CN108251390A - A kind of glucose oxidase mutant - Google Patents
A kind of glucose oxidase mutant Download PDFInfo
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- CN108251390A CN108251390A CN201710711533.4A CN201710711533A CN108251390A CN 108251390 A CN108251390 A CN 108251390A CN 201710711533 A CN201710711533 A CN 201710711533A CN 108251390 A CN108251390 A CN 108251390A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- 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)
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Abstract
The present invention relates to genetic modification technical fields, specifically provide a kind of glucose oxidase mutant.The heat resistance of the glucose oxidase single-point mutants is generally higher than wild type, and remnant enzyme activity improves 19.1 63.8% after 60 DEG C of 10 min of processing, and remnant enzyme activity improves 27.1 82.6% after 65 DEG C of 5 min of processing.So as to illustrate that above-mentioned simple point mutation causes the heat resistance of glucose oxidase to be increased dramatically, feed addictive is more suitable as than wild type, is conducive to extensive use of the glucose oxidase in feed, thus wide market.
Description
Technical field
The invention belongs to genetic modification technical fields, and in particular to a kind of glucose oxidase mutant.
Background technology
Glucose oxidase is a kind of aerobic dehydrogenase, exclusively can generate gluconic acid and peroxide by oxidation of beta-D-Glucose
Change hydrogen.Glucose oxidase usually forms an oxidation-reduction system, the oxidation of beta-D- in the presence of molecular oxygen with catalase
Glucose generates D-Glucose acid lactone, while consumes oxygen generation hydrogen peroxide.Hydrogen peroxide is decomposed and generated by catalase
Water and 1/2 oxygen, then water is combined again with glucolactone generates gluconic acid.Glucose oxidase is to β-D- glucopyranoses
Show strong specificity, the hydroxyl on glucose molecule C1 is most important to the catalytic activity of enzyme, and when hydroxyl is in β
It is 160 times higher than at α.Glucose oxidase is to L- glucose and 2-O- methyl-D-glucoses completely without activity.
Deoxygenation and antioxidation due to glucose oxidase make it in food, medicine, feed etc. using very
Extensively.In the food industry, glucose oxidase is preventing beer aging, is keeping product original as a kind of food preservative
Flavor, the aspect that extends the shelf life have remarkable result, are alternatively arranged as flour improver and Bread improver improves food quality.
In field of medicaments, hospital of China is generally using glucose oxidase electrode method, glucose oxidase-peroxidase coupling method etc.
Detect blood, glucose in serum content.As a kind of novel feed addictive, glucose oxidase can improve animal intestine
Road environment adjusts diet digestion, promotes growth of animal.
Glucose oxidase is distributed widely in animal, plant and microbial body, it is industrial mainly using black-koji mould or
Penicillium notatum is produced, but often occurs that enzyme activity is not high, stability is poor, foreign protein pollution and isolates and purifies cumbersome etc. ask
Topic.Because there are one 80~90 DEG C of short duration of hot stages during particle manufacture at present.And the grape of Aspergillus niger origin
Carbohydrate oxidase thermal stability is poor, and aqueous solution keeps the temperature 5 minutes remaining enzymatic activitys less than 40% at 65 DEG C, makes the enzyme in particle
The application of feed is restricted.At present using after feed granulating by the method in glucose oxidase liquid spray to feed not only
Increase equipment investment, and can not ensure the distribution uniformity of the stability and enzyme preparation of enzyme preparation in feed.Therefore, it carries
High glucose oxidation enzyme heat stability has important practical significance to current feed with glucose oxidase.
Invention content
The object of the present invention is to provide a kind of glucose oxidase mutant, heat resistance is significantly improved, is conducive to
Its extensive use in field of fodder.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme:
The present invention provides a kind of glucose oxidase mutant, have the amino acid sequence shown in (I), (II) or (III)
Any one in row:
(I) with the amino acid sequence SEQ ID NO of glucose oxidase:1 has the sequence of at least 95% homology;
(II) there is at least one immune epitope of the glucose oxidase described in (I), and the grape glycosyloxy
Change the amino acid sequence that the amino acid sequence of enzyme is obtained through modifying, replacing, lacking or adding one or several amino acid;
(III) by such as SEQ ID NO:When nucleotide sequence or its complementary series shown in 2 or the degeneracy because genetic code
With such as SEQ ID NO:The amino acid of the different sequential coding of the nucleotide sequence of nucleotide sequence or its complementary series shown in 2
Sequence;
In some embodiments of the invention, it is described to be substituted by 1 amino acid of substitution.
