
preservationmechanism referred to as CGMCC.
The preparation method of the pichia pastoris phaff Pichiapastoris utilizing glucose oxidase gene GOD to transform, the step in its preparation method is as follows:
A, by PCR method design primer by the glucose oxidase gene sequence of a mould signal coding sequence remove, primer sequence is as follows: GodF:5 ' GGCTGAAGCTTACGTA
gAATTCtATAGCCCGGCCGAGCAGATCGAC3 ', in sequence, underscore part is that upstream primer adds restriction enzyme EcoRI recognition site; GodR:5 ' GAGATGAGTTTTTGTTCTAGA
gCGGCCGCcTAAGCTTTCTTGGCATAGTCTTC3 ', in sequence, underscore part is that restriction enzyme NotI recognition site is added in downstream;
The purified recovery of B, PCR primer is adopted GIBSON and is connected on EcoRI and the NotI site of expression vector pMD-AOX, is transformed into E.coliDH5 α, digestion verification screening positive clone, and send order-checking;
C, correct recombinant plasmid pMD-AOX-GOD linearizing after PmeI enzyme is cut of checking order.With the electroporated 80 μ l Pichia pastoris X33 competent cells of 2 μ g linearizing pMD-AOX-GOD plasmids, then transformant is applied on the YPD flat board containing 100 μ g/mlG418, cultivates 96h for 30 DEG C; The positive bacterium colony switching of random choose is containing the liquid YPCS substratum of 100 μ g/mLG418, and shaking culture 3d under temperature 30 DEG C, rotating speed 200r/min condition, period adds the methyl alcohol of 1% every 24h, measures the glucose oxidase activity in fermented liquid supernatant.
Concrete technology contents of the present invention also has:
In the preparation method of described glucose oxidase gene GOD be utilize designed by Auele Specific Primer, with a mould DNA for template, according to 98 DEG C of denaturations 5 minutes on amplification instrument; 30 PCR circulation: 98 DEG C of sex change 30 seconds, 65 DEG C of annealing 30 seconds, 72 DEG C extend 1 minute; 72 DEG C of programs extending 10 minutes carry out pcr amplification; PCR reaction is all undertaken by following composition and consumption: reaction system is 50 μ l: genomic dna 100ng, the each 0.2 μm of ol/L of primer, dNTPs250 μm of ol/L, 5 × Q5High-FidelityDNAPolymeraseBuffer10 μ l, Q5High-FidelityDNAPolymerase0.5 μ l.PCR primer runs the agarose electrophoresis observations of 1%; PCR primer is connected on plasmid pMD18-T carrier by T4DNA ligase enzyme, and transformation of E. coli DH5 α, be then applied to and have on the LB flat board of penbritin, 37 DEG C of incubated overnight are until bacterium colony grows, random picking 2 clones check order, and sequential analysis obtains GOD gene order.
Substantive distinguishing features acquired by the present invention and significant technical progress are:
1, the present invention obtains the GOD gene order of a total length by round pcr, and this full length gene is 1815bp, GC content is 55.4%.This gene of homogeneous assays has certain difference with the gene order of the GOD of report in the past, proves that this gene is a new GOD gene.
2, prepared in the present invention pichia pastoris phaff PichiapastorisCGMCCNo.11626 breaches in traditional aspergillus niger and Penicillium notatum fermentative production GOD process, is mingled with the assorted enzymes such as a large amount of catalase, cellulase and amylase, the problem of difficult purifying.Utilize the transgenic Pichia yeast engineering that the present invention obtains, under 10L fermentation tank level testing conditions, methanol induction 144 hours, the enzyme in fermented liquid is lived and is reached 594U/ml, significantly improves fermenting enzyme and lives; And produce bacterial strain by genetic engineering means transformation glucose oxidase, can also obtain the production of grape carbohydrate oxidase precise and high efficiency, improve yield of enzyme (9g/L), the suitability for industrialized production for the later stage provides guiding basis, increases economic efficiency.
