CN109666657B - Glucose oxidase for improving heat resistance - Google Patents
Glucose oxidase for improving heat resistance Download PDFInfo
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- CN109666657B CN109666657B CN201710962735.6A CN201710962735A CN109666657B CN 109666657 B CN109666657 B CN 109666657B CN 201710962735 A CN201710962735 A CN 201710962735A CN 109666657 B CN109666657 B CN 109666657B
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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
The application relates to glucose oxidase with improved heat resistance, and the amino acid sequence is that glutamic acid at the 129 th position of the sequence number 2 is mutated into proline, and/or glutamic acid at the 243 rd position is mutated into valine.
Description
[ technical field ] A method for producing a semiconductor device
The present application relates to glucose oxidase, and more particularly, to glucose oxidase with improved heat resistance.
[ Prior Art ] A method for producing a semiconductor device
Glucose oxidase (Glucose oxidase) belongs to an oxidoreductase (EC 1.1.3.4). Can specifically catalyze the oxidation of beta-D-glucose under the aerobic condition to generate gluconic acid (gluconic acid) and simultaneously generate hydrogen peroxide (H)2O2). Glucose oxidationThe enzyme was first found to be present in an extract of Aspergillus niger (Aspergillus niger) in 1928 by Muller's laboratory. Glucose oxidase is widely found in animals, plants and microorganisms, and most of them, the oxidase derived from microorganisms, which mainly exists in Aspergillus niger and Penicillium spp. At present, most of industrial glucose oxidase is produced by using the two natural strains, but the production mode has the problems of low yield and low activity. In addition, they also produce many hetero proteins other than oxidase at the same time, resulting in difficulty in purification in subsequent processes, and ultimately, an increase in production cost. Therefore, in recent years, the production of oxidase by other microbial systems, especially the Pichia pastoris (Pichia pastoris) system, which is commonly used in industry, has been studied.
The application range of the glucose oxidase is very wide. In the food industry, glucose oxidase can be used as an antioxidant food preservative to keep freshness of vegetables, fruits and the like and maintain flavor of beer beverages. Glucose oxidase can also be used for improving the gluten degree of dough and improving the quality of bread. Glucose oxidase is also used in the medical field to detect the amount of glucose in blood. The textile industry utilizes hydrogen peroxide produced by glucose oxidase, which has a bleaching function, and can also be added to laundry detergents. In recent years, glucose oxidase has been more used in the feed industry. Glucose oxidase, because of its effects of reducing oxygen content, lowering the pH in the environment and producing hydrogen peroxide, can further inhibit the growth of certain bacteria or fungi. Therefore, the intestinal environment of animals can be improved by adding the glucose oxidase into the feed. In other respects, glucose oxidase has even the possibility of being used in bio-energy (biofuel). Thus, glucose oxidase has certain importance in various industrial applications. Therefore, studies on the improvement of the activity yield and properties of glucose oxidase have been increasing.
In many related studies, existing enzyme proteins are modified in order to obtain better enzymes, in addition to being screened in nature. The present application intends to improve the heat resistance of glucose oxidase by logically designing mutations, thereby increasing the value and potential of glucose oxidase in industrial applications.
[ summary of the invention ]
The application aims to modify the existing glucose oxidase, and the heat resistance of the glucose oxidase is effectively improved by utilizing structural analysis and point mutation technology, so that the industrial application value and potential of the glucose oxidase are increased.
To achieve the above objects, one of the broader embodiments of the present application provides a glucose oxidase, which has an amino acid sequence in which glutamic acid at the 129 th position of SEQ ID No. 2 is mutated to proline, and glutamic acid at the 243 rd position is mutated to valine.
In one embodiment, the gene encoding SEQ ID NO 2 is the AngOD gene isolated from Aspergillus niger.
In one embodiment, the amino acid sequence of the glucose oxidase is shown in SEQ ID No. 10.
Another broader embodiment of the present application provides a glucose oxidase having the amino acid sequence of the 129 th position of the amino acid sequence of SEQ ID NO. 2 mutated to proline.
In one embodiment, the gene encoding SEQ ID NO 2 is the AngOD gene isolated from Aspergillus niger.
In one embodiment, the amino acid sequence of the glucose oxidase is shown in seq id No. 6.
In yet another broad embodiment, the present application provides a glucose oxidase having the amino acid sequence of glutamic acid mutation at position 243 of SEQ ID No. 2 to valine.
In one embodiment, the gene encoding SEQ ID NO 2 is the AngOD gene isolated from Aspergillus niger.
In one embodiment, the amino acid sequence of the glucose oxidase is shown in SEQ ID No. 8.
[ description of the drawings ]
FIG. 1 shows the nucleotide sequence and amino acid sequence of wild-type glucose oxidase AngOD.