In other embodiments of the present invention, the substitution includes amino acid sequence for SEQ ID NO:1 glucose
The 387th of oxidizing ferment, 388,402,407,409,419,456,467,471,477,479,480,493,494,502,532,
Any one in 533,547,579 amino acids is replaced.
The present invention other embodiments in, it is described substitution include the 387th amino acids from T become A or E or F or
P。
In other embodiments of the present invention, the substitution includes the 388th amino acids and becomes E or P from T.
In other embodiments of the present invention, the substitution includes the 402nd amino acids and becomes F from V.
In other embodiments of the present invention, the substitution includes the 407th amino acids and becomes P from A.
In other embodiments of the present invention, the substitution includes the 409th amino acids and becomes A from S.
In other embodiments of the present invention, the substitution includes the 419th amino acids and becomes L from A.
In other embodiments of the present invention, the substitution includes the 456th amino acids and becomes D from E.
In other embodiments of the present invention, the substitution includes the 467th amino acids and becomes K from Q.
In other embodiments of the present invention, the substitution includes the 471st amino acids and becomes E or F or K from N.
In other embodiments of the present invention, the substitution includes the 477th amino acids and becomes P from A.
In other embodiments of the present invention, the substitution includes the 479th amino acids and becomes K from Q.
In other embodiments of the present invention, the substitution includes the 480th amino acids and becomes D from T.
In other embodiments of the present invention, the substitution includes the 493rd amino acids and becomes P from A.
In other embodiments of the present invention, the substitution includes the 494th amino acids and becomes P from Y.
The present invention other embodiments in, it is described substitution include the 502nd amino acids from T become E or K or L or
R, or Y.
In other embodiments of the present invention, the substitution includes the 532nd amino acids and becomes F or P from N.
In other embodiments of the present invention, the substitution includes the 533rd amino acids and becomes R from A.
In other embodiments of the present invention, the substitution includes the 547th amino acids and becomes A from G.
The present invention other embodiments in, it is described substitution include the 579th amino acids from S become F or K or R or
Y。
In some embodiments of the invention, the missing is missing 1 or 2 amino acid.
In other embodiments of the present invention, the missing includes the 581st amino acids missing.
In other embodiments of the present invention, the missing includes the 580th and the 581st amino acids lack simultaneously.
The present invention also provides application of the above-mentioned glucose oxidase mutant in feed.
The present invention also provides the recombinant expression carriers with above-mentioned DNA molecular.
The present invention also provides a kind of host cells, include above-mentioned recombinant expression carrier.
In some embodiments of the invention, host cell is Pichia pastoris.
The heat resistance of glucose oxidase mutant provided by the invention is generally higher than wild type, after 60 DEG C handle 10min
Remnant enzyme activity improves 19.1-63.8%, and remnant enzyme activity improves 27.1-82.6% after 65 DEG C of processing 5min.On illustrating
Stating mutation causes the heat resistance of glucose oxidase to be increased dramatically, and is more suitable as feed addictive than wild type, favorably
In extensive use of the glucose oxidase in feed, thus wide market.
Specific embodiment:
The routine techniques and method that the present invention has used genetic engineering and biology field uses, such as
MOLECULAR CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT
Recorded method in PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).These general bibliography
Provide definition well known by persons skilled in the art and method.But those skilled in the art can be recorded in the present invention
Technical solution on the basis of, using the other conventional methods in this field, experimental program and reagent, and be not limited to of the invention specific
The restriction of embodiment.
A in the present invention, R, D, C, Q, E, H, I, G, N, L, K, M, F, P, S, T, W, Y, V are alanine Ala respectively, arginine
Arg, aspartic acid Asp, cysteine Cys, glutamine Gln, glutamic acid Glu, histidine, isoleucine Ile, sweet ammonia
Sour Gly, asparagine Asn, leucine Leu, lysine Lys, methionine Met, phenylalanine Phe, proline Pro, silk ammonia
Sour Ser, threonine Thr, tryptophan Trp, tyrosine Tyr, the abbreviation of valine Val.
Experiment material used in the specific embodiment of the invention and reagent are as follows:
Bacterial strain and carrier:Bacillus coli DH 5 alpha, Pichia pastoris GS115, carrier pPIC9K, Amp, G418 are purchased from
Invitrogen companies.