Pichia pastoris phaff Pichiapastoris applicant in the present invention submits Chinese microorganism strain to
preservationmanagement
the councilcommon micro-organisms center
preservation,
preservationbe numbered CGMCCNo.11626, should
preservationthe address of mechanism is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, should
preservationmechanism referred to as CGMCC.
Accompanying drawing explanation
Of the present invention
accompanying drawinghave:
fig. 1it is the sequence table of glucose oxidase gene GOD and the albumen that utilizes it to encode in the present invention.
fig. 2point mould GOD full length gene result.
With a mould genomic dna for template, primer P1 and P2 carries out pcr amplification, the special band of amplification appearance one treaty 1800bp.The sequencing results shows, long 1815bp, the GC content of its genomic dna is 55.4%, the GOD gene fragment containing total length.M is DNAMarker, and molecular weight is followed successively by 10000,8000,6000,5000,4000,3000bp from big to small, 2000bp, 1000bp; 1 and 2 is GOD gene amplification product.
fig. 3it is expression plasmid pMD-AOX-GOD double digestion detected result.
Pcr amplification will be carried out and the some mould GOD gene that purifying reclaims carries out Gibson with the carrier pMD-AOX through EcoRI with NotI double digestion and is connected with construction recombination plasmid through primer GodF with GodF.Use recombinant plasmid transformed competence colibacillus bacillus coli DH 5 alpha, and falling containing the LB flat board of Amp screens positive bacteria.Positive bacterium colony plasmid, after EcoRI and NotI double digestion, obtains two DNA fragmentations of about 6.5.0kb and 1.8kb, also conforms to expection size.
in figurem is DNAMarker, and molecular weight is followed successively by 10000,8000,6000,5000,4000,3000bp from big to small, and 2000bp, 1000bp, 1-6 are expression plasmid pMD-AOX-GOD double digestion products.
fig. 4the SDS-PAGE detected result of glucose oxidase in the expression of YPCS.
By the expression plasmid pMD-AOX-GOD of restructuring after PmeI enzyme is cut after linearizing, electricity transforms pichia spp X33 competence, after YPD plated growth 3d, random choose positive bacterium colony switching liquid YPCS, after methanol induction 72h, measure the glucose oxidase activity in fermented liquid supernatant, from 32 transformants, pick out the transformant that 6 strains have glucose oxidase activity, expression rate is 16.7%.Fermented liquid supernatant liquid does protein s DS-PAGE electrophoresis, the protein band of the about 90kD of a visible dense dye simultaneously.
in figurethe different Pichi strains of M for being albumen Marker, 1-8 screening, wherein No. 5 bacterial strain enzymes are lived the highest, 24U/ml.
fig. 5the expression SDS-PAGE detected result of glucose oxidase at 10L fermentor tank.
In 10L fermentor tank, before methyl alcohol is not induced, be in strain culturing and carbon source feeding period, within these two stages, thalline increases in a large number, now without the expression of glucose oxidase zymoprotein.Along with the induction of methyl alcohol, glucose oxidase activity in fermented liquid increases gradually, in induction 144h secondary fermentation liquid, glucose oxidase activity is 496U/ml, and protein expression amount reaches 9.4g, and SDS-PAGE shows that in fermented liquid, glucose oxidase expressing quantity is also in continuous accumulation simultaneously.
in figurem is through methanol induction 0h for being albumen Marker, 1-12,24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h, 120h, 132h, 144h glucose oxidase expression amount.
Embodiment
Below in conjunction with
accompanying drawingembodiments of the invention are further described, but not as a limitation of the invention, and the content that protection scope of the present invention is recorded with claim is as the criterion, and any equivalent technical elements of making according to specification sheets is replaced, and does not all depart from protection category of the present invention.
Embodiment 1
The structure of pichia pastoris phaff Pichiapastoris
Glucose oxidase gene GOD sequence in the present embodiment and the protein sequence of coding
as Fig. 1shown in, it is specifically prepared and glucose oxidase gene GOD Function Identification comprises:
The complete sequence amplification of A, glucose oxidase gene GOD and clone;
B, glucose oxidase gene GOD transform pichia spp.
