CN108034642B - Glucose oxidase CnGOD19 and improved enzyme, gene and application thereof - Google Patents
Glucose oxidase CnGOD19 and improved enzyme, gene and application thereof Download PDFInfo
- Publication number
- CN108034642B CN108034642B CN201711111804.9A CN201711111804A CN108034642B CN 108034642 B CN108034642 B CN 108034642B CN 201711111804 A CN201711111804 A CN 201711111804A CN 108034642 B CN108034642 B CN 108034642B
- Authority
- CN
- China
- Prior art keywords
- ala
- leu
- gly
- val
- thr
- 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)
-
- 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)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the field of genetic engineering, in particular to glucose oxidase CnGOD19, and an improved enzyme, a gene and application thereof. The amino acid sequence of the improved enzyme is shown in SEQ ID NO.5 or SEQ ID NO.6, and the improved glucose oxidase CnGOD19A has good enzymological properties and can be widely applied to food, chemical industry, medicine and other biotechnology industries.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to glucose oxidase CnGOD19, and an improved enzyme, a gene and application thereof.
Background
Glucose oxidase (GOD, E.C.1.1.3.4) is a homodimer consisting of two identical subunits with molecular weight of 60-90kDa, is a flavin-dependent aerobic dehydrogenase, belongs to GMC (glucose/methane/choleline) oxidoreductase family, and is widely applied to food, chemical, pharmaceutical and other biotechnology industries, particularly the application in medical diagnosis.
At present, the industrial enzyme preparation of the glucose oxidase produced in China has few sources, low purity and extremely unstable enzymological properties, and depends on import for a long time, so that the method has important significance for searching for a substitute product of an imported enzyme preparation.
Disclosure of Invention
The new source glucose oxidase gene excavated from Cladosporium neoplasms SL-19 strain not only expands the excavation range of the glucose oxidase gene, but also obtains the glucose oxidase CnGOD19A with better enzymological property by improving and designing the gene, and further promotes the excavation and improvement work of the glucose oxidase.
The invention aims to provide a novel source of unreported glucose oxidase.
It is still another object of the present invention to provide a gene encoding the above-mentioned glucose oxidase.
It is still another object of the present invention to provide an improved enzyme of the above-mentioned glucose oxidase.
It is still another object of the present invention to provide a gene of the above glucose oxidase-modifying enzyme.
It is still another object of the present invention to provide a recombinant vector comprising the gene encoding the glucose oxidase-improving enzyme.
It is still another object of the present invention to provide a recombinant strain comprising a gene encoding the above-mentioned glucose oxidase-improving enzyme.
It is a further object of the present invention to provide a method for producing a new source of a glucose oxidase-modified enzyme.
It is still another object of the present invention to provide the above glucose oxidase-modified enzyme.
The glucose oxidase Cngodd 19 gene provided by the invention is derived from Cladosporium neopysychrotolers SL-19 strain, and the cDNA sequence of the gene is shown as SEQ ID NO. 1.
The comparison shows that the gene is a new gene, and the peptide chain corresponding to the gene has the activity of glucose oxidase.
The corresponding amino acid sequence of the glucose oxidase CnGOD19 obtained according to the nucleotide sequence is shown as SEQ ID NO. 2.
The theoretical isoelectric point of the protein corresponding to the sequence is 4.68, the theoretical molecular weight is about 61.58KDa, and the 1 st to 16 th amino acids are signal peptides. In order to eliminate the influence of the theoretical signal peptide on the later expression and secretion of the protein, the nucleotide sequence corresponding to the signal peptide can be removed when a recombinant expression vector is constructed, and then the amino acid sequence corresponding to the expression is shown as SEQ ID NO. 3.
Experiments verify that the polypeptide expressed by the glucose oxidase Cnggod 19 gene has glucose oxidase activity, but the activity is low, a plurality of schemes are designed to modify the Cnggod 19 gene, and an effective modification scheme is finally obtained through optimization to obtain the Cngod19A gene, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 4.
The corresponding glucose oxidase modified enzyme CnGOD19A can be obtained according to the nucleotide sequence, and the amino acid sequence of the enzyme is shown as SEQ ID NO. 5.
According to the specific embodiment of the invention, when the recombinant expression vector is constructed, the nucleotide sequence corresponding to the signal peptide can be removed, and then the amino acid sequence corresponding to the expression is shown as the following SEQ ID NO. 6.
The isoelectric point of the protein corresponding to the sequence is 4.81, and the molecular weight is about 64.68 kDa. Experiments prove that the product expressed by the Cngod19A gene has glucose oxidase enzyme activity, the enzyme activity before improvement is extremely low, the enzyme activity after improvement is improved to 33.34U/mg, the optimum reaction temperature of the improved enzyme is 20 ℃, the enzyme activity is still more than 60 percent at 0-50 ℃, the treatment is carried out for 60min at 40 ℃, and the residual enzyme activity is nearly 70 percent.
Drawings
FIG. 1 shows the optimum pH of the recombinant glucose oxidase of the present invention.
FIG. 2 shows the optimum reaction temperature of the glucose oxidase of the present invention.
FIG. 3 shows the pH stability of the glucose oxidase of the present invention.
FIG. 4 shows the thermostability of the glucose oxidase of the present invention.
Detailed Description
Test materials and reagents
1. Bacterial strain and carrier: pichia pastoris (Pichiapastoris GS 115); the pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitrogen.
2. Enzymes and other biochemical reagents: the endonuclease was purchased from TaKaRa, the ligase was purchased from Invitrogen, the Peasy-Uni Seamless cloning and Assembly Kit was purchased from Beijing Quanjin Biotechnology Co., Ltd, the RNA extraction Kit was purchased from Promega Biotechnology Co., Ltd, the plasmid extraction Kit was purchased from Tiangen Biochemical technology Co., Ltd, the nucleic acid recovery Kit was purchased from OMEGA bio-tek, and the others were all made-by-home reagents (all available from general Biochemical reagents Co., Ltd.).
3. Culture medium:
(1) enzyme production medium (/ L): 172.11g of glucose, 11.05g of corn steep liquor, 52.29g of calcium carbonate, 3.5g of beef peptone (NH)4)H2PO40.5g，MgSO4·7H2O 0.125g，FeSO4·7H2O 0.125g。
(2) Coli LB medium: 1% peptone, 0.5% yeast extract, 1% NaCI, pH 7.0.