Enzyme and kit:PCR enzymes and ligase purchase are from Takara companies, and restriction enzyme is purchased from Fermentas public affairs
Department, plasmid extraction kit and glue purification QIAquick Gel Extraction Kit are purchased from Omega companies, the purchase of GeneMorph II Random Mutagenesis Kits
From Beijing Bo Maisi bio tech ltd.
Culture medium prescription:
Escherichia coli culture medium (LB culture mediums):0.5% yeast extract, 1% peptone, 1%NaCl, pH7.0);
LB-AMP culture mediums:LB culture mediums add 100 μ g/mL ampicillins;
Yeast culture medium (YPD culture mediums):1% yeast extract, 2% peptone, 2% glucose;
Yeast screening assay culture medium (MD culture mediums):2% glucose, 2% agarose, 1.34%YNB, 4 × 10-5Biotin;
BMGY culture mediums:2% peptone, 1% yeast extract, 100mM kaliumphosphate buffers (pH6.0), 1.34%
YNB, 4 × 10-5Biotin, 1% glycerine;
BMMY culture mediums:2% peptone, 1% yeast extract, 100mM kaliumphosphate buffers (pH6.0), 1.34%
YNB, 4 × 10-5Biotin, 0.5% methanol.
The present invention is described in detail with reference to embodiment.
The acquisition of the heat-resisting mutant of 1 glucose oxidase of embodiment
The amplification of 1.1 glucose oxidase genes
PCR amplification, PCR primer GOD-F1, GOD- are carried out by template of aspergillus niger (Aspergillus niger) genome
R1 is as follows:
GOD-F1:GGTATTGAGGCATCTTTGTTGAC
GOD-R1:TTATTGCATAGAAGCGTAATC
Glue recycle PCR product, connect pEASY-T carriers, convert into bacillus coli DH 5 alpha, the correct transformant of picking into
Row sequencing.Sequencing result shows that the nucleotides sequence of the genetic fragment expanded is classified as SEQ ID NO:2, the amino of coding
Acid sequence is SEQ ID NO:1.It is compared and found by NCBI BLAST, SEQ ID NO:1 glucose with deriving from aspergillus niger
Oxidase sequence similitude is up to 100%, so that it is determined that being glucose oxidase gene by the gene that PCR is obtained, is named as
GOD。
The amplification and synthesis of 1.2 glucose oxidase mutant genes
In order to improve the heat resistance of above-mentioned glucose oxidase GOD, applicant carries out the enzyme by directed evolution technologies
The screening of mass mutation, design PCR primer GOD-F2, GOD-R2 are as follows:
GOD-F2:GGCGAATTC(underscore identifies GGTATTGAGGCATCTTTGTTGAC for restriction enzyme EcoRI
Site)
GOD-R2:ATAGCGGCCGC(underscore identifies TTATTGCATAGAAGCGTAATC for restriction enzyme Not I
Site)
Using GOD genes as template, with above-mentioned primer GeneMorph II random mutations PCR kits (Stratagene)
Carry out PCR amplification, glue recycling PCR product, EcoRI, Not I carry out digestion processing after with the pET21a carriers after similary digestion
Connection, converts into e. coli bl21 (DE3), is coated on LB+Amp tablets, and 37 DEG C are inverted culture, after sub- appearance to be transformed,
It is chosen one by one with toothpick to 96 orifice plates, adds in the LB+Amp culture mediums that 150ul contains 0.1mM IPTG in each hole, 37 DEG C
220rpm cultures 6h or so, supernatant is abandoned in centrifugation, and thalline is resuspended with buffer solution, multigelation broken wall, is obtained glycoxidative containing grape
The Bacillus coli cells lysate of enzyme.
10 μ L lysates are taken out respectively to two pieces of 96 new orifice plates, one of after 70 DEG C handle 5min, two piece of 96 hole
Plate all adds in 40 μ L substrates, and after 30 DEG C are reacted 30min, DNS methods measure the reduced sugar of generation, calculate the enzyme solution phase of high-temperature process
Than in the opposite enzyme activity of untreated enzyme solution.The experimental results showed that some mutation do not have shadow to the heat resistance of glucose oxidase GOD
It rings, some mutation even make its heat resistance or enzyme activity become worse;In addition also some mutation, although it is glycoxidative to improve grape
Enzyme is to the tolerance of temperature, but significant change has occurred in its zymologic property after being mutated, these are undesirable.Finally, Shen
It asks someone to screen the heat resistance that can significantly improve glucose oxidase GOD and its enzyme activity and original zymologic property
Mutational site:T387A/E/F/P, T388E/P, V402F, A407P, S409A, A419L, E456D, Q467K, N471E/F/K,
A477P, Q479K, T480D, A493P, Y494P, T502E/K/L/R/Y, N532F/P, A533R, G547A, S579F/K/R/Y,
And Q581 missings, M580 and Q581 are lacked simultaneously.