The preparation of steps A glucose oxidase gene GOD, its preparation method is for special primer with 5 ' end ATGAAGTCCACTATTATCACCTCCA and 3 ' end CTAGGCACTTTTGGCATAGTCTTCA, with a mould DNA for template, according to 98 DEG C of denaturations 5 minutes on amplification instrument; 30 PCR circulation: 98 DEG C of sex change 30 seconds, 65 DEG C of annealing 30 seconds, 72 DEG C extend 1 minute; 72 DEG C of programs extending 10 minutes carry out pcr amplification; PCR reaction is all undertaken by following composition and consumption: reaction system is 50 μ L: genomic dna 100ng, the each 0.2 μm of ol/L of primer, dNTPs250 μm of ol/L, 5 × Q5High-FidelityDNAPolymeraseBuffer10 μ L, Q5High-FidelityDNAPolymerase0.5 μ L.PCR primer runs the agarose electrophoresis observations of 1%; PCR primer is connected on plasmid pMD18-T carrier by T4DNA ligase enzyme, and transformation of E. coli DH5 α, then be applied to and have on the LB flat board of penbritin, 37 DEG C of incubated overnight are until bacterium colony grows, random picking 2 clones check order, and sequential analysis obtains glucose oxidase gene GOD sequence.
The step of step B is as follows:
A, by PCR method design primer by the glucose oxidase gene sequence of a mould signal coding sequence remove, primer sequence is as follows: GodF:5 ' GGCTGAAGCTTACGTA
gAATTCtATAGCCCGGCCGAGCAGATCGAC3 ', in sequence, underscore part is that upstream primer adds restriction enzyme EcoRI recognition site; GodR:5 ' GAGATGAGTTTTTGTTCTAGA
gCGGCCGCcTAAGCTTTCTTGGCATAGTCTTC3 ', in sequence, underscore part is that restriction enzyme NotI recognition site is added in downstream;
The purified recovery of B, PCR primer is adopted GIBSON and is connected on EcoRI and the NotI site of expression vector pMD-AOX, is transformed into E.coliDH5 α, digestion verification screening positive clone, and send order-checking;
C, correct recombinant plasmid pMD-AOX-GOD linearizing after PmeI enzyme is cut of checking order.With the electroporated 80 μ L Pichia pastoris X33 competent cells of 2 μ g linearizing pMD-AOX-GOD plasmids, then transformant is applied on the YPD flat board containing 100 μ g/mlG418, cultivates 96h for 30 DEG C; Obtain pichia pastoris phaff Pichiapastoris.
Embodiment 2
The pichia pastoris phaff Pichiapastoris checking that enzyme is lived in test tube
The pichia pastoris phaff Pichiapastoris obtained with embodiment 1 is for producing bacterium, after activation temperature be 30 DEG C, rotating speed cultivate under being 200rpm condition OD600 about 1.2 seed culture fluid with 2% inoculum size proceed to YPCS substratum, in temperature be 30 DEG C, rotating speed cultivates under being 200rpm condition; When to be cultured to OD600 value in YPCS substratum be about 1.2, yeast cell proceeded to YPCS inducing culture, add 1% methanol induction every day and express 3 days.
Substratum: seed and slant medium are YPD substratum: Tryptones 2%, yeast extract 1%, glucose 2%; Slant medium adds agar 2%.Containing the YPCS substratum of 100 μ g/mLG418 containing, for example the raw material of lower mass percentage: Tryptones 2%, yeast extract 1%, casein hydrolysate 2%, sorbyl alcohol 0.5%.
One is obtained by protein electrophoresis (SDS-PAGE)
as Fig. 4shown molecular size range is about the protein band of 90kDa, induces the ability of malaga carbohydrate oxidase in shaking flask with 1% methanol concentration simultaneously, and the highest enzyme is lived as 25U/mL, improves nearly 0.8 times than the enzyme (14U/mL) alive of wild mushroom.
