CN109666657A - Promote the glucose oxidase of heat resistance - Google Patents
Promote the glucose oxidase of heat resistance Download PDFInfo
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- CN109666657A CN109666657A CN201710962735.6A CN201710962735A CN109666657A CN 109666657 A CN109666657 A CN 109666657A CN 201710962735 A CN201710962735 A CN 201710962735A CN 109666657 A CN109666657 A CN 109666657A
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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
- 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
- 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
About a kind of glucose oxidase for promoting heat resistance, amino acid sequence system is by the glutamic acid mutation of the 129th position of sequence number 2 into proline for the application system, and/or the paddy amic acid of the 243rd position is mutated into the amino acid sequence of valine.
Description
[technical field]
The application system is about a kind of glucose oxidase, espespecially a kind of glucose oxidase for promoting heat resistance.
[prior art]
Glucose oxidase (Glucose oxidase) belongs to a kind of oxidoreducing enzyme (EC 1.1.3.4).Aerobic
Under the conditions of, it can be catalyzed the oxidation of β-D-Glucose in specific manner, and then generate gluconic acid (gluconic acid), simultaneously
Generate hydrogen peroxide (H2O2).Glucose oxidase is in nineteen twenty-eight first by the laboratory of Muller in aspergillus niger
Its presence is found in the extract of (Aspergillus niger).Glucose oxidase be widely present in animal, plant and
Microorganism it is internal, be in the middle most with the correlative study of microbe-derived oxidizing ferment, and microbe-derived be primarily present in
Aspergillus niger and Penicillium (Penicillium spp.) strain.At present most of crude dextrose oxidizing ferment also with
Above two Natural strains production obtains, however such production method but has that yield and activity are relatively low.In addition,
They can also produce many foreign proteins other than oxidizing ferment simultaneously, cause the purifying of follow-up process to be not easy, ultimately cause life
Produce the raising of cost.Therefore, research and utilization others microflora also produces oxidizing ferment in recent years, especially industrial normal
Good progress is obtained in fungi Pichia pastoris (Pichia pastoris) system.
The application range of glucose oxidase is very extensive.In food industry, glucose oxidase can be used as anti-oxidant
Food preservative, for keep the freshnesses such as vegetables and fruits and maintain beer beverage flavor.Glucose oxidase, which is also used in, to be changed
The muscle degree of good dough, improves the quality of bread.In medical domain glucose in blood can also be detected with glucose oxidase
Content.Then using hydrogen peroxide caused by glucose oxidase in textile industry, have the function of to bleach (bleaching),
Also laundry detergents be may be added to that.In recent years, glucose oxidase is more used in feed industry.Due to glucose oxidase
Be reduced oxygen content, reduce the pH value in environment and generate the effect of hydrogen peroxide, thus can inhibit in turn certain bacteriums or
The growth of fungi.Therefore, addition glucose oxidase can improve the intestinal environment of animal inside feed.In other respects, Portugal
Grape carbohydrate oxidase is even also used in a possibility that in bioenergy (biofuel).It follows that glucose oxidase exists
Different industry all have certain importance using inner.Therefore, for the active yield of glucose oxidase and characteristic into
The research of row improvement is also increasing.
At present in many relevant researchs, more preferably enzyme in order to obtain, other than screening in nature, just
It is that existing zymoprotein is transformed.The application is intended to by logicality design mutation to promote the resistance to of glucose oxidase
It is hot, and then increase its value and potentiality in industrial application.
[summary of the invention]
The purpose of the application is that existing glucose oxidase is transformed, using structural analysis and point mutation technology, with effective
The heat resistance of glucose oxidase is promoted, and then increases the industrial application value and potentiality of glucose oxidase.
In order to achieve the above object, the broader embodiment of one of the application is to provide a kind of glucose oxidase, amino acid
Sequence system is by the glutamic acid mutation of the 129th position of sequence number 2 into proline, and by the paddy acyl ammonia of the 243rd position
Acid mutation at valine amino acid sequence.
In one embodiment, the gene line for encoding the sequence number 2 is separated from aspergillus niger (Aspergillus niger)
AnGOD gene out.