PCR amplification is carried out respectively to above-mentioned mutant with primer GOD-F2, GOD-R2, and primer both ends introduce EcoRI, Not
I site.PCR reaction conditions are:94 DEG C of denaturation 5min;Then 94 DEG C of denaturation 30s, 56 DEG C of renaturation 30s, 72 DEG C of extension 1min, 30
After a cycle, 72 DEG C of heat preservation 10min.Agarose gel electrophoresis is the results show that the mutant gene that amplification obtains is size
The segment of 1800bp or so.
It expands to obtain the genetic fragment of wild type glucose oxidase GOD by above-mentioned same PCR method.
The structure of 1.3 pichia pastoris engineered strains
The glucose oxidase mutant gene that above-mentioned clone obtains is passed through into EcoRI and NotI sites and expression vector
PPIC9K is connected, construction of expression vector.
Expression vector is linearized with Sal I, expression vector linearized fragment is converted by electroporation finishes red ferment
Female GS115, screening obtains Pichia pastoris recombinant bacterial strain respectively on MD tablets, then respectively in the Geneticin containing various concentration
The transformant of multicopy is screened on YPD tablets.
The obtained switching of the transformant containing above-mentioned mutant will be screened in BMGY culture mediums, 30 DEG C, 250rpm oscillation trainings
Support 1d;It is transferred to again in BMMY culture mediums, 30 DEG C, 250rpm shaken cultivations;The methanol of addition 0.5% daily, induced expression 4d;From
The heart removes thalline, obtains the fermented supernatant fluid of the mutant containing glucose oxidase;Carried out SDS-PAGE electrophoresis detection analyses.
The results show that the molecular size range of glucose oxidase mutant is about 64kDa in fermented supernatant fluid, with theoretical molecular weight size
It is identical.
It builds to obtain the Pichia yeast engineering of recombinant expression wild type glucose oxidase by above-mentioned same method.
Shaking flask level is fermented, 30 DEG C, 250rpm shaken cultivations;The methanol of addition 0.5% daily, induced expression 4d;Centrifugation removal thalline,
Obtain the fermented supernatant fluid of the GOD of glucose oxidase containing wild type.
(1) definition of glucose oxidase enzyme-activity unit
It is per minute β-D-Glucose of 1 μm of ol to be oxidized to maltonic acid and peroxide under the conditions of pH6.0,30 DEG C
Change the required enzyme amount of hydrogen, be defined as 1 enzyme activity unit (IU).
(2) enzyme activity determination method
Crude enzyme liquid is directly diluted to about 10U/mL with buffer solution.Take the test tube of 4 150*15, add in 2ml buffer solutions,
0.3ml glucose, 0.4ml phenol, 0.1ml 4- amino antipyrine, 0.1ml horseradish peroxidases, 30 DEG C of preheating 5min.
A pipe adds in 0.1ml distilled water thereto, returns to zero as blank.Water-bath is placed on to be conveniently operated by spectrophotometer, to sample
0.1ml sample solutions are added in quality control, start timing at this time, after vortex mixing immediately at 500nm wavelength with 1cm cuvettes ratio
Color.Absorbance value is A0 when reading 0.5min, then after reacting 1min, reads absorbance value A1, obtains Δ A500=A1-A0.
Enzyme activity calculation formula:
Enzyme activity X1 (U/mL or U/g) is calculated according to equation below in sample:
X1=Δs A500 × f × B × 1000/ (887 × t × A × d)=33.82 × Δ A500 × f
In formula:
F--------------------- enzyme solution extension rates
B-------------------- reaction solutions volume (3ml)
1000---------------- extinction coefficient unit conversion factors
887----------------- extinction coefficients (Lmol-1cm-1)
T--------------------- reaction time (min), i.e., the time difference 1min between reading A1 and A0.
A-------------------- adds in sample volume (0.1ml)
The thickness (cm) of d-------------------- cuvettes
(3) enzyme activity determination result
The enzyme activity determination of fermented supernatant fluid is carried out according to the method described above, is as a result shown:Recombinantly express wild type grape glycosyloxy
The enzyme activity for changing the Pichia pastoris fermented supernatant fluid of enzyme is 105U/ml, and recombinantly expresses the complete red ferment of glucose oxidase mutant
The enzyme activity of female fermented supernatant fluid is about 111-165U/mL.