Embodiment 3
The pichia pastoris phaff Pichiapastoris checking that enzyme is lived on 10L fermentor tank
The recombinant bacterial strain activated is inoculated in YPD substratum, temperature be 30 DEG C, rotating speed be 200-250rpm cultivate OD600 >=2 time with 3% inoculum size be inoculated in BMGY substratum, temperature be 30 DEG C, rotating speed is that 250rpm is cultured to OD600 and reaches about 10.By cultured BMGY fermented liquid with 10% inoculum size access 10L automatic fermenter.Initial loading liquid measure 6L, and add 4 ‰ PTM1, add aseptic VC6mL every 24h aseptic technique, after induction 48h, aseptic VB212ml is added in aseptic technique.
In the yeast culture stage: 18-24h is cultivated in 30 DEG C of aeration-agitations, and the glycerine in substratum consumes gradually, when thalline not regrowth after glycerine approach exhaustion, now DO can rise suddenly, stablizes constant.Maintain pH value about 5.0 with ammoniacal liquor in culturing process, now thalline OD600 is about about 90.
Carbon source feeding period: after yeast culture stage glycerol depletion, Continuous Flow adds 50% glycerine, containing 12mLPTM1 in often liter of glycerine, flow acceleration is 12mL/h/L, and cultivate 4h for 30 DEG C, dissolved oxygen maintains 0, pH ammoniacal liquor and maintains about 5.0.During this EOS, OD600 is about between 150-170.
Hungry cultivation stage: before inducing culture, stops feed supplement 0.5-1h, confirms that glycerine exhausts completely.PH ammoniacal liquor maintains 5.0.Thalline not regrowth after glycerine runs out of, now DO can rise suddenly, stablizes constant.Now thalline OD600 is about 180.
In the abduction delivering stage: stream adds inductor methyl alcohol, containing 12mlPTM1 in often liter of methyl alcohol, flow acceleration is 3.0ml-5.0ml/h/L, improves flow acceleration after 10 hours.Sorbyl alcohol adds as carbon source stream, and stream dosage is 1/20 of methyl alcohol, and speed is 0.3ml-0.5ml/h/L, after 10 hours, improve flow acceleration, and now dissolved oxygen maintains the level of 0, cultivates under temperature is 30 DEG C of conditions.Induction time reaches 120-144h.At induction period, when raising medium pH is to 6.0-6.5, the expression amount of glucose oxidase can further improve, but the probability of living contaminants can increase greatly.
After the abduction delivering stage terminates, the enzyme work of pichia pastoris phaff Pichiapastoris on 10L fermentor tank can reach 594U/ml.
Substratum and reagent
BMGY substratum: yeast extract 1%, peptone 2%, potassiumphosphate pH=6.0100mM, YNB1.34%, vitamin H 4 × 10-5%, glycerine 1% or methyl alcohol 0.5%; Trace element comprises: copper sulfate 6.0g/L, sodium iodide 0.08g/L, manganous sulfate 3.0g/L, Sodium orthomolybdate 0.2g/L, boric acid 0.02g/L, cobalt chloride 0.5g/L, zinc chloride 20g/L, ferrous sulfate 65g/L, sour 5.0mL/L, vitamin H 0.2g/L, VC80mg/L, VB2:0.5M, surplus is water.
Sequence table
Claims (5)
1. glucose oxidase gene GOD, is characterized in that its gene order is SEQIDNo.1.
2. the preparation method of glucose oxidase gene GOD, it is characterized in that with a mould DNA for template, employing PCR amplification method obtains with 5 ' end ATGAAGTCCACTATTATCACCTCCA and 3 ' end CTAGGCACTTTTGGCATAGTCTTCA for special primer.
3. the albumen utilizing glucose oxidase gene GOD to encode, is characterized in that its sequence is SEQIDNo.2.
4. utilize the pichia pastoris phaff Pichiapastoris that glucose oxidase gene GOD transforms, it is characterized in that its deposit number is CGMCCNo.11626.