In one embodiment, the amino acid sequence of the glucose oxidase is as shown in sequence number 10.
Another broader embodiment of the application is to provide a kind of glucose oxidase, and amino acid sequence system is by sequence
The glutamic acid mutation of the 129th position of column number 2 at proline amino acid sequence.
In one embodiment, the gene line for encoding the sequence number 2 is separated from aspergillus niger (Aspergillus niger)
AnGOD gene out.
In one embodiment, the amino acid sequence of the glucose oxidase is as shown in sequence number 6.
The another broader embodiment of the application is to provide a kind of glucose oxidase, and amino acid sequence system is by sequence
The paddy amic acid of the 243rd position of column number 2 is mutated into the amino acid sequence of valine.
In one embodiment, the gene line for encoding the sequence number 2 is separated from aspergillus niger (Aspergillus niger)
AnGOD gene out.
In one embodiment, the amino acid sequence of the glucose oxidase is as shown in sequence number 8.
[Detailed description of the invention]
1st figure shows the nucleotide sequence and amino acid sequence of wild type glucose oxidase AnGOD.
2nd figure shows primer sequence used by point mutation technology.
3rd figure shows the nucleotide sequence and amino acid sequence of E129P saltant type glucose oxidase.
4th figure shows the nucleotide sequence and amino acid sequence of Q243V saltant type glucose oxidase.
5th figure shows the nucleotide sequence and amino acid sequence of E129P/Q243V saltant type glucose oxidase.
6th figure shows the Analysis of Heat Tolerance of wild type and saltant type glucose oxidase.
[embodiment]
The some exemplary embodiments for embodying the application features and advantages will describe in detail in the explanation of back segment.It should be understood that
It is that the application there can be various variations in different aspects, does not all depart from scope of the present application, and explanation therein
And schema is to be illustrated as being used in itself, rather than to limit the application.
The glucose oxidase (AnGOD) of the application is the institute from fungus Aspergillus niger (Aspergillus niger) bacterial strain
The gene separated.It is pointed out according to previous pertinent literature, the optimal activity of glucose oxidase is about at 37 DEG C and pH 6
Under condition.The application constructs this glucose oxidase gene on carrier, and is sent into the common Pichia pastoris of industry
Its albumen is expressed in (Pichia pastoris).In order to promote the heat resistance of this glucose oxidase, the application is further analyzed
Its protein structure is selected the amino acid of tool transformation potentiality, is recycled point mutation technology (site-directed mutagenesis)
It is transformed.
The stability of protein steric structure and its heat resistance have very big association, and hydrophobicity active force is to influence albumen
One of an important factor for stability.Therefore, the application further analyzes the protein structure of glucose oxidase, it is intended to by increase
Hydrophobicity active force promotes its heat resistance to reinforce the stability of protein structure.Position is picked out after analyzing in ring-shaped area
(loop) glutamic acid (glutamate) of the 129th amino acid and it is located on secondary structure β-plate (β-sheet) the on
The paddy amic acid (glutamine) of 243 amino acid is transformed, single respectively to be mutated into proline using point mutation technology
(proline) and valine (valine), more catastrophe points in pairs two catastrophe points are combined thereafter, and obtaining the application has
Promote the glucose oxidase of heat resistance.
Will be described below the application be transformed glucose oxidase method and its acquired improvement glucose oxidase.
1st figure shows the nucleotide sequence and amino acid sequence of wild type glucose oxidase AnGOD.Such as the 1st figure institute
Show, AnGOD gene includes 1749 bases (nucleotide sequence is with the mark of sequence number 1) and 583 amino acid (amino acid sequences
With the mark of sequence number 2).Firstly, constructing AnGOD gene on the carrier of pPICZ α A.Then by the plasmid after linearisation
DNA turns to grow in Pichia pastoris, then the bacterium solution after turning to grow is applied on the YPD disk containing 0.1mg/ml zeocin antibiotic,
Cultivated 2 days in 30 DEG C, filter out successful conversion turn grow yeast cells.Choosing colony later is inoculated into individually in YPD culture medium
Culture hyperplasia is carried out, yet further separates thallus, is transferred in the BMMY culture medium containing 0.5% methanol, in turn
Induce the expression of target protein.Its bacterium solution is separated via centrifugation, collection, which contains, to be expressed and secrete to extracellular target
The supernatant of albumen.
