CN107012130B - Glucose oxidase mutant and coding gene and application thereof - Google Patents
Glucose oxidase mutant and coding gene and application thereof Download PDFInfo
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- CN107012130B CN107012130B CN201710409350.7A CN201710409350A CN107012130B CN 107012130 B CN107012130 B CN 107012130B CN 201710409350 A CN201710409350 A CN 201710409350A CN 107012130 B CN107012130 B CN 107012130B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- 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
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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 invention discloses a glucose oxidase mutant GOD2, and a coding gene and application thereof. The glucose oxidase GOD2 mutant provided by the invention is obtained by taking glucose oxidase GODA derived from Aspergillus niger as a female parent and carrying out three point mutations of Asn159Asp/Ala160Pro/Val418 Glu. The mutant enzyme activity provided by the invention is improved from 228.0U/mg of a wild type to 273.7U/mg, and the improvement range is 20%; after the treatment is carried out for 30min at the temperature of 60 ℃, the residual enzyme activity of GODA is 130.8U/mg, the residual enzyme activity of GOD2 is 180.5U/mg, and the improvement amplitude is 40%; therefore, the glucose oxidase mutant GOD2 provided by the invention can well meet the application requirements in the fields of food, medicine, feed, textile industry and the like, and has a very wide application prospect.
Description
Technical Field
The invention belongs to the field of genetic engineering and genetic engineering, and particularly relates to a glucose oxidase mutant and a coding gene and application thereof.
Background
Glucose Oxidase (GOD) is an aerobic dehydrogenase, has an EC number of 1.1.3.4, can highly specifically oxidize β -D-glucose into glucolactone and hydrogen peroxide under the aerobic condition, and is also called β -D-glucose oxidoreductase, most GODs in the market at present are realized by heterologous expression in a concerned pichia pastoris eukaryotic expression system, strains for industrially producing the GOD mainly come from aspergillus niger and penicillium, relatively speaking, the GOD produced by the aspergillus niger has better thermal stability, and the enzyme activity of the GOD produced by the penicillium is higher.
In order to meet the application requirements of characteristic industries on GOD (good quality of service), namely to obtain GOD with the characteristics of good stability, high activity and the like, the current common means are to excavate novel gene resources, protein engineering, optimize application environments and the like. The research of improving the catalytic performance of GOD by molecular improvement through protein engineering means is rapidly developed. For example, three catalytic residues of GOD, i.e., glutamic acid at position 412, and histidines at positions 516 and 559, were investigated by site-directed mutagenesis. Statistics of the current report on the research aspect of enzyme stability improvement, rational design strategies on protein surface electrostatic interaction, B-factor value, water transport interaction, hydrogen bond, salt bond, cation-pi interaction, disulfide bond and the like are widely applied to the improvement of protein stability. However, most of the above mentioned strategies improve protein stability at the expense of enzyme catalytic activity. Therefore, it is very meaningful to explore a protein modification strategy which can improve protein stability and does not lose enzyme activity. The invention tries to improve the thermal stability of GOD on the premise of not losing enzyme activity, and has important theoretical research significance and application significance.
Disclosure of Invention
An object of the present invention is to provide a glucose oxidase GOD2 mutant obtained by mutating Asn159Asp/Ala160Pro/Val418Glu at three points using glucose oxidase GODA derived from Aspergillus niger (Aspergillus niger) as a parent, wherein the protein of the present invention is the protein of 1), 2), or 3) as follows:
1) protein with an amino acid sequence shown in SEQ ID No. 2 in a sequence table;
2) a protein derived from 1) by replacing and/or deleting and/or adding one or more amino acid residues of the amino acid residue sequence of SEQ ID No. 2 in the sequence table while the 159 th aspartic acid, the 160 th proline and/or the 418 th glutamic acid in the amino acid residue sequence of SEQ ID No. 2 in the sequence table are/is remained unchanged;
3) protein derived from 1) and/or 2) and obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid residue sequence of SEQ ID No. 2 in the sequence table, wherein the enzyme activity and the thermal stability of the protein are enhanced compared with those of wild enzyme.
The amino acid sequence shown in SEQ ID No. 2 in the sequence table is composed of 581 amino acid residues.
The protein of 1), 2) or 3) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out recombinant heterologous expression. The coding gene of the protein described in 1), 2) or 3) can be obtained by deleting one or more codons for coding amino acid residues from the DNA sequence shown in SEQ ID No. 1 in the sequence table and/or carrying out missense mutation of one or more base pairs.