(3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.000049< Biotin, 1% glycerol (v/v).
(4) BMMY medium: the composition was identical to BMGY except that 0.5% methanol was used instead of glycerol.
Description of the drawings: the above culture media and the devices for culturing the strains in the experiments are used without special instructions and need to be sterilized, and the molecular biology experimental methods which are not specifically described in the following examples are performed according to the specific methods listed in molecular cloning experimental manual (third edition) J. SammBruk book, or according to the kit and the product instructions.
EXAMPLE 1 cloning of the Cngod19 Gene
(1) Extraction of Cladosporium neophyllum neopysychrotolers SL-19 total RNA
First, the bacteria cultured in the enzyme-producing medium for 3 days were collected on a filter paper and press-dried, added into a mortar sterilized at high temperature, rapidly ground to powder with liquid nitrogen, and then total RNA of the strain was extracted according to the instructions of the RNA extraction kit.
(2) Obtaining of glucose oxidase cDNA
Using oligo (dT)20And reverse transcriptase to reverse transcribe the gene, then designing synthetic primers Z19# F and Z19# R (see Table 1), and performing PCR amplification on the coding region of the mature protein (signal peptide removal sequence) of CnGOD19 to obtain the cDNA sequence of glucose oxidase.
TABLE 1Cngod19 cloning primer
Example 2 construction of recombinant Strain GS 115/Cngold 19
(1) Construction of the recombinant vector pPIC9-Cngod19
The pPIC9 expression vector and the target product amplified by the primers in Table 1 were mixed and seamlessly spliced according to the specifications of the Peasy-Uni Seamless cloning and Assembly Kit to construct the yeast expression vector pPIC9-Cngod 19. The pPIC 9-Cnggod 19 plasmid is transferred into Escherichia coli competence Trans1-T1, the Escherichia coli competence Trans1-T1 is coated on an LB solid plate with ampicillin resistance for overnight culture, colony PCR verification is carried out, positive transformants are selected for DNA sequencing, and the transformants with correct sequencing sequence are used for preparing a large amount of recombinant plasmids. The recombinant plasmid is linearized by restriction endonuclease Bgl II, the enzyme digestion product is recovered, then the enzyme digestion product is transferred into yeast GS115 competent cells by electric shock, the yeast GS115 competent cells are coated on an MD plate and cultured for 2-3 days at the temperature of 30 ℃, and transformants growing on the MD plate are selected for further expression experiments, wherein the specific operation refers to a pichia pastoris expression operation manual.
(2) Screening of GS 115/Cnggod 19 high-yield enzyme transformant
And selecting a single colony of a transformant from the MD plate by using the sterilized toothpick, sequentially dotting the single colony on the MD plate, numbering and marking the single colony on the MD plate, and culturing the MD plate in an incubator at 30 ℃ for 1-2 days until the colony grows out. Selecting transformants from the MD plate according to the number, inoculating the transformants into a culture tube filled with 3mL of BMGY medium, and carrying out shake culture at 30 ℃ and 220rpm for 48 hours; centrifuging the bacterial solution cultured by shaking for 48h at 3,000 Xg for 15min, removing supernatant, adding 1mL BMMY culture medium containing 0.5% methanol into the culture tube, and performing induction culture at 30 deg.C and 220 rpm; after the induction culture is carried out for 48h, centrifuging for 5min at 3,000 Xg, taking the supernatant for enzyme activity detection, and screening out a high-activity transformant of the glucose oxidase from the supernatant, wherein the specific operation refers to a pichia pastoris expression operation manual.
The enzyme activity detection result shows that the CnGOD19 enzyme expressed by the GS115/CnGOD19 recombinant strain has glucose oxidase activity but lower activity, so the construction process of the recombinant strain GS115/CnGOD19 after gene modification and the expression and purification process of the CnGOD19A enzyme in later period are explained in the following embodiment.
Example 3 construction of the recombinant Strain GS 115/Cnggod 19A
(1) Construction of the recombinant vector pPIC9-Cngod19A
The research of the invention finds that the CnGOD19 encoding gene has structural change due to evolution, thereby influencing the structural change of the expressed protein and finally causing low enzyme activity. In order to solve the above problems, the present invention optimizes various schemes, and finally designs segment substitution of carbon terminal, and replaces the peptide fragment from 550 th to 568 th (including signal peptide sequence when counting) in CnGOD19 with the peptide fragment of amino acid sequence "YIATLGMMKEELGGVVDSRLKVYGIENVRAVDASVLPIQLSAHLSSSLYGIAEKAAMMIKEDQGH".
(2) Construction of GS 115/Cnggod 19A high-enzyme-producing Strain
Referring to the procedure of example 2, the pPIC 9-Cnggod 19A recombinant plasmid was finally transferred into GS115 yeast competent cells and the GS 115/Cnggod 19A high enzyme producing strain was selected.
In the same manner, a recombinant vector containing a signal peptide sequence was constructed and the host strain was transformed.
Example 4 fermentation of recombinant glucose oxidase in Pichia pastoris
(1) Mass expression of recombinant glucose oxidase at shake flask level
Inoculating the screened transformant with higher enzyme-producing activity into 30mL YPD seed culture medium for culturing for 48h, then transferring into 300mL BMGY liquid culture medium, carrying out shake culture at 30 ℃, 220rpm for 48h, and carrying out thallus enrichment; centrifuging at 4,500rpm for 5min, discarding supernatant, transferring the thallus into 100mL BMMY liquid culture medium containing 0.5% methanol, and inducing culture at 30 deg.C and 220rpm for 72 h. During the induction culture period, the methanol solution is replenished every 24 hours to compensate the loss of methanol, so that the final concentration of the methanol is kept at about 0.5 percent; after induction culture, centrifuging at 12,000 Xg for 10min, collecting supernatant fermentation liquor, detecting enzyme activity and carrying out SDS-PAGE protein electrophoresis analysis.
(2) Purification of recombinant glucose oxidase
Collecting the supernatant of recombinant glucose oxidase expressed in a shake flask, firstly concentrating by using a 5kDa membrane package, replacing the culture medium in the fermentation liquor by low-salt buffer solution in the process, and then further concentrating by using a 5kDa ultrafiltration tube. Concentrating until the enzyme solution can be diluted by a certain multiple, and purifying by ion exchange chromatography. Specifically, the CnGOD19A concentrated solution was injected into HiTrap Q Sepharose XL anion column pre-equilibrated with 20mM PBS (pH 6.5), and then subjected to linear gradient elution with 0-1mol/L NaCl-containing PBS buffer solution, and the enzyme activity of the eluate collected stepwise was detected and the protein concentration was measured.