FIG. 2 shows the primer sequences used in the point mutation technique.
FIG. 3 shows the nucleotide and amino acid sequence of E129P mutant glucose oxidase.
FIG. 4 shows the nucleotide sequence and amino acid sequence of Q243V mutant glucose oxidase.
FIG. 5 shows the nucleotide and amino acid sequence of E129P/Q243V mutant glucose oxidase.
FIG. 6 shows the analysis of heat resistance of wild-type and mutant glucose oxidases.
[ embodiment ] A method for producing a semiconductor device
Some exemplary embodiments that embody features and advantages of the present application will be described in detail in the description that follows. It is to be understood that the present application is capable of various modifications without departing from the scope of the application, and that the description and drawings are to be taken as illustrative in nature and not as limiting the application.
The glucose oxidase enzyme (AngOD) of the present application is a gene isolated from the fungus Aspergillus niger strain. According to the prior related literature, the optimum activity of glucose oxidase is about 37 ℃ and pH 6. The glucose oxidase gene is constructed on a carrier and is delivered into industrial common Pichia pastoris (Pichia pastoris) to express the protein. In order to improve the heat resistance of the glucose oxidase, the protein structure of the glucose oxidase is further analyzed, amino acid with modification potential is selected, and then point-directed mutagenesis technology (site-directed mutagenesis) is utilized for modification.
The stability of the protein three-dimensional structure is greatly related to the heat resistance, and the hydrophobic acting force is one of the important factors influencing the protein stability. Therefore, the present application further analyzes the protein structure of glucose oxidase, and attempts to enhance the stability of the protein structure by increasing the hydrophobic interaction force, thereby improving the heat resistance. After analysis, 129 th amino acid glutamic acid (glutamate) positioned on a loop region (loop) and 243 th amino acid glutamic acid (glutamate) positioned on a secondary structure beta-plate (beta-sheet) are selected for transformation, a point mutation technology is utilized to perform single mutation to proline (proline) and valine (valine), and then the two mutation points are combined to form a double mutation point, so that the glucose oxidase with improved heat resistance is obtained.
The method of modifying glucose oxidase and the modified glucose oxidase obtained by the method are described in detail below.
FIG. 1 shows the nucleotide sequence and amino acid sequence of wild-type glucose oxidase AngOD. As shown in FIG. 1, the AngOD gene comprises 1749 bases (nucleotide sequence is denoted by SEQ ID NO: 1) and 583 amino acids (amino acid sequence is denoted by SEQ ID NO: 2). First, the AngOD gene was constructed on a vector of pPICZ α A. Then transferring the linearized plasmid DNA into pichia pastoris, coating the transferred bacterial liquid on a YPD disk containing 0.1mg/ml zeocin antibiotic, culturing for 2 days at 30 ℃, and screening the successfully transformed transferred yeast cells. Then selecting colonies, inoculating the colonies into YPD culture medium for culture proliferation, further separating thalli, transferring the thalli into BMMY culture medium containing 0.5% methanol, and further inducing the expression of target protein. The resulting suspension was separated by centrifugation, and the supernatant containing the target protein expressed and secreted outside the cells was collected.
The three mutant genes of AngOD are obtained by point mutation technology, and polymerase chain reaction is performed using wild-type AngOD gene as template, wherein the mutation primers used in the method are shown in FIG. 2, wherein E129P means that the 129 th amino acid of AngOD is mutated from glutamic acid (glutamate) to proline (proline), the E129P mutation primer sequence is shown in SEQ ID No. 3, the Q243V is that the 243 th amino acid is mutated from glutamic acid (glutamate) to valine (valine), and the Q243V mutation primer sequence is shown in SEQ ID No. 354. Therefore, the three mutant genes of AngOD obtained by the point mutation technology are E129P, Q243V and E129P/Q243V.
FIGS. 3 to 5 show the nucleotide and amino acid sequences of three mutants constructed according to the present invention. FIG. 3 shows the nucleotide sequence and amino acid sequence of E129P mutant glucose oxidase, wherein the nucleotide sequence is shown in SEQ ID No. 5, the amino acid sequence is shown in SEQ ID No. 6, and the 129 amino acid position is mutated from glutamic acid (glutamate) to proline (proline). FIG. 4 shows the nucleotide sequence and amino acid sequence of Q243V mutant glucose oxidase, wherein the nucleotide sequence is labeled with SEQ ID No. 7, the amino acid sequence is labeled with SEQ ID No. 8, and the amino acid at position 243 is mutated from glutamine (glutamine) to valine (valine). FIG. 5 shows the nucleotide sequence and amino acid sequence of E129P/Q243V mutant glucose oxidase, wherein the nucleotide sequence is shown as SEQ ID No. 9, the amino acid sequence is shown as SEQ ID No. 10, and the 129 amino acid position is mutated from glutamic acid (glutamate) to proline (proline), and the 243 amino acid position is mutated from glutamic acid (glutamate) to valine (valine).