5. the preparation method of pichia pastoris phaff Pichiapastoris according to claim 4, is characterized in that the step in its preparation method is as follows:
A, by PCR method design primer by the glucose oxidase gene sequence of a mould signal coding sequence remove, primer sequence is as follows: GodF:5 ' GGCTGAAGCTTACGTA
gAATTCtATAGCCCGGCCGAGCAGATCGAC3 ', in sequence, underscore part is that upstream primer adds restriction enzyme EcoRI recognition site; GodR:5 ' GAGATGAGTTTTTGTTCTAGA
gCGGCCGCcTAAGCTTTCTTGGCATAGTCTTC3 ', in sequence, underscore part is that restriction enzyme NotI recognition site is added in downstream;
The purified recovery of B, PCR primer is adopted GIBSON and is connected on EcoRI and the NotI site of expression vector pMD-AOX, is transformed into E.coliDH5 α, digestion verification screening positive clone, and send order-checking;
C, correct recombinant plasmid pMD-AOX-GOD linearizing after PmeI enzyme is cut of checking order, with the electroporated 80 μ L Pichia pastoris X33 competent cells of 2 μ g linearizing pMD-AOX-GOD plasmids, then transformant is applied on the YPD flat board containing 100 μ g/mlG418, cultivates 96h for 30 DEG C; Obtain pichia pastoris phaff PichiapastorisCGMCCNo.11626.
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Cited By (11)
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| CN107937360A (en) * | 2017-12-22 | 2018-04-20 | 河北省微生物研究所 | A kind of fermentation process in high density of glucose oxidase in Pichia pastoris |
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| CN108070532A (en) * | 2016-11-16 | 2018-05-25 | 福建力多利生物科技有限公司 | A kind of method for producing glucose oxidase |
| CN109880809A (en) * | 2019-02-28 | 2019-06-14 | 大连大学 | A kind of genetic engineering bacterium and preparation method thereof producing low temperature glucose oxidase |
| CN110894219A (en) * | 2019-12-26 | 2020-03-20 | 河北省微生物研究所 | Pichia pastoris transcription factor HAC1, protein, pichia pastoris and preparation and application thereof |
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| CN114058637A (en) * | 2021-03-31 | 2022-02-18 | 山西大禹生物工程股份有限公司 | Efficient expression method of aspergillus niger glucose oxidase gene |
| CN115369049A (en) * | 2021-05-17 | 2022-11-22 | 北京化工大学 | Genetically engineered bacterium secreting glucose oxidase, and construction method and application thereof |
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| CN108070532A (en) * | 2016-11-16 | 2018-05-25 | 福建力多利生物科技有限公司 | A kind of method for producing glucose oxidase |
| CN108004256A (en) * | 2017-12-14 | 2018-05-08 | 河北省微生物研究所 | Glucose oxidase gene Glox, albumen, pichia pastoris yeast and its preparation and application |
| CN107937360B (en) * | 2017-12-22 | 2020-04-17 | 河北省微生物研究所 | High-density fermentation method of glucose oxidase in pichia pastoris |
| CN107937360A (en) * | 2017-12-22 | 2018-04-20 | 河北省微生物研究所 | A kind of fermentation process in high density of glucose oxidase in Pichia pastoris |
| CN109880809A (en) * | 2019-02-28 | 2019-06-14 | 大连大学 | A kind of genetic engineering bacterium and preparation method thereof producing low temperature glucose oxidase |
| WO2021103123A1 (en) * | 2019-11-25 | 2021-06-03 | 中国海洋大学 | Glucose oxidase m5god, coding gene thereof and application thereof |
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| CN115369049B (en) * | 2021-05-17 | 2023-12-15 | 北京化工大学 | Genetically engineered bacterium secreting glucose oxidase, construction method and application thereof |
| CN113717872A (en) * | 2021-09-27 | 2021-11-30 | 河北省微生物研究所有限公司 | Method for screening high-expression FAD-GDH recombinant pichia pastoris strain |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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