1.4 fermentation verifications
Carry out the fermentation for the Pichia yeast engineering that above-mentioned structure obtains, the training used of fermenting respectively on 10 liters of fermentation tanks
Foster based formulas is:Calcium sulfate 1.1g/L, potassium dihydrogen phosphate 5.5g/L, ammonium dihydrogen phosphate 55g/L, potassium sulfate 20.3g/L, magnesium sulfate
16.4g/L, potassium hydroxide 1.65g/L, antifoaming agent 0.05%.
Zymotechnique:PH value 5.0,30 DEG C of temperature, stir speed (S.S.) 300rpm, ventilation quantity 1.0-1.5 (v/v), dissolved oxygen control
More than 20%.
Entire fermentation process is divided into three phases:First stage be thalline cultivation stage, in 7% ratio access seed, 30
DEG C culture 24-26h, using mended glucose for indicate;Second stage is the hungry stage, after glucose has been mended, does not flow plus appoints
What carbon source, terminates, by a definite date about 30-60min when dissolved oxygen rose to for 80% stage indicated above;Phase III is induced expression rank
Section, stream plus methanol induction, and keep dissolved oxygen incubation time is between 150-180h more than 20%.After fermentation, it ferments
Liquid obtains crude enzyme liquid after being handled by flame filter press.
Enzyme activity assay is carried out to crude enzyme liquid using in the embodiment 1 1.3 glucose oxidase enzyme activity determination methods, as a result
It has been shown that, the fermentation enzyme activity that the Pichia pastoris of recombinant expression wild type glucose oxidase is final is 3050U/ml, and is recombinantly expressed
The fermentation enzyme activity that the Pichia pastoris of glucose oxidase mutant is final reaches 3155-3561U/ml.
1.5 glucose oxidase zymologic properties measure
1st, most suitable action pH
It is respectively 2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0 phosphorus using pH value
Sour disodium hydrogen-citrate buffer solution, it is glycoxidative to carry out grape for fermentation crude enzyme liquid described in embodiment 1 1.4 under the conditions of 30 DEG C
Enzyme activity determination using highest enzyme activity as 100%, calculates opposite enzyme activity, as a result shows wild type glucose oxidase GOD and mutation
The Optimun pH of body is all 6.0, and the horizontal difference of opposite enzyme activity under condition of different pH is little.
2nd, thermal stability analysis
After the acetic acid-sodium acetate buffer solution dilution of pH 6.0 of above-mentioned crude enzyme liquid, at 60 DEG C of processing 10min, 65 DEG C
After managing 5min, enzyme activity is measured respectively, using the enzyme activity of untreated samples as 100%, calculates remnant enzyme activity.The results are shown in table below.
It can be seen that compared with wild type from the data in table, the heat resistance of 35 mutant provided by the invention has
It significantly improves, remnant enzyme activity improves 19.1-63.8% after 60 DEG C of processing 10min, and remnant enzyme activity improves after 65 DEG C of processing 5min
27.1-82.6%.So as to illustrate that above-mentioned mutation causes the heat resistance of glucose oxidase to be increased dramatically, than wild type more
Feed addictive is suitable as, is conducive to extensive use of the glucose oxidase in feed, wide market.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Qingdao Weilan Biology Group Co., Ltd.
<120>A kind of glucose oxidase mutant
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gaaaaggctc atgaattgtt gaacactaag ttggaacaat gggctgaaga agctgttgct 1140
agaggtggtt ttcataatac tactgctttg ttgattcaat acgaaaacta cagagactgg 1200
attgttaacc ataacgttgc ctattctgag ttgtttttgg acaccgctgg tgttgcttct 1260
tttgatgttt gggatttgtt gccatttaca agaggttacg ttcacatttt ggataaagat 1320
ccatacttgc atcactttgc atacgatcca caatactttt tgaacgaatt ggacttgttg 1380
ggtcaagctg ctgctactca attggctaga aacatttcta actctggtgc aatgcaaact 1440
tactttgccg gtgaaactat cccaggagat aacttggctt acgatgctga tttgtctgct 1500
tggactgaat acattccata ccatttcaga ccaaactacc acggtgtcgg tacttgttct 1560
atgatgccaa aggaaatggg aggtgttgtc gataacgctg caagagtcta cggagttcaa 1620
ggtttgagag ttattgatgg ttctattcca ccaactcaaa tgtcttctca tgttatgact 1680
gttttttacg ctatggcttt gaagatttct gatgctatct tggaagatta cgcttctatg 1740
caataa 1746
Claims (10)
1. a kind of glucose oxidase mutant, with any of amino acid sequence shown in (I), (II) or (III):
(I) with the amino acid sequence SEQ ID NO of glucose oxidase:1 has the sequence of at least 95% homology;
(II) there is at least one immune epitope of glucose oxidase described in (I), and the amino of the glucose oxidase
The amino acid sequence that acid sequence is obtained through modifying, replacing, lacking or adding one or several amino acid;
(III) by such as SEQ ID NO:When nucleotide sequence or its complementary series shown in 2 or the degeneracy because genetic code with such as
SEQ ID NO:The amino acid sequence of the different sequential coding of the nucleotide sequence of nucleotide sequence or its complementary series shown in 2
Row.