Nucleic acid molecules encoding such proteins are also within the scope of the invention.
The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be an RNA, such as an mRNA, hnRNA, or tRNA, and the like.
It is still another object of the present invention to provide a coding gene.
The coding gene has one of the following nucleotide sequences:
1) polynucleotide sequences encoding the above proteins;
2) SEQ ID No: 1;
3) SEQ ID No:2 a polynucleotide sequence of a protein sequence;
4) and SEQ ID No: 1, 475-480 site and/or 1252-1254 site nucleotide sequence is identical with the sequence table SEQ ID No: 1 to a DNA sequence defined in the specification;
5) can be mixed with the DNA of SEQ ID No: 1 to a DNA sequence defined in the specification;
6) a DNA sequence which has more than 90% of homology with the DNA sequence defined by 1), 2), 3), 4) or 5) and encodes the same functional protein; specifically, the homology is 95% or more; more specifically more than 96%; more specifically more than 97%; more specifically more than 98%; more specifically, it is 99% or more.
The high stringency conditions can be hybridization with a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS, and 1 XSSC, 0.1% SDS.
Wherein, SEQ ID No: 1 consists of 1743 nucleotides, the Open Reading Frame (ORF) of the gene is the 1 st to 1743 rd nucleotides from the 5' end, and the coded sequence table SEQ ID No:2, namely the GOD2 protein.
Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the nucleic acid molecules also belong to the scope of protection of the invention.
The recombinant vector can be a recombinant expression vector and can also be a recombinant cloning vector.
The starting strain of the recombinant strain comprises escherichia coli, saccharomycetes, bacillus and/or lactobacillus.
The yeast comprises pichia pastoris cells, beer yeast cells and/or polytype senecio cells.
The recombinant expression vector can be constructed using existing expression vectors. The expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters can be added before the transcription initiation nucleotide, and can be used alone or combined with other promoters; in addition, when a recombinant expression vector is constructed using the gene of the present invention, an enhancer, including a translation enhancer or a transcription enhancer, may also be used. In order to facilitate the identification and screening of recombinant bacteria, expression vectors used may be processed, for example, by adding genes (GUS gene, GFP gene, luciferase gene, etc.) expressing an enzyme or a luminescent compound which produces a color change in the host bacteria, antibiotic markers having resistance (gentamicin marker, kanamycin marker, etc.). The transformed strain can also be screened directly in a stress environment without adding any selective marker gene.
The starting vector of the recombinant expression vector is specifically pPIC 9; the starting strain of the recombinant strain is GS115 specifically;
the recombinant expression vector is specifically pPIC9-GOD 2; the recombinant strain is GS115/GOD 2.
Primer pairs for amplifying the full length of the coding gene or any fragment thereof are also within the protection scope of the invention.
Another object of the present invention is to provide a method for preparing a glucose oxidase mutant, which is 1) or 2) the method comprising:
1) the method comprises changing asparagine at position 159, alanine at position 160, and/or valine at position 418 of the amino acid sequence of wild-type glucose oxidase to aspartic acid, proline, and/or glutamic acid, respectively;
2) the method comprises changing 159 th, 160 th and/or 418 th amino acid of an amino acid sequence homologous to a wild-type glucose oxidase amino acid sequence to aspartic acid, proline and/or glutamic acid, respectively;
in the method, the amino acid sequence of the wild type glucose oxidase is an amino acid sequence obtained by respectively changing 159 th, 160 th and/or 418 th amino acids of an amino acid sequence shown by SEQ ID No. 2 in a sequence table into asparagine, alanine and valine; the amino acid sequence homologous with the amino acid sequence of the wild-type glucose oxidase is specifically the amino acid sequence of the protein which has more than 90 percent of homology with the amino acid sequence of the wild-type glucose oxidase and has the same function; specifically, the homology is 95% or more; more specifically more than 96%; more specifically more than 97%; more specifically more than 98%; more specifically, it is 99% or more.
Compared with the wild-type glucose oxidase, the glucose oxidase mutant has at least one of the following characters: 1) the activity of the glucose oxidase enzyme is enhanced; 2) the thermal stability of the glucose oxidase is enhanced.
It is still another object of the present invention to provide a method for preparing a glucose oxidase mutant, the method comprising:
1) preparing the coding gene;
2) preparing a recombinant expression vector containing the coding gene of the step 1);
3) expressing the expression vector in the step 2) to obtain the target protein glucose oxidase mutant.
Specifically, the coding gene has a sequence shown in SEQ ID No: 1; more specifically, the coding gene has a sequence table with SEQ ID No: 1, 1-1743.
Still another object of the present invention is to provide the use of the encoding gene, the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium, the primer pair, and/or the preparation method in at least one of the following 1) to 4):
1) preparing glucose oxidase and/or related products containing glucose oxidase;
2) preparing a glucose oxidase mutant and/or a related product containing the glucose oxidase mutant;
3) preparing a glucose oxidase mutant with enhanced enzyme activity compared with a wild type and/or a related product containing the glucose oxidase mutant with enhanced enzyme activity compared with the wild type;
4) preparing a glucose oxidase mutant with enhanced thermostability compared with a wild type and/or a related product containing the glucose oxidase mutant with enhanced thermostability compared with the wild type.
Still another object of the present invention is to provide the use of the encoding gene, the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium, the primer pair, and/or the preparation method in preparing additives applied in the fields including food, medicine, animal feed and/or textile industry.
The invention also aims to provide the application of the protein, the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium, and/or the glucose oxidase mutant prepared by the preparation method in at least one of the following 1) to 4):
1) preparing a product containing and/or directly acting as glucose oxidase related product per se;
2) preparing related products containing and/or directly serving as glucose oxidase mutants per se;
3) preparing a product related to a glucose oxidase mutant containing and/or directly serving as enzyme activity which is enhanced compared with a wild type;
4) preparing related products containing and/or directly using as a glucose oxidase mutant with enhanced heat stability compared with a wild type.
The invention further aims to provide application of the protein, the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium, and/or the glucose oxidase mutant prepared by the preparation method in preparation of additives applied to the fields of food, medicine, animal feed and/or textile industry.
The invention overcomes the defects of the prior art and provides the glucose oxidase which has high enzyme activity and excellent thermal stability and is suitable for being applied in the fields of food, medicine, feed, textile industry and the like.
The mutant enzyme activity provided by the invention is improved from 228.0U/mg of a wild type to 273.7U/mg, and the improvement range is 20%; after the wild GODA is treated for 120min at 60 ℃, the enzyme activity is reduced to 89.2U/mg, and the residual enzyme activity is equal to 37.6 percent before treatment; and after the mutant GOD2 is treated at 60 ℃ for 120min, the residual enzyme activity is 105.3U/mg, which is improved by 1.18 times compared with the wild type. After the treatment is carried out for 30min at the temperature of 60 ℃, the residual enzyme activity of GODA is 130.8U/mg, the residual enzyme activity of GOD2 is 180.5U/mg, and the improvement amplitude is 40%; after the wild GODA is treated for 2min at 70 ℃, the enzyme activity is reduced to 116.9U/mg, and the residual enzyme activity is equivalent to 50% of that before treatment; and after the mutant GOD2 is treated at 70 ℃ for 2min, the residual enzyme activity is 186.4U/mg, which is improved by 1.59 times compared with the wild type. Therefore, the glucose oxidase mutant GOD2 provided by the invention can well meet the application requirements in the fields of food, medicine, feed, textile industry and the like, and has a very wide application prospect.
Drawings
FIG. 1 is a graph comparing the enzymatic activities of wild type GODA and mutant GOD 2.
FIG. 2 is a graph comparing the stability of wild type GODA and mutant GOD2 at 60 ℃.
FIG. 3 is a graph comparing the stability of wild type GODA and mutant GOD2 at 70 ℃.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Test materials and reagents
Test materials and reagents
1. Bacterial strain and carrier: the expression host Pichia pastoris GS115, expression plasmid vector pPIC9(Invitrogen) for the laboratory storage.
2. Enzymes and other biochemical reagents: the endonuclease was purchased from Fermentas, and the ligase was purchased from Promaga. The others are domestic analytical pure reagents (all can be purchased from common biochemical reagents).
3. Culture medium:
LB culture medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0
YPD medium: 1% yeast extract, 2% peptone, 2% glucose
MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin
MM solid medium: 1.5% agarose, 1.34% YNB, 0.00004% Biotin, 0.5% methanol
BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (V/V), 1.34% YNB, 0.00004% Biotin
BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 0.5% methanol (V/V)
4. The molecular biological experiments, which are not described in detail in this example, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to the kit and product instructions.
Example 1 site-directed mutagenesis of glucose oxidase-encoding Gene
The glucose oxidase GODA was homologously modeled, and the mutation sites were designed such that asparagine at position 159, alanine at position 160, and valine at position 418 were mutated to aspartic acid, proline, and glutamic acid, respectively. Introducing mutation sites by using an Over-lap PCR method, and performing sequencing verification on the mutation sites to obtain a mutant gene GOD 2. The primers used in the Over-lap PCR are shown in Table 1:
TABLE 1
Sequencing results show that the Over-lap PCR amplification can obtain a DNA sequence with a sequence shown in SEQ ID No: 1, total 1743bp, wherein the length of the coding region is 1743bp, and the sequence of the coding region is shown as SEQ ID No: 1, 1-1743 th nucleotides, and a coding sequence table SEQ ID No:2, and a total of 581 amino acid residues. The titanium alloy with the titanium alloy of SEQ ID No: 1 is named as mutant gene GOD 2; the titanium alloy with the sequence shown in SEQ ID No:2 is named as glucose oxidase mutant GOD 2.
Example 2 preparation of glucose oxidase mutant GOD2
Preparation of recombinant plasmid pPIC9-GOD2
Carrying out double enzyme digestion on the expression vector pPIC9 (Eco RI + Not I); meanwhile, the titanium dioxide powder prepared in the embodiment 1 and having a sequence shown in SEQ ID No: 1 (2) double digestion of the nucleic acid fragment with the nucleotide sequence of (Eco RI + Not I); and connecting the two cut nucleic acid fragments to obtain a recombinant plasmid containing the mutant gene GOD 2.
And (3) sequencing the obtained recombinant plasmid to verify the correctness of the sequence. The sequence of the exogenous gene inserted into the obtained plasmid is SEQ ID No: 1, and is named pPIC9-GOD 2.
Preparation of (II) recombinant bacterium GS115/GOD2
The recombinant plasmid pPIC9-GOD2 is transformed into Pichia pastoris GS115 cells to obtain a recombinant yeast strain GS115/GOD 2. The plasmid of the recombinant bacteria is extracted and sequenced, and the recombinant bacteria containing the plasmid pPIC9-GOD2 with correct sequencing is named as GS115/GOD 2.
Preparation of (tri) glucose oxidase mutant GOD2
Inoculating the recombinant yeast strain GS115/GOD2 into a 1L triangular flask of 300mL BMGY medium, and performing shake culture at 30 ℃ and 220rpm for 48 h; after this time, the culture broth was centrifuged at 3000g for 5min, the supernatant was discarded, and the pellet was resuspended in 100mL BMMY medium containing 0.5% (0.5mL/100mL medium) methanol and again placed at 30 ℃ under 220rpm for induction culture. And (3) supplementing 0.5mL of methanol every 12h to keep the volume concentration of the methanol in the bacterial liquid at 0.5%, and simultaneously taking the supernatant for recycling and purifying the glucose oxidase mutant GOD2 by affinity chromatography for enzyme activity detection.
Example 3 comparison of the Properties of the glucose oxidase mutant GOD2 and the wild type
Enzyme activity assay comparison
And (3) measuring the GOD enzyme activity by adopting an ultraviolet spectrophotometer method. The specific method comprises the following steps: carrying out an enzymatic reaction under given conditions, wherein the enzymatic reaction system is as follows: a200. mu.L reaction system comprised 50. mu.L of the appropriate diluted enzyme solution, 20. mu.L of 10mM ABTS solution, 20. mu.L of 50U/mL HRP solution, 90. mu.L of disodium hydrogenphosphate-citric acid Buffer, and 20. mu.L of 1M glucose solution. And measuring the increase curve of the optical density along with time within 3min at the wavelength of 420nm, recording the increase curve every 30s, and obtaining the enzyme activity according to the slope of a straight line. 1 enzyme activity unit (U) is defined as the amount of enzyme required to produce 1. mu. mol of oxidized ABTS per unit time under given conditions.
The glucose oxidase mutant GOD2 prepared in example 2 was purified and then enzymatically reacted with wild-type glucose oxidase at pH 6.5 and 30 ℃ to determine its enzymatic activity.
The enzyme activity determination result is shown in figure 1, the activity of the wild enzyme GODA is 228.0U/mg, and the enzyme activity of the glucose oxidase mutant GOD2 is 273.7U/mg, which is improved by 20% compared with the wild enzyme.
(II) comparison of thermal stability analysis
The glucose oxidase mutant GOD2 prepared in example 2 was purified and then measured for thermal stability at 60 ℃ or 70 ℃ together with wild-type glucose oxidase by the following method:
the thermostability of the mutant and the wild type was determined by treating the mutant and the wild type in a 0.1mol/L citrate-disodium hydrogen phosphate buffer (pH 6.5) buffer system at different temperatures (60 ℃ or 70 ℃) for different times (2, 5, 10, 20, 30, 60, 90 and 120min at 60 ℃ and 2, 5, 10, 15 and 20min at 70 ℃), and then determining the residual enzyme activity at 30 ℃.
As shown in figure 2, after wild GODA is treated at 60 ℃ for 120min, the enzyme activity is reduced to 89.2U/mg, and the residual enzyme activity is equal to 37.6% before treatment; and after the mutant GOD2 is treated at 60 ℃ for 120min, the residual enzyme activity is 105.3U/mg, which is improved by 1.18 times compared with the wild type. After the treatment for 30min at the temperature of 60 ℃, the residual enzyme activity of GODA is 130.8U/mg, the residual enzyme activity of GOD2 is 180.5U/mg, and the improvement amplitude is 40%. Can well meet the application requirements in the fields of food, medicine, feed, textile industry and the like, and has very wide application prospect.
As shown in figure 3, after the wild GODA is treated at 70 ℃ for 2min, the enzyme activity is reduced to 116.9U/mg, and the residual enzyme activity is equivalent to 50% of that before treatment; and after the mutant GOD2 is treated at 70 ℃ for 2min, the residual enzyme activity is 186.4U/mg, which is improved by 1.59 times compared with the wild type.
Sequence listing
<110> feed institute of Chinese academy of agricultural sciences
<120> glucose oxidase mutant and coding gene and application thereof
<160>2
<210>1
<211>1743
<212>DNA
<213> Aspergillus niger (Aspergillus niger)
<400>1
ggtattgagg cttccttgtt gactgaccca aaggaggtcg ccggtagaac tgttgactac 60
atcattgctg gtggtggatt gactggtttg actaccgctg ccagattgac tgagaaccca 120
gacatcaccg ttttggtcat tgagtccggt tcttacgaat ctgatagagg tcctatcatt 180
gaagacttga acgcttacgg tgacatcttc ggatcttccg ttgaccacgc ttacgagact 240
gtcgaacttg ccactaacaa tcaaaccgct ttgattagat ccggtaacgg tttgggtggt 300
tctactttgg ttaacggagg tacttggacc agaccacaca aggctcaagt tgactcttgg 360
gagaccgtct tcggtaacga aggttggaat tgggattctg tcgcagctta ctccttgcag 420
gccgagagag cccgtgctcc aaacgctaag caaatcgccg caggtcacta cttcgaccca 480
tcctgtcacg gtattaacgg aactgttcac gctggtccaa gagacaccgg tgacgattac 540
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caggttttga ccggtcaata cgttggtaag gtccttttgt ctcaaaacgc cactacccca 780
agagctgttg gtgtcgagtt cggaactcac aagggtaaca cccacaatgt ttacgctaaa 840
cacgaagtcc ttttggcagc tggttccgct gtttctccaa ctatcttgga gtactctggt 900
atcggaatga agtccatttt ggaaccactt ggtattgaca ccgtcgttga cttgcctgtt 960
ggtctgaact tgcaagacca gactacctct actgtcagat cccgtattac ctccgccggt 1020
gctggacagg gtcaggctgc ctggtttgct actttcaacg agaccttcgg tgactacact 1080
gagaaggctc acgaattgct taacaccaaa ttggaacaat gggctgagga agccgttgct 1140
agaggtggtt tccacaacac taccgctctt ttgatccaat acgagaacta cagagactgg 1200
attgttaagg ataacgtcgc ttactctgaa ttgttcttgg acactgccgg tgaggcttcc 1260
ttcgacgtct gggacttgct gccattcact agaggatacg ttcacatctt ggacaaggac 1320
ccatacttga gacacttcgc ttacgatcct caatacttct tgaacgagtt ggacttgctt 1380
ggtcaggctg ccgctactca attggctaga aacatctcta actccggtgc catgcaaact 1440
tactttgctg gtgaaaccat tccaggtgac aacttggcct acgatgctga cttgagagct 1500
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atgatgccaa aggagatggg tggtgtcgtt gacaacgccg ctagagttta cggtgtccag 1620
ggattgagag ttatcgacgg ttctatccca cctactcaaa tgtcctctca cgttatgacc 1680
gtcttctacg ctatggcttt gaagatcgca gacgctgttt tggctgacta cgcctccatg 1740
caa 1743
<210>2
<211>581
<212>PRT
<213> Aspergillus niger (Aspergillus niger)
<400>2
Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg
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Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Thr
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Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu
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Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
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Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
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Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn
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Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro
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His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
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Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
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Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asp Pro
145 150 155 160
Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
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Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
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Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His
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Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295300
Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala
405 410 415
Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr Asn Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp
565 570 575
Tyr Ala Ser Met Gln
580
Claims (9)
1. A glucose oxidase mutant is characterized in that the glucose oxidase mutant has an amino acid sequence shown as SEQ ID NO. 2 in a sequence table.
2. A glucose oxidase mutant coding gene is characterized in that the glucose oxidase mutant coding gene codes a polypeptide with the amino acid sequence shown as SEQ ID NO:2 in sequence shown in the specification.
3. The glucose oxidase mutant encoding gene as claimed in claim 2, wherein the glucose oxidase mutant encoding gene has the amino acid sequence shown as SEQ ID NO: 1.
4. A recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium, wherein the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium comprises the glucose oxidase mutant encoding gene of claim 2.
5. A method for preparing a glucose oxidase mutant, comprising:
1) preparing a gene encoding the glucose oxidase mutant of claim 2;
2) preparing a recombinant expression vector containing the glucose oxidase mutant coding gene in the step 1);
3) expressing the recombinant expression vector in the step 2) to obtain the target protein glucose oxidase mutant.
6. The use of the glucose oxidase mutant encoding gene of claim 2 in at least one of the following 1) -4):
1) preparing glucose oxidase and related products containing glucose oxidase;
2) preparing a glucose oxidase mutant and related products containing the glucose oxidase mutant;
3) preparing a glucose oxidase mutant with enhanced enzyme activity compared with a wild type and a related product containing the glucose oxidase mutant with enhanced enzyme activity compared with the wild type;
4) preparing glucose oxidase mutant with enhanced thermal stability compared with wild type and related products containing glucose oxidase mutant with enhanced thermal stability compared with wild type.
7. The use of the glucose oxidase mutant coding gene of claim 2 in the preparation of additives for use in the fields of food, medicine, animal feed and textile industry.
8. The use of the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of claim 4 in at least one of 1) to 4) below:
1) preparing glucose oxidase and related products containing glucose oxidase;
2) preparing a glucose oxidase mutant and related products containing the glucose oxidase mutant;
3) preparing a glucose oxidase mutant with enhanced enzyme activity compared with a wild type and a related product containing the glucose oxidase mutant with enhanced enzyme activity compared with the wild type;
4) preparing glucose oxidase mutant with enhanced thermal stability compared with wild type and related products containing glucose oxidase mutant with enhanced thermal stability compared with wild type.
9. Use of the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of claim 4 for the preparation of an additive for use in the fields of food, medicine, animal feed and textile industry.
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| CN108251391A (en) * | 2017-08-18 | 2018-07-06 | 青岛蔚蓝生物集团有限公司 | Novel grape carbohydrate oxidase mutant |
| CN108034642B (en) * | 2017-11-13 | 2020-12-11 | 中国农业科学院北京畜牧兽医研究所 | Glucose oxidase CnGOD19 and improved enzyme, gene and application thereof |
| CN108893453B (en) * | 2018-06-04 | 2020-05-22 | 中国农业科学院饲料研究所 | Glucose oxidase GOD mutant and gene and application thereof |
| CN112301009B (en) * | 2019-07-26 | 2022-12-09 | 中国农业科学院北京畜牧兽医研究所 | Glucose oxidase mutant GOD with improved heat stability as well as gene and application thereof |
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| CN113862233B (en) * | 2021-12-03 | 2022-03-25 | 中国农业科学院北京畜牧兽医研究所 | Method for improving acid stability of glucose oxidase, mutant Q241E/R499E, gene and application |
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| CN114736881B (en) * | 2022-06-09 | 2022-09-27 | 中国农业科学院北京畜牧兽医研究所 | Glucose oxidase GoxM10 mutant A4D with improved acid stability and derivative mutant and application thereof |
| CN114736879B (en) * | 2022-06-09 | 2022-09-27 | 中国农业科学院北京畜牧兽医研究所 | Glucose oxidase GoxM10 mutant E361P with improved heat stability and derivative mutant and application thereof |
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Effective date of registration: 20200824 Address after: 100193 Beijing Old Summer Palace West Road, Haidian District, No. 2 Patentee after: Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences Address before: 100081 Beijing, Zhongguancun, South Street, No. 12, No. Patentee before: FEED Research Institute CHINESE ACADEMY OF AGRICULTURAL SCIENCES |