Example 5 analysis of the properties of recombinant glucose oxidase moiety
The enzyme activity of the glucose oxidase is measured by a spectrophotometric method. The specific method comprises the following steps: 5mL of the reaction system at 30 ℃ and pH6.0 contained 2.5mL of o-dianisidine buffer, 0.3mL of 18% glucose solution, 0.1mL of 90U/mL horseradish peroxidase, 0.1mL of an appropriate diluted enzyme solution, reaction 3 rmin, addition of 2mL of 2M sulfuric acid to stop the reaction, and measurement of OD540 absorbance after cooling. Glucose oxidase activity unit definition: under certain conditions, the amount of enzyme capable of oxidizing l μmol β -D-glucose to D-gluconic acid and hydrogen peroxide per minute is 1 activity unit (U).
(1) Optimum pH and temperature of glucose oxidase CnGOD19A
The enzyme activities of the purified glucose oxidase samples of example 4 were measured under different pH buffer conditions to determine their optimum pH buffer conditions. Preparing buffers for different pH values: citric acid-disodium hydrogen phosphate buffer solution with the pH value of 3.0-8.0, glycine-sodium hydroxide buffer solution with the pH value of 9.0-l 2.0 and buffer solutions with different pH values are used for preparing o-dianisidine solution. The results of the pH optima determined at 30 ℃ (FIG. 1) show that: the optimum pH value of CnGOD19A is 7.0, and the enzyme activity can be maintained above 60% in the pH range of 6.0-8.0.
And (3) measuring the enzyme activity of the purified glucose oxidase sample at different temperatures (0-60 ℃) under the condition of pH 7.0. The analysis experiment result shows that the optimal reaction temperature of the enzyme is 20 ℃, and the enzyme activity is still more than 60% between 0 and 50 ℃ (figure 2).
(2) pH stability and thermal stability of glucose oxidase CnGOD19A
The enzyme solution was treated in buffers of different pH values at 25 ℃ for 60min, and the enzyme activity was measured at the optimum pH to investigate the pH stability of the enzyme. The analysis result shows (figure 3), the enzyme is basically stable between pH6.0-pH9.0, can maintain more than 80% of enzyme activity, and the enzyme activity basically disappears after the treatment of pH5.0 and pH10.0.
The thermal stability is measured by treating glucose oxidase samples at different temperatures (30 ℃, 40 ℃ and 50 ℃) for different times and then measuring the enzyme activity at 30 ℃. The thermal stability experiment shows (figure 4) that the glucose oxidase is treated at 30 ℃ for 60min, the enzyme activity is basically unchanged, the residual enzyme activity is about 70 percent when the glucose oxidase is treated at 40 ℃ for 60min, but only about 60 percent of the enzyme activity is remained after the glucose oxidase is treated at 50 ℃ for 5 min.
(3) Metal ion resistance of glucose oxidase CnGOD19A
TABLE 2 results of ion resistance experiments after purification of CnGOD19A
As can be seen from Table 2, the substrate contained 5mM Mn as compared with the blank control2+、Fe3+Or Co2+When the substrate contains 5mM of beta-mercaptoethanol, the loss of the enzyme activity of CnGOD19A is serious, and other metal ions and organic reagents with the concentration of 5mM have small promotion effects on the enzyme activity. When the substrate contains 10mM Cu2 +、Ni2+Or Na+When the metal ions are used, the enzyme activity of CnGOD19A is slightly improved, and when the substrate contains 10mM of beta-mercaptoethanol, the enzyme activity of CnGOD19A is seriously lost, and other metal ions with the concentration of 10mM have inhibition effect on the enzyme activity.
Sequence listing
<110> institute of feed of Chinese academy of agricultural sciences
<120> glucose oxidase CnGOD19 and improved enzyme, gene and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1707
<212> DNA
<213> Cladosporium species (Cladosporium neopysylchromerolers SL-19)
<400> 1
atggtttacc cggctgctat tctcacattt ctcctcgcca ctctcgcaca atcattaccc 60
cagaagcaac gagcggacaa ggcacgcgga atcatcaccg acgccaacca tgtcgccaac 120
aagacctttg actacatcgt ctgcggtggc ggactgacag gactagtcgt agcgagtcgt 180