Three kinds of mutated genes of AnGOD then utilize point mutation technology to obtain, and are carried out with wild type AnGOD gene as template
Polymerase chain reaction, the mutant primer used in are listed in the 2nd figure, wherein E129P mean the 129th position AnGOD it
Amino acid is mutated into proline (proline) by glutamic acid (glutamate), and E129P mutant primer sequence is with sequence number 3
Mark, and the amino acid that Q243V is the 243rd position is mutated into valine (valine) by paddy amic acid (glutamine), and
Q243V mutant primer sequence is with the mark of sequence number 4.Therefore, the application obtains three kinds of mutation of AnGOD using point mutation technology
Gene is E129P, Q243V and E129P/Q243V respectively.
3rd figure to the 5th figure is the nucleotide and amino acid sequence for showing three kinds of saltant types constructed by the application.3rd figure
Show that the nucleotide sequence and amino acid sequence of E129P saltant type glucose oxidase, nucleotide sequence are compiled with sequence
Numbers 5 marks, amino acid sequence is with the mark of sequence number 6, and the amino acid of its 129th position is by glutamic acid (glutamate)
It is mutated into proline (proline).4th figure shows the nucleotide sequence and amino acid of Q243V saltant type glucose oxidase
Sequence, nucleotide sequence is with the mark of sequence number 7, and amino acid sequence is indicated with sequence number 8, and its 243rd position
Amino acid valine (valine) is mutated by paddy amic acid (glutamine).5th figure shows E129P/Q243V saltant type
The nucleotide sequence and amino acid sequence of glucose oxidase, nucleotide sequence is with the mark of sequence number 9, amino acid sequence
Column are with the mark of sequence number 10, and the amino acid of its 129th position is mutated into proline by glutamic acid (glutamate)
(proline) and the amino acid of the 243rd position by paddy amic acid (glutamine) is mutated into valine (valine).
Then, original DNA profiling is removed using DpnI enzyme.Three kinds of mutated genes are respectively fed in Escherichia coli multiple
System amplification, then by DNA sequencing, confirm the success or not of mutant nucleotide sequence.Finally, three kinds of mutated genes are respectively fed to Pichia pastoris
Middle its mutain of expression, such as abovementioned steps.And then to carry out grape respectively to wild-type protein and mutain glycoxidative
The determination of activity of enzyme and Analysis of Heat Tolerance.
The determination of activity of glucose oxidase is hydrogen peroxide to be generated while oxidizing glucose by this enzyme, then pass through
Under catalysis by catalase (horseradish peroxidase), dianisidine (o-dianisidine) color developing agent
Meeting and hydroperoxidation, and then change its colour generation after being oxidized, the activity of glucose oxidase is extrapolated whereby.Substantially and
Speech, after 2.5ml dianisidine, 18% glucose of 0.3ml and 0.1ml catalase (90unit/ml) three mixing,
It is put into 37 DEG C of water-baths and preheats.The enzyme solution that 0.1ml suitably diluted is added, after reacting 3min, adds 2ml sulfuric acid to terminate
Reaction.Its light absorption value is finally detected under OD540nm wavelength, and then calculates the activity of glucose oxidase.
In terms of as albumen Analysis of Heat Tolerance, after the protein content of wild-type protein and mutein is quantified, respectively
It is placed in 64 DEG C, 66 DEG C, 68 DEG C and 70 DEG C of environment, carries out heat treatment 2min.It is subsequently put on 5min cooling on ice, then is placed in
5min is replied at room temperature.Finally, measure simultaneously the sample not being heat-treated enzymatic activity (as a control group 100%) and
The heat treated remaining enzymatic activity of albumen sample institute.
6th figure shows the Analysis of Heat Tolerance of wild type and saltant type glucose oxidase.As shown in Fig. 6, different
Under the conditions of heat treatment temperature (64 DEG C -70 DEG C), the heat resistance of three kinds of muteins E129P, Q243V and E129P/Q243V
All it is higher than wild-type protein.By taking the result that 68 DEG C are heat-treated as an example: the residual activity of wild-type protein remaining 43.7%, and be mutated
The residual activity of type albumen E129P and Q243V are respectively 49.8% and 50.2%, double mutation eggs that two catastrophe points are combined
Then there are also 55.2% for the residual activity of white E129P/Q243V.It follows that single catastrophe point E129P and Q243V just can succeed
Promote the heat resistance of AnGOD.And after combining two catastrophe points, further improve at least one one-tenth of heat resistance.
In conclusion in order to promote the heat resistance of glucose oxidase AnGOD, the application further analyzes its albumen knot
Structure is selected the amino acid of tool transformation potentiality, is reasonably transformed.The results show that three kinds of muteins being transformed
The heat resistance of E129P, Q243V and E129P/Q243V are all higher than wild-type protein.Therefore, the application successfully promotes grape
The heat resistance of carbohydrate oxidase AnGOD also further increases the industrial application value of this glucose oxidase and expands its work
A possibility that industry application range.
Even if the present invention described in detail as above-described embodiment and can as be familiar with this those skilled in the art appoint apply craftsman think and be it is all as
Modification, it is so neither de- as attached claims are intended to Protector.
Sequence table
<110>Dongguan Fanyatai Biological Sci-Tech Co., Ltd.
<120>glucose oxidase of heat resistance is promoted
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Thr Ala Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu His His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr
485 490 495
Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu
565 570 575
Glu Asp Tyr Ala Ser Met Gln
580
<210> 7
<211> 1749
<212> DNA
<213>artificial sequence
<220>
<223>mutant
<400> 7
tctaacggaa tcgaggcttc tttgttgaca gaccctaaag acgtttctgg aagaactgtc 60
gattacatca ttgccggtgg tggtttgacc ggattgacaa ctgccgctag attgaccgaa 120
aatccaaata tctctgtttt ggtcatcgag tctggatctt acgaatctga cagaggacca 180
attatcgagg atttgaacgc atacggtgac atcttcggtt cttctgttga tcatgcatac 240
gaaacagttg aattggctac taacaatcaa acagctttga ttagatctgg taacggattg 300
ggtggttcta cattggtcaa cggaggtact tggactagac cacataaggc ccaggtcgat 360
tcttgggaaa ctgtcttcgg aaacgaaggt tggaattggg ataatgttgc tgcatattct 420
ttgcaggcag aaagagctag agccccaaac gctaagcaaa ttgctgctgg tcattacttt 480
aatgcttctt gtcatggagt taacggtact gttcatgccg gaccaagaga tacaggtgac 540
gattactctc caattgttaa agccttgatg tctgctgttg aggatagagg tgtccctact 600
aagaaagact ttggatgtgg tgacccacac ggtgtctcta tgttccctaa cacattgcat 660
gaggatcagg tcagatctga tgctgctaga gaatggttgt tgcctaatta ccaaagacca 720
aacttggttg ttttgactgg tcagtatgtt ggaaaggtct tgttgtctca aaacggaact 780
accccaagag ccgttggagt cgaatttggt actcataagg gtaacactca caacgtttat 840
gctaaacatg aagttttgtt ggcagctggt tctgcagttt ctccaactat cttggaatac 900
tctggtattg gtatgaaatc tattttggag ccattgggta ttgatactgt cgttgatttg 960
ccagttggat tgaatttgca agaccagaca accgctacag ttagatctag aattacttct 1020
gccggagcag gacaaggaca ggctgcctgg ttcgctactt ttaacgagac ttttggtgac 1080
tattctgaga aggcacatga gttgttgaat actaagttgg aacagtgggc tgaagaggct 1140
gtcgcaagag gtggattcca caacactaca gcattgttga ttcaatatga gaactacaga 1200
gactggattg tcaaccataa cgttgcttac tctgaattgt ttttggatac agccggtgtt 1260
gcatctttcg acgtctggga tttgttgcca ttcacaagag gatacgttca catcttggat 1320
aaggatccat acttgcacca tttcgcctat gatccacaat acttcttgaa cgaattggac 1380
ttgttgggtc aagccgctgc aactcagttg gctagaaaca tttctaattc tggagctatg 1440
caaacttatt tcgcaggtga aactattcct ggtgacaatt tggcatatga cgcagatttg 1500
tctgcatgga ctgaatacat tccataccat tttagaccta actatcatgg tgtcggaact 1560
tgttctatga tgcctaagga aatgggtgga gttgtcgata acgccgccag agtctacggt 1620
gttcaaggtt tgagagttat tgatggatct attccaccaa cacaaatgtc ttctcatgtt 1680
atgacagtct tctacgctat ggcattgaaa atctctgacg caattttgga agattacgct 1740
tctatgcag 1749
<210> 8
<211> 583
<212> PRT
<213>artificial sequence
<220>
<223>mutant
<400> 8
Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Asp Val Ser
1 5 10 15
Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu
20 25 30
Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro Asn Ile Ser Val Leu Val
35 40 45
Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Asn Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ser Cys His Gly Val Asn Gly Thr Val His Ala Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Phe Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
Asn Leu Val Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Gly Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Gly Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala
275 280 285
Ala Gly Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ala Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Asn His Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Ala Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu His His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr
485 490 495
Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu
565 570 575
Glu Asp Tyr Ala Ser Met Gln
580
<210> 9
<211> 1749
<212> DNA
<213>artificial sequence
<220>
<223>mutant
<400> 9
tctaacggaa tcgaggcttc tttgttgaca gaccctaaag acgtttctgg aagaactgtc 60
gattacatca ttgccggtgg tggtttgacc ggattgacaa ctgccgctag attgaccgaa 120
aatccaaata tctctgtttt ggtcatcgag tctggatctt acgaatctga cagaggacca 180
attatcgagg atttgaacgc atacggtgac atcttcggtt cttctgttga tcatgcatac 240
gaaacagttg aattggctac taacaatcaa acagctttga ttagatctgg taacggattg 300
ggtggttcta cattggtcaa cggaggtact tggactagac cacataaggc ccaggtcgat 360
tcttgggaaa ctgtcttcgg aaacccaggt tggaattggg ataatgttgc tgcatattct 420
ttgcaggcag aaagagctag agccccaaac gctaagcaaa ttgctgctgg tcattacttt 480
aatgcttctt gtcatggagt taacggtact gttcatgccg gaccaagaga tacaggtgac 540
gattactctc caattgttaa agccttgatg tctgctgttg aggatagagg tgtccctact 600
aagaaagact ttggatgtgg tgacccacac ggtgtctcta tgttccctaa cacattgcat 660
gaggatcagg tcagatctga tgctgctaga gaatggttgt tgcctaatta ccaaagacca 720
aacttggttg ttttgactgg tcagtatgtt ggaaaggtct tgttgtctca aaacggaact 780
accccaagag ccgttggagt cgaatttggt actcataagg gtaacactca caacgtttat 840
gctaaacatg aagttttgtt ggcagctggt tctgcagttt ctccaactat cttggaatac 900
tctggtattg gtatgaaatc tattttggag ccattgggta ttgatactgt cgttgatttg 960
ccagttggat tgaatttgca agaccagaca accgctacag ttagatctag aattacttct 1020
gccggagcag gacaaggaca ggctgcctgg ttcgctactt ttaacgagac ttttggtgac 1080
tattctgaga aggcacatga gttgttgaat actaagttgg aacagtgggc tgaagaggct 1140
gtcgcaagag gtggattcca caacactaca gcattgttga ttcaatatga gaactacaga 1200
gactggattg tcaaccataa cgttgcttac tctgaattgt ttttggatac agccggtgtt 1260
gcatctttcg acgtctggga tttgttgcca ttcacaagag gatacgttca catcttggat 1320
aaggatccat acttgcacca tttcgcctat gatccacaat acttcttgaa cgaattggac 1380
ttgttgggtc aagccgctgc aactcagttg gctagaaaca tttctaattc tggagctatg 1440
caaacttatt tcgcaggtga aactattcct ggtgacaatt tggcatatga cgcagatttg 1500
tctgcatgga ctgaatacat tccataccat tttagaccta actatcatgg tgtcggaact 1560
tgttctatga tgcctaagga aatgggtgga gttgtcgata acgccgccag agtctacggt 1620
gttcaaggtt tgagagttat tgatggatct attccaccaa cacaaatgtc ttctcatgtt 1680
atgacagtct tctacgctat ggcattgaaa atctctgacg caattttgga agattacgct 1740
tctatgcag 1749
<210> 10
<211> 583
<212> PRT
<213>artificial sequence
<220>
<223>mutant
<400> 10
Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Asp Val Ser
1 5 10 15
Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu
20 25 30
Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro Asn Ile Ser Val Leu Val
35 40 45
Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Pro Gly Trp Asn Trp Asp Asn Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ser Cys His Gly Val Asn Gly Thr Val His Ala Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Phe Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
Asn Leu Val Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Gly Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Gly Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala
275 280 285
Ala Gly Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ala Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Asn His Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Ala Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu His His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr
485 490 495
Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu
565 570 575
Glu Asp Tyr Ala Ser Met Gln
580
Claims (9)
1. a kind of glucose oxidase, amino acid sequence system is by the glutamic acid mutation of the 129th position of sequence number 2 into dried meat
Propylhomoserin, and the paddy amic acid of the 243rd position is mutated into the amino acid sequence of valine.
2. glucose oxidase described in claim 1, wherein encoding the gene line of the sequence number 2 from aspergillus niger
(Aspergillus niger) separated AnGOD gene come out.
3. glucose oxidase described in claim 1, the wherein amino acid sequence of the glucose oxidase such as sequence number 10
It is shown.
4. grape carbohydrate oxidase, amino acid sequence system be by the glutamic acid mutation of the 129th position of sequence number 2 at proline it
Amino acid sequence.
5. glucose oxidase described in claim 4, wherein encoding the gene line of the sequence number 2 from aspergillus niger
(Aspergillus niger) separated AnGOD gene come out.
6. glucose oxidase described in claim 4, the wherein amino acid sequence of the glucose oxidase such as 6 institute of sequence number
Show.
7. grape carbohydrate oxidase, amino acid sequence system is that the paddy amic acid of the 243rd position of sequence number 2 is mutated into valine
Amino acid sequence.
8. glucose oxidase described in claim 7, wherein encoding the gene line of the sequence number 2 from aspergillus niger
(Aspergillus niger) separated AnGOD gene come out.
9. glucose oxidase described in claim 7, the wherein amino acid sequence of the glucose oxidase such as 8 institute of sequence number
Show.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628738A (en) * | 2019-09-27 | 2019-12-31 | 华东理工大学 | Method for improving activity of glucose oxidase, mutant and application thereof |
| CN113874498A (en) * | 2019-05-31 | 2021-12-31 | 南京百斯杰生物工程有限公司 | Thermostable glucose oxidase |
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105950577A (en) * | 2016-07-06 | 2016-09-21 | 青岛红樱桃生物技术有限公司 | Glucose oxidase mutant with improved thermal stability as well as encoding genes and application thereof |
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2017
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105950577A (en) * | 2016-07-06 | 2016-09-21 | 青岛红樱桃生物技术有限公司 | Glucose oxidase mutant with improved thermal stability as well as encoding genes and application thereof |
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| REILLY, PETER J.ET AL.: "Increased thermostability of Asn182 → Ala mutant Aspergillus awamori glucoamylase", 《BIOTECHNOLOGY AND BIOENGINEERING》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113874498A (en) * | 2019-05-31 | 2021-12-31 | 南京百斯杰生物工程有限公司 | Thermostable glucose oxidase |
| CN110628738A (en) * | 2019-09-27 | 2019-12-31 | 华东理工大学 | Method for improving activity of glucose oxidase, mutant and application thereof |
| CN110628738B (en) * | 2019-09-27 | 2021-01-12 | 华东理工大学 | Method for improving activity of glucose oxidase, mutant and 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 |
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|---|---|
| CN109666657B (en) | 2022-01-21 |
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