The original DNA template was then removed using the DpnI enzyme. The three mutant genes are respectively sent into escherichia coli for replication and amplification, and the success of the mutant sequence is confirmed by DNA sequencing. Finally, the three mutant genes are respectively sent into pichia pastoris to express the mutant proteins, and the steps are the same as the steps. Then, the wild-type protein and the mutant protein were subjected to glucose oxidase activity measurement and heat resistance analysis, respectively.
The activity of glucose oxidase is determined by oxidizing glucose with the enzyme to generate hydrogen peroxide, reacting o-dianisidine color-developing agent with hydrogen peroxide under the catalysis of catalase (catalase), and changing the color after oxidation, thereby calculating the activity of glucose oxidase. Approximately, 2.5ml of o-dianisidine, 0.3ml of 18% glucose and 0.1ml of catalase (90 units/ml) were mixed and then placed in a 37 ℃ water bath to preheat. 0.1ml of an appropriately diluted enzyme solution was added thereto, and after reacting for 3min, 2ml of sulfuric acid was added thereto to terminate the reaction. Finally, the absorbance of the glucose oxidase was measured at OD540nm wavelength to calculate the activity of the glucose oxidase.
For protein thermostability analysis, the amounts of wild-type protein and mutant protein were quantified and then heat-treated at 64 ℃, 66 ℃, 68 ℃ and 70 ℃ for 2 min. Then cooling on ice for 5min, and standing at room temperature for 5min for recovery. Finally, the enzyme activity of the samples not subjected to the heat treatment (100% as a control group) and the enzyme activity remaining in the heat-treated protein samples were measured at the same time.
FIG. 6 shows the analysis of heat resistance of wild-type and mutant glucose oxidases. As shown in FIG. 6, the heat resistance of the three mutant proteins E129P, Q243V and E129P/Q243V was higher than that of the wild-type protein under different heat treatment temperatures (64 ℃ -70 ℃). Taking the results of the heat treatment at 68 ℃ as an example: the residual activity of the wild-type protein remained 43.7%, while the residual activities of the mutant proteins E129P and Q243V were 49.8% and 50.2%, respectively, and the residual activity of the double mutant protein E129P/Q243V, which binds the two mutations, was 55.2%. Thus, the single mutation points E129P and Q243V successfully improved the heat resistance of AngOD. In addition, the heat resistance of at least one of the mutants is further improved by combining the two mutations.
In conclusion, in order to improve the heat resistance of the glucose oxidase AngOD, the protein structure of the glucose oxidase AngOD is further analyzed, and amino acid with modification potential is selected and reasonably modified. The results show that the heat resistance of the three mutant proteins E129P, Q243V and E129P/Q243V which are modified are all higher than that of the wild type protein. Therefore, the application successfully improves the heat resistance of the glucose oxidase AngOD, and further increases the industrial application value of the glucose oxidase and the possibility of expanding the industrial application range of the glucose oxidase.
While the present invention has been described in detail with respect to the above embodiments, it will be apparent to those skilled in the art that various modifications can be made without departing from the scope of the invention as defined in the appended claims.
Sequence listing
<110> Dongguan Panya Tai Biotech Co., Ltd
<120> glucose oxidase for improving thermal endurance
<160> 10
<170> PatentIn version 3.5
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<213> Artificial sequence
<220>
<223> mutant
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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 (3)
1. A glucose oxidase, whose amino acid sequence is an amino acid sequence obtained by mutating glutamic acid at position 129 of SEQ ID No. 2 to proline and mutating glutamic acid at position 243 to valine.
2. A glucose oxidase, wherein the amino acid sequence of the glucose oxidase is an amino acid sequence obtained by mutating the 129 th position of glutamic acid in the sequence number 2 into proline.
3. A glucose oxidase, wherein the amino acid sequence of the glucose oxidase is an amino acid sequence obtained by mutating the glutamic acid at the 243 position of SEQ ID No. 2 to valine.
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| 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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| 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 |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| 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 |
Non-Patent Citations (2)
| Title |
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| Increased thermostability of Asn182 → Ala mutant Aspergillus awamori glucoamylase;Reilly, Peter J.et al.;《Biotechnology and Bioengineering》;19941231;全文 * |
| 葡萄糖氧化酶在毕赤酵母中的高效表达和计算机辅助设计提高热稳定性;牟庆璇;《中国优秀博硕士学位论文全文数据库(硕士)基础科学辑》;20170515;全文 * |
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