2. glucose oxidase mutant as described in claim 1, which is characterized in that described is substituted by 1 amino of substitution
Acid.
3. glucose oxidase mutant as claimed in claim 2, which is characterized in that the substitution includes amino acid sequence
For SEQ ID NO:The 387th of 1 glucose oxidase, 388,402,407,409,419,456,467,471,477,479,
Any one in 480,493,494,502,532,533,547,579 amino acids is replaced.
4. glucose oxidase mutant as claimed in claim 3, which is characterized in that the substitution includes T387A,
T387E, T387F, T387P, T388E, T388P, V402F, A407P, S409A, A419L, E456D, Q467K, N471E,
N471F, N471K, A477P, Q479K, T480D, A493P, Y494P, T502E, T502K, T502L, T502R, T502Y,
Any one in N532F, N532P, A533R, G547A, S579F, S579K, S579R, S579Y.
5. glucose oxidase mutant as described in claim 1, which is characterized in that the missing is lacks 1 or 2
Amino acid.
6. glucose oxidase mutant as claimed in claim 5, which is characterized in that the missing includes the 581st ammonia
Base acid lacks or the 580th and the 581st amino acids lack simultaneously.
7. encode the DNA molecular of claim 1-6 any one of them glucose oxidase mutant.
8. a kind of recombinant expression carrier, which is characterized in that the recombinant expression carrier carries the DNA described in claim 7
Molecule.
9. a kind of host cell, which is characterized in that the host cell includes recombinant expression according to any one of claims 8 and carries
Body.
10. application of the claim 1-6 any one of them glucose oxidase mutant in feed preparation.
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Cited By (3)
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CN114181916A (en) * | 2021-11-16 | 2022-03-15 | 华东师范大学 | Artificially modified enzyme based on glucose oxidase and expression application thereof |
CN115181734A (en) * | 2022-08-29 | 2022-10-14 | 上海茵肽信息科技有限公司 | Novel glucose oxidase with high thermal stability based on saturation mutation and composite evaluation design |
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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WO2015099112A1 (en) * | 2013-12-27 | 2015-07-02 | キッコーマン株式会社 | Flavin-binding glucose dehydrogenase exhibiting improved heat stability |
CN105950578A (en) * | 2016-07-06 | 2016-09-21 | 青岛红樱桃生物技术有限公司 | Heat-resisting glucose oxidase mutant as well as encoding gene and application thereof |
CN108118038B (en) * | 2016-11-28 | 2021-08-31 | 青岛蔚蓝生物集团有限公司 | Glucose oxidase mutant |
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WO2015099112A1 (en) * | 2013-12-27 | 2015-07-02 | キッコーマン株式会社 | Flavin-binding glucose dehydrogenase exhibiting improved heat stability |
CN105950578A (en) * | 2016-07-06 | 2016-09-21 | 青岛红樱桃生物技术有限公司 | Heat-resisting glucose oxidase mutant as well as encoding gene and application thereof |
CN108118038B (en) * | 2016-11-28 | 2021-08-31 | 青岛蔚蓝生物集团有限公司 | Glucose oxidase mutant |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN114181916A (en) * | 2021-11-16 | 2022-03-15 | 华东师范大学 | Artificially modified enzyme based on glucose oxidase and expression application thereof |
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 |
CN115181734A (en) * | 2022-08-29 | 2022-10-14 | 上海茵肽信息科技有限公司 | Novel glucose oxidase with high thermal stability based on saturation mutation and composite evaluation design |
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