ctctcagaag accccgagat atcagtgcta gtgatcgaga acggcaacga cgaccacgag 240
gaccctcgtg tcaacgatgt caggacctac ggagcagcat tcgaatccga gctcgacttc 300
aatcttacat cgacgcctgt cccgtggcag aacgacaccg gcctgctcct agttgcgggc 360
aggactctag gcggaagtgg cagcatcaac ggcgctagct ggacgaaagg cgacaagacg 420
cagtatgatt tgctaccagt gctgacgggg gatgactctt ggtctttcga cgccctgaac 480
gagatcatgc tcagcatcga ggacttccac gagccaacgg aagagcacat cgccaaaggc 540
gcgaacttca caagcgagtt ccacggccgg gatgggatgg tgcaggtttc gtttccgaga 600
ggcatgtttg gcggaatcca agttccagcg ttggaagcgt ccacggccat ctggaagggg 660
ttggggatag ttgcggattt cgcagcggga ctcacgaacg gcgctacaat cattcctaac 720
atgattgaag acaacgagga tcagaaccgc tcttcgcctt tcactgtgta tgcaaaggac 780
cagacgcagc agcgggataa tttcgtgatc ctgacaggtc accgcgtcac atcgatcaac 840
tggcgtccgg gcaaagacat ggccgccgag ggcgtgaact tccaggcatg tcgcagctgc 900
gagaaacacg tcgcgacaac gaagcgcgaa gtactgctcg ccgcaggctc tctacaaagc 960
ccacagctgc tcgagctgtc aggcgtagga gacccagaag tcctcgccgc cgccggcgta 1020
cccgtgaaga tggcatcccc caacgtcggc aagaacatgc aagaacaaac caaaaacacc 1080
ctctggttcg accccgtcag caccgacttc gacggcaccg gccccccaaa cgccatcgcc 1140
ttccccaccg tcaaccaact cttcaaaaac aacagcgcat ccttgtacca aagcatcaca 1200
tccagcctct ggcaatacgc cctcgacctc aaagcctctg gcctcgtaac aaacgccacc 1260
gcaacccacc taatcctctc agcccaagtc aacaacctgt ggaaagactc cgccggcgcc 1320
gcagaagtct tcttcgtcac ctcgcccgcc accggccaaa tcggcatcga cctctggaac 1380
ctcatcgtgc tgtccagggg ttacgtgcac atcacctcca actcctcctg ggaccacccg 1440
gagatcgagc cctcgtactt cggccacccc ttcgacctcg gggtccagct ggcagcggcc 1500
cagcaatcgc gcgagatctt tcagacggca ccactcgcag ctcacgtcgc gtcggagacg 1560
ttccctggtc ttgaaaccgt tcctcaaaac gccactacgg aagtctggga ggagtgggtc 1620
aaaggtactt tcacttctgt gtggcattat atcgcgacgt tggggatgat gaaagaggag 1680
ctgggggagt tgtggatcac agactga 1707
<210> 2
<211> 568
<212> PRT
<213> Cladosporium species (Cladosporium neopysylchromerolers SL-19)
<400> 2
Met Val Tyr Pro Ala Ala Ile Leu Thr Phe Leu Leu Ala Thr Leu Ala
1 5 10 15
Gln Ser Leu Pro Gln Lys Gln Arg Ala Asp Lys Ala Arg Gly Ile Ile
20 25 30
Thr Asp Ala Asn His Val Ala Asn Lys Thr Phe Asp Tyr Ile Val Cys
35 40 45
Gly Gly Gly Leu Thr Gly Leu Val Val Ala Ser Arg Leu Ser Glu Asp
50 55 60
Pro Glu Ile Ser Val Leu Val Ile Glu Asn Gly Asn Asp Asp His Glu
65 70 75 80
Asp Pro Arg Val Asn Asp Val Arg Thr Tyr Gly Ala Ala Phe Glu Ser
85 90 95
Glu Leu Asp Phe Asn Leu Thr Ser Thr Pro Val Pro Trp Gln Asn Asp
100 105 110
Thr Gly Leu Leu Leu Val Ala Gly Arg Thr Leu Gly Gly Ser Gly Ser
115 120 125
Ile Asn Gly Ala Ser Trp Thr Lys Gly Asp Lys Thr Gln Tyr Asp Leu
130 135 140
Leu Pro Val Leu Thr Gly Asp Asp Ser Trp Ser Phe Asp Ala Leu Asn
145 150 155 160
Glu Ile Met Leu Ser Ile Glu Asp Phe His Glu Pro Thr Glu Glu His
165 170 175
Ile Ala Lys Gly Ala Asn Phe Thr Ser Glu Phe His Gly Arg Asp Gly
180 185 190
Met Val Gln Val Ser Phe Pro Arg Gly Met Phe Gly Gly Ile Gln Val
195 200 205
Pro Ala Leu Glu Ala Ser Thr Ala Ile Trp Lys Gly Leu Gly Ile Val
210 215 220
Ala Asp Phe Ala Ala Gly Leu Thr Asn Gly Ala Thr Ile Ile Pro Asn
225 230 235 240
Met Ile Glu Asp Asn Glu Asp Gln Asn Arg Ser Ser Pro Phe Thr Val
245 250 255
Tyr Ala Lys Asp Gln Thr Gln Gln Arg Asp Asn Phe Val Ile Leu Thr
260 265 270
Gly His Arg Val Thr Ser Ile Asn Trp Arg Pro Gly Lys Asp Met Ala
275 280 285
Ala Glu Gly Val Asn Phe Gln Ala Cys Arg Ser Cys Glu Lys His Val
290 295 300
Ala Thr Thr Lys Arg Glu Val Leu Leu Ala Ala Gly Ser Leu Gln Ser
305 310 315 320
Pro Gln Leu Leu Glu Leu Ser Gly Val Gly Asp Pro Glu Val Leu Ala
325 330 335
Ala Ala Gly Val Pro Val Lys Met Ala Ser Pro Asn Val Gly Lys Asn
340 345 350
Met Gln Glu Gln Thr Lys Asn Thr Leu Trp Phe Asp Pro Val Ser Thr
355 360 365
Asp Phe Asp Gly Thr Gly Pro Pro Asn Ala Ile Ala Phe Pro Thr Val
370 375 380
Asn Gln Leu Phe Lys Asn Asn Ser Ala Ser Leu Tyr Gln Ser Ile Thr
385 390 395 400
Ser Ser Leu Trp Gln Tyr Ala Leu Asp Leu Lys Ala Ser Gly Leu Val
405 410 415
Thr Asn Ala Thr Ala Thr His Leu Ile Leu Ser Ala Gln Val Asn Asn
420 425 430
Leu Trp Lys Asp Ser Ala Gly Ala Ala Glu Val Phe Phe Val Thr Ser
435 440 445
Pro Ala Thr Gly Gln Ile Gly Ile Asp Leu Trp Asn Leu Ile Val Leu
450 455 460
Ser Arg Gly Tyr Val His Ile Thr Ser Asn Ser Ser Trp Asp His Pro
465 470 475 480
Glu Ile Glu Pro Ser Tyr Phe Gly His Pro Phe Asp Leu Gly Val Gln
485 490 495
Leu Ala Ala Ala Gln Gln Ser Arg Glu Ile Phe Gln Thr Ala Pro Leu
500 505 510
Ala Ala His Val Ala Ser Glu Thr Phe Pro Gly Leu Glu Thr Val Pro
515 520 525
Gln Asn Ala Thr Thr Glu Val Trp Glu Glu Trp Val Lys Gly Thr Phe
530 535 540
Thr Ser Val Trp His Tyr Ile Ala Thr Leu Gly Met Met Lys Glu Glu
545 550 555 560
Leu Gly Glu Leu Trp Ile Thr Asp
565
<210> 3
<211> 552
<212> PRT
<213> Cladosporium species (Cladosporium neopysylchromerolers SL-19)
<400> 3
Gln Ser Leu Pro Gln Lys Gln Arg Ala Asp Lys Ala Arg Gly Ile Ile
1 5 10 15
Thr Asp Ala Asn His Val Ala Asn Lys Thr Phe Asp Tyr Ile Val Cys
20 25 30
Gly Gly Gly Leu Thr Gly Leu Val Val Ala Ser Arg Leu Ser Glu Asp
35 40 45
Pro Glu Ile Ser Val Leu Val Ile Glu Asn Gly Asn Asp Asp His Glu
50 55 60
Asp Pro Arg Val Asn Asp Val Arg Thr Tyr Gly Ala Ala Phe Glu Ser
65 70 75 80
Glu Leu Asp Phe Asn Leu Thr Ser Thr Pro Val Pro Trp Gln Asn Asp
85 90 95
Thr Gly Leu Leu Leu Val Ala Gly Arg Thr Leu Gly Gly Ser Gly Ser
100 105 110
Ile Asn Gly Ala Ser Trp Thr Lys Gly Asp Lys Thr Gln Tyr Asp Leu
115 120 125
Leu Pro Val Leu Thr Gly Asp Asp Ser Trp Ser Phe Asp Ala Leu Asn
130 135 140
Glu Ile Met Leu Ser Ile Glu Asp Phe His Glu Pro Thr Glu Glu His
145 150 155 160
Ile Ala Lys Gly Ala Asn Phe Thr Ser Glu Phe His Gly Arg Asp Gly
165 170 175
Met Val Gln Val Ser Phe Pro Arg Gly Met Phe Gly Gly Ile Gln Val
180 185 190
Pro Ala Leu Glu Ala Ser Thr Ala Ile Trp Lys Gly Leu Gly Ile Val
195 200 205
Ala Asp Phe Ala Ala Gly Leu Thr Asn Gly Ala Thr Ile Ile Pro Asn
210 215 220
Met Ile Glu Asp Asn Glu Asp Gln Asn Arg Ser Ser Pro Phe Thr Val
225 230 235 240
Tyr Ala Lys Asp Gln Thr Gln Gln Arg Asp Asn Phe Val Ile Leu Thr
245 250 255
Gly His Arg Val Thr Ser Ile Asn Trp Arg Pro Gly Lys Asp Met Ala
260 265 270
Ala Glu Gly Val Asn Phe Gln Ala Cys Arg Ser Cys Glu Lys His Val
275 280 285
Ala Thr Thr Lys Arg Glu Val Leu Leu Ala Ala Gly Ser Leu Gln Ser
290 295 300
Pro Gln Leu Leu Glu Leu Ser Gly Val Gly Asp Pro Glu Val Leu Ala
305 310 315 320
Ala Ala Gly Val Pro Val Lys Met Ala Ser Pro Asn Val Gly Lys Asn
325 330 335
Met Gln Glu Gln Thr Lys Asn Thr Leu Trp Phe Asp Pro Val Ser Thr
340 345 350
Asp Phe Asp Gly Thr Gly Pro Pro Asn Ala Ile Ala Phe Pro Thr Val
355 360 365
Asn Gln Leu Phe Lys Asn Asn Ser Ala Ser Leu Tyr Gln Ser Ile Thr
370 375 380
Ser Ser Leu Trp Gln Tyr Ala Leu Asp Leu Lys Ala Ser Gly Leu Val
385 390 395 400
Thr Asn Ala Thr Ala Thr His Leu Ile Leu Ser Ala Gln Val Asn Asn
405 410 415
Leu Trp Lys Asp Ser Ala Gly Ala Ala Glu Val Phe Phe Val Thr Ser
420 425 430
Pro Ala Thr Gly Gln Ile Gly Ile Asp Leu Trp Asn Leu Ile Val Leu
435 440 445
Ser Arg Gly Tyr Val His Ile Thr Ser Asn Ser Ser Trp Asp His Pro
450 455 460
Glu Ile Glu Pro Ser Tyr Phe Gly His Pro Phe Asp Leu Gly Val Gln
465 470 475 480
Leu Ala Ala Ala Gln Gln Ser Arg Glu Ile Phe Gln Thr Ala Pro Leu
485 490 495
Ala Ala His Val Ala Ser Glu Thr Phe Pro Gly Leu Glu Thr Val Pro
500 505 510
Gln Asn Ala Thr Thr Glu Val Trp Glu Glu Trp Val Lys Gly Thr Phe
515 520 525
Thr Ser Val Trp His Tyr Ile Ala Thr Leu Gly Met Met Lys Glu Glu
530 535 540
Leu Gly Glu Leu Trp Ile Thr Asp
545 550
<210> 4
<211> 1845
<212> DNA
<213> Cladosporium species (Cladosporium neopysylchromerolers SL-19)
<400> 4
atggtttacc cggctgctat tctcacattt ctcctcgcca ctctcgcaca atcattaccc 60
cagaagcaac gagcggacaa ggcacgcgga atcatcaccg acgccaacca tgtcgccaac 120
aagacctttg actacatcgt ctgcggtggc ggactgacag gactagtcgt agcgagtcgt 180
ctctcagaag accccgagat atcagtgcta gtgatcgaga acggcaacga cgaccacgag 240
gaccctcgtg tcaacgatgt caggacctac ggagcagcat tcgaatccga gctcgacttc 300
aatcttacat cgacgcctgt cccgtggcag aacgacaccg gcctgctcct agttgcgggc 360
aggactctag gcggaagtgg cagcatcaac ggcgctagct ggacgaaagg cgacaagacg 420
cagtatgatt tgctaccagt gctgacgggg gatgactctt ggtctttcga cgccctgaac 480
gagatcatgc tcagcatcga ggacttccac gagccaacgg aagagcacat cgccaaaggc 540
gcgaacttca caagcgagtt ccacggccgg gatgggatgg tgcaggtttc gtttccgaga 600
ggcatgtttg gcggaatcca agttccagcg ttggaagcgt ccacggccat ctggaagggg 660
ttggggatag ttgcggattt cgcagcggga ctcacgaacg gcgctacaat cattcctaac 720
atgattgaag acaacgagga tcagaaccgc tcttcgcctt tcactgtgta tgcaaaggac 780
cagacgcagc agcgggataa tttcgtgatc ctgacaggtc accgcgtcac atcgatcaac 840
tggcgtccgg gcaaagacat ggccgccgag ggcgtgaact tccaggcatg tcgcagctgc 900
gagaaacacg tcgcgacaac gaagcgcgaa gtactgctcg ccgcaggctc tctacaaagc 960
ccacagctgc tcgagctgtc aggcgtagga gacccagaag tcctcgccgc cgccggcgta 1020
cccgtgaaga tggcatcccc caacgtcggc aagaacatgc aagaacaaac caaaaacacc 1080
ctctggttcg accccgtcag caccgacttc gacggcaccg gccccccaaa cgccatcgcc 1140
ttccccaccg tcaaccaact cttcaaaaac aacagcgcat ccttgtacca aagcatcaca 1200
tccagcctct ggcaatacgc cctcgacctc aaagcctctg gcctcgtaac aaacgccacc 1260
gcaacccacc taatcctctc agcccaagtc aacaacctgt ggaaagactc cgccggcgcc 1320
gcagaagtct tcttcgtcac ctcgcccgcc accggccaaa tcggcatcga cctctggaac 1380
ctcatcgtgc tgtccagggg ttacgtgcac atcacctcca actcctcctg ggaccacccg 1440
gagatcgagc cctcgtactt cggccacccc ttcgacctcg gggtccagct ggcagcggcc 1500
cagcaatcgc gcgagatctt tcagacggca ccactcgcag ctcacgtcgc gtcggagacg 1560
ttccctggtc ttgaaaccgt tcctcaaaac gccactacgg aagtctggga ggagtgggtc 1620
aaaggtacgt tcacatctgt ttggcactac atagctacat tgggcatgat gaaagaggaa 1680
ttgggtggtg ttgtggacag caggctgaag gtatatggca ttgagaatgt acgagcggtg 1740
gatgctagtg tgctgccgat ccagctttcg gcgcacttga gctcttcgct gtacggcatt 1800
gcggagaagg ctgctatgat gattaaggaa gatcagggac attga 1845
<210> 5
<211> 614
<212> PRT
<213> Cladosporium species (Cladosporium neopysylchromerolers SL-19)
<400> 5
Met Val Tyr Pro Ala Ala Ile Leu Thr Phe Leu Leu Ala Thr Leu Ala
1 5 10 15
Gln Ser Leu Pro Gln Lys Gln Arg Ala Asp Lys Ala Arg Gly Ile Ile
20 25 30
Thr Asp Ala Asn His Val Ala Asn Lys Thr Phe Asp Tyr Ile Val Cys
35 40 45
Gly Gly Gly Leu Thr Gly Leu Val Val Ala Ser Arg Leu Ser Glu Asp
50 55 60
Pro Glu Ile Ser Val Leu Val Ile Glu Asn Gly Asn Asp Asp His Glu
65 70 75 80
Asp Pro Arg Val Asn Asp Val Arg Thr Tyr Gly Ala Ala Phe Glu Ser
85 90 95
Glu Leu Asp Phe Asn Leu Thr Ser Thr Pro Val Pro Trp Gln Asn Asp
100 105 110
Thr Gly Leu Leu Leu Val Ala Gly Arg Thr Leu Gly Gly Ser Gly Ser
115 120 125
Ile Asn Gly Ala Ser Trp Thr Lys Gly Asp Lys Thr Gln Tyr Asp Leu
130 135 140
Leu Pro Val Leu Thr Gly Asp Asp Ser Trp Ser Phe Asp Ala Leu Asn
145 150 155 160
Glu Ile Met Leu Ser Ile Glu Asp Phe His Glu Pro Thr Glu Glu His
165 170 175
Ile Ala Lys Gly Ala Asn Phe Thr Ser Glu Phe His Gly Arg Asp Gly
180 185 190
Met Val Gln Val Ser Phe Pro Arg Gly Met Phe Gly Gly Ile Gln Val
195 200 205
Pro Ala Leu Glu Ala Ser Thr Ala Ile Trp Lys Gly Leu Gly Ile Val
210 215 220
Ala Asp Phe Ala Ala Gly Leu Thr Asn Gly Ala Thr Ile Ile Pro Asn
225 230 235 240
Met Ile Glu Asp Asn Glu Asp Gln Asn Arg Ser Ser Pro Phe Thr Val
245 250 255
Tyr Ala Lys Asp Gln Thr Gln Gln Arg Asp Asn Phe Val Ile Leu Thr
260 265 270
Gly His Arg Val Thr Ser Ile Asn Trp Arg Pro Gly Lys Asp Met Ala
275 280 285
Ala Glu Gly Val Asn Phe Gln Ala Cys Arg Ser Cys Glu Lys His Val
290 295 300
Ala Thr Thr Lys Arg Glu Val Leu Leu Ala Ala Gly Ser Leu Gln Ser
305 310 315 320
Pro Gln Leu Leu Glu Leu Ser Gly Val Gly Asp Pro Glu Val Leu Ala
325 330 335
Ala Ala Gly Val Pro Val Lys Met Ala Ser Pro Asn Val Gly Lys Asn
340 345 350
Met Gln Glu Gln Thr Lys Asn Thr Leu Trp Phe Asp Pro Val Ser Thr
355 360 365
Asp Phe Asp Gly Thr Gly Pro Pro Asn Ala Ile Ala Phe Pro Thr Val
370 375 380
Asn Gln Leu Phe Lys Asn Asn Ser Ala Ser Leu Tyr Gln Ser Ile Thr
385 390 395 400
Ser Ser Leu Trp Gln Tyr Ala Leu Asp Leu Lys Ala Ser Gly Leu Val
405 410 415
Thr Asn Ala Thr Ala Thr His Leu Ile Leu Ser Ala Gln Val Asn Asn
420 425 430
Leu Trp Lys Asp Ser Ala Gly Ala Ala Glu Val Phe Phe Val Thr Ser
435 440 445
Pro Ala Thr Gly Gln Ile Gly Ile Asp Leu Trp Asn Leu Ile Val Leu
450 455 460
Ser Arg Gly Tyr Val His Ile Thr Ser Asn Ser Ser Trp Asp His Pro
465 470 475 480
Glu Ile Glu Pro Ser Tyr Phe Gly His Pro Phe Asp Leu Gly Val Gln
485 490 495
Leu Ala Ala Ala Gln Gln Ser Arg Glu Ile Phe Gln Thr Ala Pro Leu
500 505 510
Ala Ala His Val Ala Ser Glu Thr Phe Pro Gly Leu Glu Thr Val Pro
515 520 525
Gln Asn Ala Thr Thr Glu Val Trp Glu Glu Trp Val Lys Gly Thr Phe
530 535 540
Thr Ser Val Trp His Tyr Ile Ala Thr Leu Gly Met Met Lys Glu Glu
545 550 555 560
Leu Gly Gly Val Val Asp Ser Arg Leu Lys Val Tyr Gly Ile Glu Asn
565 570 575
Val Arg Ala Val Asp Ala Ser Val Leu Pro Ile Gln Leu Ser Ala His
580 585 590
Leu Ser Ser Ser Leu Tyr Gly Ile Ala Glu Lys Ala Ala Met Met Ile
595 600 605
Lys Glu Asp Gln Gly His
610
<210> 6
<211> 598
<212> PRT
<213> Cladosporium species (Cladosporium neopysylchromerolers SL-19)
<400> 6
Gln Ser Leu Pro Gln Lys Gln Arg Ala Asp Lys Ala Arg Gly Ile Ile
1 5 10 15
Thr Asp Ala Asn His Val Ala Asn Lys Thr Phe Asp Tyr Ile Val Cys
20 25 30
Gly Gly Gly Leu Thr Gly Leu Val Val Ala Ser Arg Leu Ser Glu Asp
35 40 45
Pro Glu Ile Ser Val Leu Val Ile Glu Asn Gly Asn Asp Asp His Glu
50 55 60
Asp Pro Arg Val Asn Asp Val Arg Thr Tyr Gly Ala Ala Phe Glu Ser
65 70 75 80
Glu Leu Asp Phe Asn Leu Thr Ser Thr Pro Val Pro Trp Gln Asn Asp
85 90 95
Thr Gly Leu Leu Leu Val Ala Gly Arg Thr Leu Gly Gly Ser Gly Ser
100 105 110
Ile Asn Gly Ala Ser Trp Thr Lys Gly Asp Lys Thr Gln Tyr Asp Leu
115 120 125
Leu Pro Val Leu Thr Gly Asp Asp Ser Trp Ser Phe Asp Ala Leu Asn
130 135 140
Glu Ile Met Leu Ser Ile Glu Asp Phe His Glu Pro Thr Glu Glu His
145 150 155 160
Ile Ala Lys Gly Ala Asn Phe Thr Ser Glu Phe His Gly Arg Asp Gly
165 170 175
Met Val Gln Val Ser Phe Pro Arg Gly Met Phe Gly Gly Ile Gln Val
180 185 190
Pro Ala Leu Glu Ala Ser Thr Ala Ile Trp Lys Gly Leu Gly Ile Val
195 200 205
Ala Asp Phe Ala Ala Gly Leu Thr Asn Gly Ala Thr Ile Ile Pro Asn
210 215 220
Met Ile Glu Asp Asn Glu Asp Gln Asn Arg Ser Ser Pro Phe Thr Val
225 230 235 240
Tyr Ala Lys Asp Gln Thr Gln Gln Arg Asp Asn Phe Val Ile Leu Thr
245 250 255
Gly His Arg Val Thr Ser Ile Asn Trp Arg Pro Gly Lys Asp Met Ala
260 265 270
Ala Glu Gly Val Asn Phe Gln Ala Cys Arg Ser Cys Glu Lys His Val
275 280 285
Ala Thr Thr Lys Arg Glu Val Leu Leu Ala Ala Gly Ser Leu Gln Ser
290 295 300
Pro Gln Leu Leu Glu Leu Ser Gly Val Gly Asp Pro Glu Val Leu Ala
305 310 315 320
Ala Ala Gly Val Pro Val Lys Met Ala Ser Pro Asn Val Gly Lys Asn
325 330 335
Met Gln Glu Gln Thr Lys Asn Thr Leu Trp Phe Asp Pro Val Ser Thr
340 345 350
Asp Phe Asp Gly Thr Gly Pro Pro Asn Ala Ile Ala Phe Pro Thr Val
355 360 365
Asn Gln Leu Phe Lys Asn Asn Ser Ala Ser Leu Tyr Gln Ser Ile Thr
370 375 380
Ser Ser Leu Trp Gln Tyr Ala Leu Asp Leu Lys Ala Ser Gly Leu Val
385 390 395 400
Thr Asn Ala Thr Ala Thr His Leu Ile Leu Ser Ala Gln Val Asn Asn
405 410 415
Leu Trp Lys Asp Ser Ala Gly Ala Ala Glu Val Phe Phe Val Thr Ser
420 425 430
Pro Ala Thr Gly Gln Ile Gly Ile Asp Leu Trp Asn Leu Ile Val Leu
435 440 445
Ser Arg Gly Tyr Val His Ile Thr Ser Asn Ser Ser Trp Asp His Pro
450 455 460
Glu Ile Glu Pro Ser Tyr Phe Gly His Pro Phe Asp Leu Gly Val Gln
465 470 475 480
Leu Ala Ala Ala Gln Gln Ser Arg Glu Ile Phe Gln Thr Ala Pro Leu
485 490 495
Ala Ala His Val Ala Ser Glu Thr Phe Pro Gly Leu Glu Thr Val Pro
500 505 510
Gln Asn Ala Thr Thr Glu Val Trp Glu Glu Trp Val Lys Gly Thr Phe
515 520 525
Thr Ser Val Trp His Tyr Ile Ala Thr Leu Gly Met Met Lys Glu Glu
530 535 540
Leu Gly Gly Val Val Asp Ser Arg Leu Lys Val Tyr Gly Ile Glu Asn
545 550 555 560
Val Arg Ala Val Asp Ala Ser Val Leu Pro Ile Gln Leu Ser Ala His
565 570 575
Leu Ser Ser Ser Leu Tyr Gly Ile Ala Glu Lys Ala Ala Met Met Ile
580 585 590
Lys Glu Asp Gln Gly His
595
Claims (10)
1. The glucose oxidase CnGOD19 is characterized in that the amino acid sequence is shown in SEQ ID NO.2 or SEQ ID NO. 3.
2. A glucose oxidase gene encoding the glucose oxidase CnGOD19 of claim 1.
3. The improved glucose oxidase CnGOD19A is characterized in that the amino acid sequence is shown in SEQ ID NO.5 or SEQ ID NO. 6.
4. A modified glucose oxidase gene encoding the modified glucose oxidase CnGOD19A of claim 3.
5. The improved glucose oxidase gene of claim 4, wherein the nucleotide sequence is as shown in SEQ ID No. 4.
6. A recombinant vector comprising the improved glucose oxidase gene of claim 4.
7. A recombinant cell comprising the improved glucose oxidase gene of claim 4.
8. A method of producing the improved glucose oxidase CnGOD19A of claim 3, comprising the steps of:
(1) transforming a host cell with the recombinant vector of claim 6 to obtain a recombinant host cell;
(2) culturing the recombinant host cell obtained in the step (1);
(3) the glucose oxidase CnGOD19A was isolated and purified.
9. The use of the improved glucose oxidase CnGOD19A for oxidizing glucose according to claim 3, wherein said use is for non-disease diagnostic and therapeutic purposes.
10. Use of the improved glucose oxidase gene of claim 4 for the oxidation of glucose, wherein said use is for non-disease diagnostic and therapeutic purposes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711111804.9A CN108034642B (en) | 2017-11-13 | 2017-11-13 | Glucose oxidase CnGOD19 and improved enzyme, gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711111804.9A CN108034642B (en) | 2017-11-13 | 2017-11-13 | Glucose oxidase CnGOD19 and improved enzyme, gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108034642A CN108034642A (en) | 2018-05-15 |
| CN108034642B true CN108034642B (en) | 2020-12-11 |
Family
ID=62092759
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711111804.9A Active CN108034642B (en) | 2017-11-13 | 2017-11-13 | Glucose oxidase CnGOD19 and improved enzyme, gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108034642B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108795891B (en) * | 2017-04-26 | 2021-03-26 | 中国农业科学院北京畜牧兽医研究所 | Glucose oxidase CnGODA as well as gene and application thereof |
| CN110358688B (en) * | 2019-07-04 | 2022-07-12 | 河南省科学院生物研究所有限责任公司 | Extracellular glucose oxidase producing strain CY-12 and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107012130A (en) * | 2017-06-02 | 2017-08-04 | 中国农业科学院饲料研究所 | A kind of glucose oxidase mutant and its encoding gene and application |
| CN107189991A (en) * | 2017-05-08 | 2017-09-22 | 中国农业科学院饲料研究所 | A kind of glucose oxidase mutant and its encoding gene and application |
| CN108893453A (en) * | 2018-06-04 | 2018-11-27 | 中国农业科学院饲料研究所 | Glucose oxidase GOD mutant and its gene and application |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995029996A1 (en) * | 1994-05-03 | 1995-11-09 | Novo Nordisk A/S | Alkaline glucose oxidase |
| US5879921A (en) * | 1996-11-07 | 1999-03-09 | Novo Nordisk A/S | Recombinant expression of a glucose oxidase from a cladosporium strain |
| CN105779401B (en) * | 2016-05-16 | 2020-03-17 | 北京科为博生物科技有限公司 | High-temperature-resistant acidic glucose oxidase GODL8, and gene and application thereof |
-
2017
- 2017-11-13 CN CN201711111804.9A patent/CN108034642B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107189991A (en) * | 2017-05-08 | 2017-09-22 | 中国农业科学院饲料研究所 | A kind of glucose oxidase mutant and its encoding gene and application |
| CN107012130A (en) * | 2017-06-02 | 2017-08-04 | 中国农业科学院饲料研究所 | A kind of glucose oxidase mutant and its encoding gene and application |
| CN108893453A (en) * | 2018-06-04 | 2018-11-27 | 中国农业科学院饲料研究所 | Glucose oxidase GOD mutant and its gene and application |
Non-Patent Citations (1)
| Title |
|---|
| glucose oxidase [Cladosporium neopsychrotolerans];Ge J;《NCBI GenBank database》;20191117;QGH51193.1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108034642A (en) | 2018-05-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108251392B (en) | Glucose oxidase mutant capable of improving specific activity and thermal stability and coding gene and application thereof | |
| CN108034642B (en) | Glucose oxidase CnGOD19 and improved enzyme, gene and application thereof | |
| CN110628738B (en) | Method for improving activity of glucose oxidase, mutant and application thereof | |
| CN114667346A (en) | EanB enzyme mutant and application thereof | |
| CN113462678B (en) | Glutamic acid decarboxylase mutant | |
| CN114395541A (en) | Glucose oxidase mutant GOx1-MUT with improved thermal stability and specific activity, and coding gene and application thereof | |
| CN110129305B (en) | Cephalosporin C acylase mutant for preparing 7-ACA | |
| CN110283797B (en) | Tyrosinase, gene, engineering bacterium and preparation method thereof | |
| CN112143717B (en) | Glucose oxidase mutant with improved specific activity | |
| CN108795891B (en) | Glucose oxidase CnGODA as well as gene and application thereof | |
| CN116064266B (en) | Recombinant saccharomyces cerevisiae with enhanced salt stress resistance, and construction method and application thereof | |
| CN115029327B (en) | Glucose oxidase mutant GOx-MUT 7-11, and encoding gene and application thereof | |
| CN114736880B (en) | Mutant D497N of glucose oxidase GoxM10 with improved acid stability as well as derivative mutant and application thereof | |
| CN114181288B (en) | Process for producing L-valine, gene used therefor and protein encoded by the gene | |
| Gregor et al. | Oxygen-regulated expression of genes for pigment binding proteins in Rhodobacter capsulatus | |
| CN115029328B (en) | Glucose oxidase mutant GOx-MUT 1-6, and coding gene and application thereof | |
| CN110791508A (en) | Codon-optimized pediocin gene, expression vector and recombinant engineering strain | |
| CN110872593A (en) | Serine hydroxymethyl transferase mutant and application thereof | |
| CN116284282A (en) | tkt gene mutant and application thereof in preparation of L-lysine | |
| CN113564151B (en) | Method for improving structural isomerism catalytic activity of CE enzyme and mutant thereof | |
| CN114349831A (en) | aspA gene mutant, recombinant bacterium and method for preparing L-valine | |
| CN109097315B (en) | Genetically engineered bacterium for high-yield lipopeptide and construction method and application thereof | |
| CN111411115B (en) | Gene and method for improving rhizobia | |
| CN114621944B (en) | Arginine deiminase mutant with improved enzyme activity | |
| CN115028694B (en) | Protein related to L-glutamic acid yield, related biological material and application thereof |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200903 Address after: 100193 Beijing Old Summer Palace West Road, Haidian District, No. 2 Applicant after: Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences Address before: 100081 Beijing, Zhongguancun, South Street, No. 12, No. Applicant before: FEED Research Institute CHINESE ACADEMY OF AGRICULTURAL SCIENCES |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |