CN112877306B - Super-heat-resistant glucose oxidase AtGOD and gene and application thereof - Google Patents
Super-heat-resistant glucose oxidase AtGOD and gene and application thereof Download PDFInfo
- Publication number
- CN112877306B CN112877306B CN202110464418.8A CN202110464418A CN112877306B CN 112877306 B CN112877306 B CN 112877306B CN 202110464418 A CN202110464418 A CN 202110464418A CN 112877306 B CN112877306 B CN 112877306B
- Authority
- CN
- China
- Prior art keywords
- glucose oxidase
- ala
- god
- leu
- gly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
-
- 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
-
- 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)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Nutrition Science (AREA)
- Biophysics (AREA)
- Animal Husbandry (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to the field of genetic engineering, in particular to a super heat-resistant glucose oxidaseAtGOD and gene and application thereof. The residual enzyme activity of the glucose oxidase reaches 90% after being treated for 2min at 90 ℃, and the residual enzyme activity reaches 60% after being treated for 2min at 100 ℃. The glucose oxidase of the invention has the property of super heat stability, and can meet the application requirements of industries such as feed, food, medicine, textile and the like.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a super heat-resistant glucose oxidaseAtGOD and gene and application thereof.
Background
Glucose oxidase (e.c. 1.1.3.4) is widely derived from plants and microorganisms. The microorganisms are various and easy to culture, so the microorganisms are the main source of glucose oxidase. It is reported in the literature that glucose oxidase is mostly derived from fungi, mainly from aspergillus and penicillium. In addition to the microbial source, the enzyme activity of glucose oxidase can be detected in saliva secreted by bees, oriental tobacco budworms, cotton bollworms, beet armyworms and pelargonium armyworms and epidermal cuticles of grasshoppers, and the enzyme can play a role in resisting harmful fungi and bacteria.
Glucose oxidase is a Flavin Adenine Dinucleotide (FAD) dependent oxidase, can specifically catalyze beta-D-glucose, and hardly has other saccharidesThe catalytic activity is mostly homodimer, and the molecular mass is about 160 kDa. Glucose oxidase is easily soluble in water, not easily soluble in organic reagents such as glycerol, butanol, chloroform, pyridine, diethyl ether, etc., and can be absorbed by Cu2+、Hg2+、Ag+And inhibition of reagents such as hydrazine. Most glucose oxidases of fungal origin are reported to have a pH optimum in the neutral (5.0-7.0) range, such as from Aspergillus niger (A. niger)A. niger) The glucose oxidase has an optimum pH of 5.0, and is derived from Penicillium chrysogenum (A)P. chrysogenum) The most suitable pH of the glucose oxidase is 6.0; the optimal reaction temperature range is 25-60 ℃, and the optimal reaction temperature range is derived from Aspergillus niger (A), (B), (C)A. niger) Is at an optimum temperature of 40 ℃ and is derived from penicillium (A)P. funiculosum) The glucose oxidase has a lower optimal temperature of 25-30 ℃. At present, the glucose oxidase gene which is commercially exploited and utilized is mainly derived from Aspergillus niger (A. niger)A. niger) And Penicillium Nakazakii (P. amagasakiense) Two kinds. The comparison of the enzymatic properties of the two glucose oxidases shows that the glucose oxidase from the Aspergillus niger has wider pH reaction range and better stability than the glucose oxidase from the Penicillium, and the glucose oxidase from the Penicillium has high substrate specificity and high catalytic efficiency. Process for preparing glucose oxidase from Aspergillus nigerK mThe value of the concentration of the active ingredient was 29 mM,k catis 190 s-1Process for producing glucose oxidase derived from penicillium nilazazakiiK mThe value was 6.2 mM of the total,k catto 2003 s-1. Although the stability of the glucose oxidase is generally low, the glucose oxidase is better in stability after immobilization, and the glucose oxidase can be stored at 0 ℃ for at least two years and can be stabilized at-15 ℃ for 8 years.
The glucose oxidase is used as a novel feeding enzyme preparation, and has very wide application prospect in animal feed. The glucose oxidase for feeding has good thermal stability, so that the heat-resistant requirement in the feed granulating process and the acid-resistant requirement for playing a function in the gastric juice environment of animals can be met. The added substances such as various vitamins, carotene, grease and the like in the feed are easily oxidized by oxygen in the air, so that the nutritional value and the palatability in the feed are reduced, even the feed is rancid and deteriorated, and peroxide harmful to animals is generated. The safe and efficient oxidant is added into the feed, so that the adverse effect of the oxidant can be effectively avoided, the utilization rate of the feed is improved, and the health of animal organisms is protected. Glucose oxidase as an antioxidant is a novel feed additive for replacing antibiotics, and belongs to one of 12 allowable feed additives.
Disclosure of Invention
The invention aims to provide a super heat-resistant glucose oxidaseAtGOD。
It is still another object of the present invention to provide a gene encoding the above-mentioned glucose oxidaseAtgod。
It is still another object of the present invention to provide a recombinant microorganism comprising the above-mentioned glucose oxidase geneAtgodThe recombinant vector of (1).
It is still another object of the present invention to provide a recombinant microorganism comprising the above-mentioned glucose oxidase geneAtgodThe recombinant strain of (1).
Still another object of the present invention is to provide a genetic engineering method for preparing glucose oxidase.
The invention further aims to provide application of the glucose oxidase.
According to a particular embodiment of the invention, the Aspergillus terreus is selected fromAspergillus terreusSeparating to obtain the super heat-resistant glucose oxidaseAtGOD, the amino acid sequence of which is shown as SEQ ID NO:1 is shown. The enzyme comprises 594 amino acids and does not contain a signal peptide sequence.
The invention provides a coding gene for coding the glucose oxidaseAtgodSpecifically, the genome sequence of the gene is shown as SEQ ID NO. 2.
The invention provides a gene containing the glucose oxidaseAtgodPreferably pPIC9-atgod。
The invention also provides glucose oxidase containing the glucose oxidaseAtThe recombinant strain of GOD gene is preferably yeast, Escherichia coli, Bacillus, Lactobacillus, and filamentous fungi.
Wherein, the host cell is preferably pichia pastoris, and the recombinant Escherichia coli expression plasmid is preferably transformed into the pichia pastorisGS115 cell, the recombinant strain GS 115-atgod。
According to an embodiment of the present invention, a hyperthermostable glucose oxidase is preparedAtMethod for GOD, comprising the steps of:
(1) using glucose oxidase containing super heat-resisting propertyAtTransforming host cells by the recombinant vector of the GOD gene to obtain a recombinant strain;
(2) culturing recombinant strain, inducing recombinant glucose oxidaseAtGOD expression;
(3) recovering and purifying the expressed glucose oxidaseAtGOD。
The invention also provides a method for coding the hyperthermostable glucose oxidaseAtGOD geneAtgod。
The invention separates and clones the hyperthermostable glucose oxidase gene by a PCR methodAtgodDNA complete sequence analysis result shows that the hyperthermostable glucose oxidase geneAtgodThe total length is 1785 bp, coding 594 amino acids and a stop codon, and no signal peptide sequence. The theoretical molecular weight of the protein is 65.8 kDa, and the isoelectric point is 5.68. The comparison result in GenBank database shows that the hyperthermostable glucose oxidaseAtGOD is a novel glucose oxidase.
The invention also provides a super heat-resistant glucose oxidase containing the super heat-resistant glucose oxidaseAtThe recombinant vector of the GOD gene is preferably pPIC9-atgod. The heat-resistant glucose oxidase of the inventionAtThe GOD gene is inserted between appropriate restriction sites of an expression vector, so that the nucleotide sequence is operably linked with an expression control sequence. As a most preferred embodiment of the present invention, it is preferred that thermostable glucose oxidase is usedAtGOD Gene insertion into plasmid pPIC9EcoRI andNoti restriction enzyme sites to obtain a recombinant expression plasmid pPIC9-atgod. The theoretical molecular weight of this glucose oxidase is 65.8 kDa. The hyperthermostable glucose oxidaseAtAfter heterologous expression and purification of the GOD by using pichia pastoris, the specific activity is 85.0U/mg.
The invention also provides the super heat-resistant glucose oxidaseAtApplication of GOD.
The invention aims to overcome the defects of the prior art and provide a novel glucose oxidase which has excellent properties and is suitable for being applied to the industries of feed, food, medicine and the like. The invention obtains the super heat-resistant glucose oxidaseAtThe optimum temperature of GOD is 30 ℃, the optimum pH value is 6.0, the residual enzyme activity after 2min treatment at 90 ℃ is 90%, and the residual enzyme activity after 2min treatment at 100 ℃ reaches 60%, which is obviously superior to the currently reported enzymological performance. The glucose oxidase has only three sites of different amino acid sequences with the glucose oxidase of the application, the enzyme activity is 52.8U/mg under the conditions of pH 6.0 and 30 ℃, and the enzyme activity is higher than that of the super heat-resistant glucose oxidase of the inventionAtThe enzyme activity of GOD is reduced by 37.9 percent, the residual enzyme activity after 2min treatment at 90 ℃ is 51.6 percent, the residual enzyme activity after 2min treatment at 100 ℃ is 16.0 percent, and the enzyme activity is obviously lower than that of the super heat-resistant glucose oxidaseAtThermal stability of GOD. These properties meet the application requirements of industries such as feed, food, medicine and the like. For example, in the feed industry, the feed can resist the high-temperature condition of feed granulation, meet the digestive physiological characteristics of livestock and poultry, improve the digestive energy and metabolic energy of the feed, reduce the formula cost and reduce the environmental pollution.
Drawings
FIG. 1 shows the results of specific activity analysis of glucose oxidase of the present invention and glucose oxidase whose sequence is similar thereto;
FIG. 2 shows the results of analysis of the thermal stability at 90 ℃ of the glucose oxidase of the present invention and glucose oxidase having an approximate sequence thereof;
FIG. 3 shows the results of the thermal stability analysis of glucose oxidase of the present invention and glucose oxidase having an approximate sequence at 100 ℃;
FIG. 4 shows hyperthermostable glucose oxidase of the present applicationAtGOD and glucose oxidaseAcGOD sequence alignment results.
Detailed Description
Test materials and reagents
1. Bacterial strain and carrier: pichia pastoris (Pichia pastorisGS115), Pichia pastoris expression vector pPIC9 and strain GS 115.
2. Enzymes and other biochemical reagents: ligase was purchased from Invitrogen, site-directed mutagenesis kit was purchased from allgold, and others were made-by-home reagents (all available from general Biochemical reagents).
3. Culture medium:
(1) coli culture medium LB (1% peptone, 0.5% yeast powder, 1% NaCl, pH7. O).
(2) BMGY medium; 1% yeast powder, 2% peptone, 1.34% YNB, 0.000049< Biotin, 1% glycerol (v/v).
(3) BMMY medium: glycerol was replaced by 0.5% methanol, and the balance was BMGY.
Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.
Example 1 hyperthermostable glucose oxidaseAtCloning of GOD encoding Gene
Extracting Aspergillus terreusAspergillus terreusGenomic DNA and total RNA of (a). The amplification primers were synthesized based on the sequence designEcoRI F/NotI R：
EcoRI F： SEQ ID NO:3，NotI R：SEQ ID NO:4。
PCR amplification was performed using the genomic DNA and total RNA as templates, respectively. The touchdown PCR reaction parameters were: denaturation at 95 deg.C for 5 min; denaturation at 94 ℃ for 30 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 5 min, 25 cycles, and incubation at 4 ℃ for 10 min. An about 1800 bp fragment was obtained, recovered and ligated with pEazy-T3 vector and sent to Huada Gene sequencer for sequencing.
Example 2 hyperthermostable glucose oxidaseAtConstruction of GOD engineered Strain
The expression vector pPIC9 was subjected to double digestion (EcoRI+NotI) Simultaneously encoding gene of hyperthermostable glucose oxidaseAtGOD double enzyme digestion: (EcoRI+NotI) Connecting the enzyme-cut glucose oxidase gene fragment with an expression vector pPIC9 to obtain the enzyme-cut glucose oxidase containing super heat resistanceAtRecombinant plasmid of GOD genepPIC9-atgodAnd transforming Pichia pastoris GS115 to obtain a recombinant Pichia pastoris strain GS115atgod。
Example 3 preparation of hyperthermostable recombinant glucose oxidase
(1) Large-scale expression of glucose oxidase in shake flask level in pichia pastoris
Screening out transformants with good thermal stability and high enzyme activity, inoculating the transformants into a 1L triangular flask of 300 mL BMGY liquid medium, and carrying out shaking culture on a shaking table at 30 ℃ and 220 rpm for 48 h; centrifuging at 4500 rpm for 5 min, removing supernatant, adding 200 mL BMMY liquid culture medium containing 0.5% methanol into thallus, and performing induction culture at 30 deg.C and 220 rpm for 48 h. During the induction culture period, the methanol solution is replenished once at intervals of 24 hours to compensate the loss of methanol, so that the concentration of the methanol is kept at about 0.5 percent; centrifuging at 12000 Xg for 10 min, collecting supernatant fermentation liquid, detecting enzyme activity and performing SDS-PAGE protein electrophoresis analysis.
(2) Purification of hyperthermostable recombinant glucose oxidase
Collecting supernatant of the hyperthermostable recombinant glucose oxidase cultured by the shake flask fermentation, concentrating the fermentation broth using a 10 kDa membrane module while replacing the medium therein with a pH 6.510 mM disodium hydrogen phosphate-citric acid buffer solution, and then purifying by passing through an anion exchange column.
Example 4 determination of enzymatic Properties of purified glucose oxidase mutants
The method for determining the activity of the glucose oxidase GOD enzyme by adopting an o-dianisidine method comprises the following steps: at pH 6.0, 3mL of the reaction system included 2.5mL of o-dianisidine buffer (0.2 mL of 1% o-dianisidine in 25M of 0.1M phosphate buffer), 300. mu.L of 18% glucose solution, 100. mu.L of 0.03% horseradish peroxidase, and 100. mu.L of an appropriate dilution. After reaction at 30 ℃ for 3min, 2mL of 2M H was used2SO4The reaction was stopped and the absorbance was measured at 540 nm. 1 enzyme activity unit (U) is defined as the amount of enzyme required to produce 1. mu. mol of gluconic acid and hydrogen peroxide per unit time under given conditions.
Glucose oxidase for determining super heat resistanceAtEnzymatic activity and thermostability of GOD:
1. glucose oxidase for determining super heat resistanceAtEnzymatic activity of GOD. Hyperthermostable glucose oxidase purified in example 3AtGOD was enzymatically reacted at pH 6.0 and 30 ℃ to determine its enzymatic activity, as shown in FIG. 1, hyperthermostable glucose oxidaseAtThe enzyme activity of GOD is 85.0U/mg.
2. Glucose oxidase for determining super heat resistanceAtStability of GOD at 90 ℃ and 100 ℃:
hyperthermostable glucose oxidase in 0.1M citric acid-disodium hydrogen phosphate buffer (pH 6.0) buffer systemAtGOD was treated at 90 deg.C and 100 deg.C for 2min, and the relative residual enzyme activity was measured at 30 deg.C. As shown in FIG. 2, hyperthermostable glucose oxidaseAtThe residual enzyme activity of GOD after being treated for 2min at 90 ℃ is 90%. As shown in FIG. 3, hyperthermostable glucose oxidaseAtThe residual enzyme activity of GOD after being treated for 2min at 100 ℃ is 60%.
Example 5 recombinant glucose oxidaseAcDetermination of the Properties of GOD
Glucose oxidaseAcGOD, the amino acid sequence of which is shown as SEQ ID NO: 5, respectively. As shown in FIG. 4, the hyperthermostable glucose oxidase of the present inventionAtThe GOD has the amino acid sequence difference of only 3 amino acids, namely the 41th amino acid, the 126 th amino acid and the 520 th amino acid, and the properties of the two glucose oxidases are obviously influenced.
The glucose oxidaseAcThe enzyme activity of GOD at pH 6.0 and 30 ℃ is 52.8U/mg, which is more than the super heat-resistant glucose oxidase of the inventionAtThe enzyme activity of GOD is 37.9% lower (figure 1); the residual enzyme activity after being treated for 2min at 90 ℃ is 51.6 percent (figure 2), and the residual enzyme activity after being treated for 2min at 100 ℃ is 16.0 percent (figure 3), which are all obviously lower than the hyperthermostable glucose oxidase of the inventionAtThermal stability of GOD.
Three different amino acid functions between the two were analyzed separately.
(1) The super heat-resistant glucose oxidase of the inventionAtMutation of amino acid Val at position 41 of GOD into glucose oxidaseAcThr in GOD (mutant Val41 Thr);
(2) the super heat-resistant glucose of the inventionOxidase enzymeAtMutation of amino acid 126 Cys of GOD into glucose oxidaseAcGln in GOD (mutant Cys126 Gln);
(3) the super heat-resistant glucose oxidase of the inventionAtMutation of 520 th amino acid His of GOD into glucose oxidaseAcArg in GOD (mutant His520 Arg);
(4) the super heat-resistant glucose oxidase of the inventionAtMutation of Val41 and Cys126 amino acids of GOD into glucose oxidaseAcThr and Gln in GOD (mutant Val41Thr/Cys126 Gln);
(5) the super heat-resistant glucose oxidase of the inventionAtMutation of Val41 and His520 of GOD into glucose oxidaseAcThr and Arg in GOD (mutant Val41Thr/His520 Arg);
(6) the glucose oxidase of the invention with super heat resistanceAtMutation of Val 126 and His520 of GOD into glucose oxidaseAcGln and Arg in GOD (mutant Cys126Gln/His520 Arg).
As shown in figure 1, the enzyme activities of the mutants Val41Thr, Cys126Gln, His520Arg, Val41Thr/Cys126Gln, Val41Thr/His520Arg and Cys126Gln/His520Arg at pH 6.0 and 30 ℃ are respectively 68.4U/mg, 68.0U/mg, 52.8U/mg, 61.6U/mg, 68.4U/mg, 83.7U/mg and 51.3U/mg, and the hyperthermostable glucose oxidase of the inventionAtThe enzyme activity of GOD is 85.0U/mg.
As shown in figure 2, the residual enzyme activities of the mutant Val41Thr, Cys126Gln, His520Arg, Val41Thr/Cys126Gln, Val41Thr/His520Arg and Cys126Gln/His520Arg after being treated for 2min at 90 ℃ are 82.8%, 75.1%, 87.3%, 72.55%, 89.5% and 80.3%, respectively, and the hyperthermostable glucose oxidase of the present inventionAtThe residual enzyme activity of GOD after being treated for 2min at 90 ℃ is 90%.
As shown in figure 3, the residual enzyme activities of the mutants Val41Thr, Cys126Gln, His520Arg, Val41Thr/Cys126Gln, Val41Thr/His520Arg and Cys126Gln/His520Arg after being treated for 2min at 100 ℃ are respectively 25.6%, 35.2%, 48.7%, 17.6%, 39.6% and 48.1%, and the hyperthermostable glucose oxidase of the present inventionAtThe residual enzyme activity of GOD after being treated for 2min at 100 ℃ is 60 percent. Indicating that the three amino acid sites all have different degrees of influence on glucose oxidase.
Sequence listing
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> hyperthermostable glucose oxidase AtGOD, gene and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 594
<212> PRT
<213> Aspergillus terreus (Aspergillus terreus)
<400> 1
Ala Leu Pro Lys Phe Pro Arg Asp His Leu Gly Val Glu Pro Gln Leu
1 5 10 15
Leu Thr Asp Pro Thr Val Leu Ala Asn Thr Thr Val Asp Tyr Ile Ile
20 25 30
Ala Gly Gly Gly Leu Thr Gly Leu Val Val Ala Ala Arg Leu Thr Glu
35 40 45
Asp Pro Asn Ile Lys Val Leu Val Ile Glu Ser Gly Tyr Phe Glu Ser
50 55 60
Asn Arg Gly Pro Ile Ile Glu Asp Leu Asn Arg Tyr Gly Glu Ile Phe
65 70 75 80
Gly Thr Glu Val Asp His Ala Phe Glu Thr Val Gln Leu Ala Val Asn
85 90 95
Asn Arg Thr Glu Ile Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr
100 105 110
Leu Ile Asn Gly Gly Thr Trp Thr Arg Pro His Lys Val Cys Val Asp
115 120 125
Ser Trp Glu Thr Val Phe Gly Asn Gln Gly Trp Asn Trp Asp Asp Leu
130 135 140
Leu Pro Tyr Met Leu Lys Ile Glu Lys Ala Arg Pro Pro Asn Gln Arg
145 150 155 160
Gln Ile Glu Ala Gly His Tyr Phe Asn Pro Gln Cys His Gly Phe Asn
165 170 175
Gly Ser Val His Ala Gly Pro Arg Asp Thr Gly Glu Pro Tyr Ser Pro
180 185 190
Ile Met Arg Ala Leu Met Asp Thr Val Ser Ala Glu Gly Val Pro Val
195 200 205
Arg Lys Asp Leu Cys Cys Gly Asp Pro His Gly Val Ser Met Phe Leu
210 215 220
Asn Thr Leu Tyr Pro Ser Gln Ile Arg Ala Asp Ala Ala Arg Glu Tyr
225 230 235 240
Leu Val Pro Asn Tyr His Arg Pro Asn Phe Gln Val Leu Thr Gly Gln
245 250 255
Arg Val Gly Lys Val Leu Leu Asp Lys Thr Val Pro Gly Ser Pro Lys
260 265 270
Ala Ile Gly Val Glu Phe Gly Thr His Arg Thr Arg Lys Tyr Glu Ala
275 280 285
Tyr Ala Arg Arg Glu Val Leu Leu Ala Ala Gly Ser Thr Ile Ser Pro
290 295 300
Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys Ser Val Leu Asp Ser
305 310 315 320
Val Gly Ile Glu Gln Val Val Glu Leu Pro Val Gly Val Asn Leu Gln
325 330 335
Asp Gln Thr Thr Leu His Val Glu Ser Arg Ile Thr Pro Ala Gly Ala
340 345 350
Gly Gln Gly Gln Ala Ala Tyr Phe Ala Thr Phe Asn Glu Thr Phe Gly
355 360 365
Asp Phe Ala Pro Gln Ala His Glu Leu Leu Asn Thr Lys Leu Asp Gln
370 375 380
Trp Ala Glu Glu Val Val Ala Arg Gly Gly Phe His Asn Ala Thr Ala
385 390 395 400
Leu Arg Ile Gln Tyr Glu Asn Tyr Arg Asn Trp Leu Val Asn Asn Asn
405 410 415
Val Ala Phe Ser Glu Leu Phe Leu Asp Thr Ala Gly Lys Ile Ser Phe
420 425 430
Asp Val Trp Asp Leu Ile Pro Phe Thr Arg Gly Tyr Val His Ile Ala
435 440 445
Asp Lys Asp Pro Tyr Leu Arg Arg Leu Tyr Asn Asn Pro Gln Tyr Phe
450 455 460
Leu Asn Glu Leu Asp Val Leu Gly Glu Ala Ala Ala Ser Lys Leu Ala
465 470 475 480
Arg Glu Leu Ser Ser Lys Gly Ala Met Ala Gln Tyr Tyr Ala Gly Glu
485 490 495
Thr Val Pro Gly Phe Asp Gln Leu Pro Ala Asp Ala Ser Leu Arg Asp
500 505 510
Trp Ala Lys Tyr Val Lys Asp His Phe Arg Pro Asn Tyr His Ala Val
515 520 525
Ser Thr Cys Ala Met Met Ser Lys Glu Leu Gly Gly Val Val Asp Ser
530 535 540
Ala Ala Arg Val Tyr Asp Val Glu Arg Leu Arg Val Val Asp Gly Ser
545 550 555 560
Ile Pro Pro Thr Gln Val Ser Ser His Val Met Thr Val Phe Tyr Gly
565 570 575
Met Ala Glu Lys Ile Ala Glu Ala Ile Leu Gln Asp Tyr His Ala Arg
580 585 590
Lys Ala
<210> 2
<211> 1785
<212> DNA
<213> Aspergillus terreus (Aspergillus terreus)
<400> 2
gctttgccaa agttcccaag agatcacctg ggtgttgaac cacagttgtt gactgaccca 60
actgttttgg ctaacaccac cgtggactac attattgctg gtggtggttt gaccggtttg 120
gttgttgctg ctagattgac tgaggaccca aacatcaagg tcttggttat cgagtccggt 180
tacttcgagt ctaacagagg tccaatcatc gaggacttga acagatacgg tgagattttc 240
ggtactgagg ttgaccacgc tttcgagact gttcagttgg ccgttaacaa cagaaccgag 300
atcatcagat ccggtaacgg tcttggtggt tccactttga ttaacggtgg tacttggacc 360
agaccacaca aggtttgtgt tgactcttgg gagactgtgt tcggtaatca aggttggaac 420
tgggacgact tgttgcccta catgttgaag atcgagaagg ccagaccacc aaaccagaga 480
caaattgaag ctggtcacta cttcaaccca cagtgccatg gttttaacgg ttccgttcat 540
gctggtccta gagacactgg tgaaccatac tctccaatca tgagagcctt gatggacact 600
gtttctgctg agggtgttcc agtcagaaag gacttgtgtt gtggtgatcc acacggtgtc 660
tccatgttct tgaacactct gtacccatct cagatcagag cagacgctgc tagagagtac 720
ttggtcccaa actaccacag accaaacttc caggttttga ctggtcagag agttggtaag 780
gtcttgttgg acaagactgt cccaggttct ccaaaggcta ttggtgttga gttcggtact 840
cacagaacca gaaagtacga ggcttacgcc agaagagagg ttttgttggc tgctggttct 900
actatctccc caaccatctt ggaatactcc ggtatcggta tgaagtccgt tttggactcc 960
gttggtatcg agcaggttgt tgaattgcca gtcggtgtta acttgcagga ccaaactact 1020
ctgcacgtcg agtccagaat tactccagct ggtgctggtc aaggtcaagc tgcttacttt 1080
gctaccttca acgagacttt cggtgacttc gctccacaag ctcacgagtt gttgaacact 1140
aagttggacc aatgggccga agaggttgtt gctagaggtg gttttcataa cgctaccgcc 1200
ttgagaatcc agtacgagaa ctacagaaac tggctggtca acaacaacgt tgccttctcc 1260
gagttgttct tggataccgc tggtaagatt tccttcgacg tctgggactt gatcccattc 1320
actagaggtt acgttcacat tgccgacaag gacccatact tgagaaggct gtacaacaac 1380
cctcagtact tcttgaacga gttggacgtt ttgggtgaag ctgctgcttc taagttggcc 1440
agagaattgt cctctaaggg tgctatggct cagtactacg ctggtgaaac tgttcctggt 1500
ttcgaccaat tgccagctga cgcttctttg agagattggg ctaagtacgt caaggaccac 1560
ttcagaccta actaccacgc tgtttccacc tgtgccatga tgtccaaaga acttggtggt 1620
gttgttgatt ccgccgccag agtttacgac gtcgaaagat tgagagtcgt ggacggttct 1680
attccaccaa ctcaagtttc ttcccacgtc atgaccgttt tctacggtat ggctgagaag 1740
atcgctgagg ctatcttgca ggattaccac gctagaaagg cgtaa 1785
<210> 3
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctgaattcgc tttgccaaag ttcccaag 28
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tagcggccgc ttacgccttt ctagcgtggt 30
<210> 5
<211> 594
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Ala Leu Pro Lys Phe Pro Arg Asp His Leu Gly Val Glu Pro Gln Leu
1 5 10 15
Leu Thr Asp Pro Thr Val Leu Ala Asn Thr Thr Val Asp Tyr Ile Ile
20 25 30
Ala Gly Gly Gly Leu Thr Gly Leu Thr Val Ala Ala Arg Leu Thr Glu
35 40 45
Asp Pro Asn Ile Lys Val Leu Val Ile Glu Ser Gly Tyr Phe Glu Ser
50 55 60
Asn Arg Gly Pro Ile Ile Glu Asp Leu Asn Arg Tyr Gly Glu Ile Phe
65 70 75 80
Gly Thr Glu Val Asp His Ala Phe Glu Thr Val Gln Leu Ala Val Asn
85 90 95
Asn Arg Thr Glu Ile Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr
100 105 110
Leu Ile Asn Gly Gly Thr Trp Thr Arg Pro His Lys Val Gln Val Asp
115 120 125
Ser Trp Glu Thr Val Phe Gly Asn Gln Gly Trp Asn Trp Asp Asp Leu
130 135 140
Leu Pro Tyr Met Leu Lys Ile Glu Lys Ala Arg Pro Pro Asn Gln Arg
145 150 155 160
Gln Ile Glu Ala Gly His Tyr Phe Asn Pro Gln Cys His Gly Phe Asn
165 170 175
Gly Ser Val His Ala Gly Pro Arg Asp Thr Gly Glu Pro Tyr Ser Pro
180 185 190
Ile Met Arg Ala Leu Met Asp Thr Val Ser Ala Glu Gly Val Pro Val
195 200 205
Arg Lys Asp Leu Cys Cys Gly Asp Pro His Gly Val Ser Met Phe Leu
210 215 220
Asn Thr Leu Tyr Pro Ser Gln Ile Arg Ala Asp Ala Ala Arg Glu Tyr
225 230 235 240
Leu Val Pro Asn Tyr His Arg Pro Asn Phe Gln Val Leu Thr Gly Gln
245 250 255
Arg Val Gly Lys Val Leu Leu Asp Lys Thr Val Pro Gly Ser Pro Lys
260 265 270
Ala Ile Gly Val Glu Phe Gly Thr His Arg Thr Arg Lys Tyr Glu Ala
275 280 285
Tyr Ala Arg Arg Glu Val Leu Leu Ala Ala Gly Ser Thr Ile Ser Pro
290 295 300
Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys Ser Val Leu Asp Ser
305 310 315 320
Val Gly Ile Glu Gln Val Val Glu Leu Pro Val Gly Val Asn Leu Gln
325 330 335
Asp Gln Thr Thr Leu His Val Glu Ser Arg Ile Thr Pro Ala Gly Ala
340 345 350
Gly Gln Gly Gln Ala Ala Tyr Phe Ala Thr Phe Asn Glu Thr Phe Gly
355 360 365
Asp Phe Ala Pro Gln Ala His Glu Leu Leu Asn Thr Lys Leu Asp Gln
370 375 380
Trp Ala Glu Glu Val Val Ala Arg Gly Gly Phe His Asn Ala Thr Ala
385 390 395 400
Leu Arg Ile Gln Tyr Glu Asn Tyr Arg Asn Trp Leu Val Asn Asn Asn
405 410 415
Val Ala Phe Ser Glu Leu Phe Leu Asp Thr Ala Gly Lys Ile Ser Phe
420 425 430
Asp Val Trp Asp Leu Ile Pro Phe Thr Arg Gly Tyr Val His Ile Ala
435 440 445
Asp Lys Asp Pro Tyr Leu Arg Arg Leu Tyr Asn Asn Pro Gln Tyr Phe
450 455 460
Leu Asn Glu Leu Asp Val Leu Gly Glu Ala Ala Ala Ser Lys Leu Ala
465 470 475 480
Arg Glu Leu Ser Ser Lys Gly Ala Met Ala Gln Tyr Tyr Ala Gly Glu
485 490 495
Thr Val Pro Gly Phe Asp Gln Leu Pro Ala Asp Ala Ser Leu Arg Asp
500 505 510
Trp Ala Lys Tyr Val Lys Asp Arg Phe Arg Pro Asn Tyr His Ala Val
515 520 525
Ser Thr Cys Ala Met Met Ser Lys Glu Leu Gly Gly Val Val Asp Ser
530 535 540
Ala Ala Arg Val Tyr Asp Val Glu Arg Leu Arg Val Val Asp Gly Ser
545 550 555 560
Ile Pro Pro Thr Gln Val Ser Ser His Val Met Thr Val Phe Tyr Gly
565 570 575
Met Ala Glu Lys Ile Ala Glu Ala Ile Leu Gln Asp Tyr His Ala Arg
580 585 590
Lys Ala
Claims (9)
1. Super-heat-resistant glucose oxidaseAtGOD is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. A glucose oxidase gene encoding the hyperthermostable glucose oxidase of claim 1AtGOD。
3. The glucose oxidase gene of claim 2, wherein the nucleotide sequence is as set forth in SEQ ID NO: 2.
4. a recombinant vector comprising the glucose oxidase gene of claim 2.
5. A recombinant strain comprising the glucose oxidase gene of claim 2.
6. The recombinant strain according to claim 5, wherein the recombinant strain is recombinant Escherichia coli, recombinant yeast, recombinant Bacillus, recombinant Lactobacillus, recombinant Trichoderma reesei filamentous fungus.
7. A method for preparing hyperthermostable glucose oxidase is characterized by comprising the following steps:
(1) using a hyperthermostable glucose oxidase containing a coding amino acid sequence shown as SEQ ID NO:1AtTransforming host cells by the recombinant vector of the GOD gene to obtain a recombinant strain;
(2) culturing recombinant strain, inducing expression of hyperthermostable glucose oxidaseAtGOD；
(3) Recovering and purifying the expressed hyperthermostable glucose oxidaseAtGOD。
8. The hyperthermostable glucose oxidase of claim 1AtUse of GOD as a feed additive or a food additive.
9. The hyperthermostable glucose oxidase of claim 1AtUse of GOD for the degradation of beta-D-glucose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110464418.8A CN112877306B (en) | 2021-04-28 | 2021-04-28 | Super-heat-resistant glucose oxidase AtGOD and gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110464418.8A CN112877306B (en) | 2021-04-28 | 2021-04-28 | Super-heat-resistant glucose oxidase AtGOD and gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112877306A CN112877306A (en) | 2021-06-01 |
| CN112877306B true CN112877306B (en) | 2021-07-20 |
Family
ID=76040710
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110464418.8A Active CN112877306B (en) | 2021-04-28 | 2021-04-28 | Super-heat-resistant glucose oxidase AtGOD and gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112877306B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
|---|---|---|---|---|
| CN110577939A (en) * | 2018-06-07 | 2019-12-17 | 青岛红樱桃生物技术有限公司 | glucose oxidase mutant with improved heat resistance as well as coding gene and application thereof |
| WO2020125700A1 (en) * | 2018-12-20 | 2020-06-25 | 南京百斯杰生物工程有限公司 | Glucose oxidase mutant and use thereof in industrial production |
-
2021
- 2021-04-28 CN CN202110464418.8A patent/CN112877306B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110577939A (en) * | 2018-06-07 | 2019-12-17 | 青岛红樱桃生物技术有限公司 | glucose oxidase mutant with improved heat resistance as well as coding gene and application thereof |
| WO2020125700A1 (en) * | 2018-12-20 | 2020-06-25 | 南京百斯杰生物工程有限公司 | Glucose oxidase mutant and use thereof in industrial production |
| CN111349622A (en) * | 2018-12-20 | 2020-06-30 | 南京百斯杰生物工程有限公司 | Glucose oxidase mutant and application thereof in industrial production |
Non-Patent Citations (3)
| Title |
|---|
| Glucose oxidase [Aspergillus cristatus],GenBank: ODM18347.1;Ge,Y.等;《GenBank》;20160831;第1-2页 * |
| Synthetic construct glucose oxidase GoxAh gene, complete cds，GenBank: MN623414.1;Mou,H.等;《GenBank》;20200721;第1-2页 * |
| 黑曲霉来源葡萄糖氧化酶的稳定性改良研究;蒋肖;《中国优秀硕士学位论文全文数据库》;20210115(第1期);D050-100 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112877306A (en) | 2021-06-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108893453B (en) | Glucose oxidase GOD mutant and gene and application thereof | |
| JP5340138B2 (en) | Cloning and expression of a novel phytase | |
| US20200277582A1 (en) | Phytase mutant | |
| CN112301009B (en) | Glucose oxidase mutant GOD with improved heat stability as well as gene and application thereof | |
| JPH09500014A (en) | Fungal protein disulfide isomerase | |
| CN113862233B (en) | Method for improving acid stability of glucose oxidase, mutant Q241E/R499E, gene and application | |
| CN105950577A (en) | Glucose oxidase mutant with improved thermal stability as well as encoding genes and application thereof | |
| CN112760299B (en) | Glucose oxidase mutant with improved thermal stability as well as coding gene and application thereof | |
| WO2023236638A1 (en) | Thermal stability-improved glucose oxidase goxm10 mutant e361p, derivative mutant thereof, and use thereof | |
| EP3978602A1 (en) | Thermostable glucose oxidase | |
| CN112877306B (en) | Super-heat-resistant glucose oxidase AtGOD and gene and application thereof | |
| CN109371004B (en) | Acid protease Bs2688 mutant K203E with improved thermal stability and gene and application thereof | |
| CN107988190B (en) | Acid protease and coding gene and application thereof | |
| CN114736880B (en) | Mutant D497N of glucose oxidase GoxM10 with improved acid stability as well as derivative mutant and application thereof | |
| CN114736881B (en) | Glucose oxidase GoxM10 mutant A4D with improved acid stability and derivative mutant and application thereof | |
| CN111944790A (en) | Neutral protease gene, neutral protease, preparation method and application thereof | |
| CN107475222A (en) | The heat-resisting human lysozyme of genetic engineering transformation | |
| CN114774396B (en) | Keratinase mutant, compound preparation of keratinase mutant and bile acid and application of compound preparation in additive | |
| CN108795891B (en) | Glucose oxidase CnGODA as well as gene and application thereof | |
| CN105420220B (en) | A kind of aspartic acid albuminoid enzyme and its encoding gene and application | |
| CN115029327A (en) | Glucose oxidase mutant GOx-MUT 7-11 and coding gene and application thereof | |
| CN109810967B (en) | Acid protease Bs2688 mutant Y282L with improved thermal stability and gene and application thereof | |
| CN110885801B (en) | Glucose oxidase M5GOD and coding gene and application thereof | |
| WO2022107885A1 (en) | Thermotolerant protein glutaminase | |
| CN105420214B (en) | A kind of alpha-amylase and its encoding gene and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |