CN107012130A - A kind of glucose oxidase mutant and its encoding gene and application - Google Patents
A kind of glucose oxidase mutant and its encoding gene and application Download PDFInfo
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- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
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Abstract
The invention discloses a kind of glucose oxidase mutant GOD2 and its encoding gene and application.The glucose oxidase GOD2 mutant that the present invention is provided is obtained using the glucose oxidase GODA from aspergillus niger (Aspergillus niger) as female parent through three point mutation Asn159Asp/Ala160Pro/Val418Glu.The mutant enzyme enzyme activity that the present invention is provided has brought up to 273.7U/mg by the 228.0U/mg of wild type, and increase rate is 20%；Handled at 60 DEG C after 30min, GODA remaining enzyme activity is 130.8U/mg, and GOD2 remaining enzyme activity is 180.5U/mg, and increase rate is 40%；Therefore, the glucose oxidase mutant GOD2 that the present invention is provided is well positioned to meet the demand applied in the fields such as food, medicine, feed and textile industry, there is boundless application prospect.
Description
Technical field
The invention belongs to genetic engineering and field of genetic engineering, and in particular to a kind of glucose oxidase mutant and its volume
Code gene and application.
Background technology
Glucose oxidase (glucose oxidase, GOD) is a kind of aerobic dehydrogenase, and No. EC is 1.1.3.4.Its energy
It is enough it is highly single-minded under aerobic conditions beta -d-glucose- is oxidized to glucolactone and hydrogen peroxide, therefore also known as β-
D-Glucose oxidoreducing enzyme.Current GOD most on the market is the Pichia pastoris eukaryotic expression system by receiving much concern
What middle heterogenous expression was realized.Industrialized production GOD bacterial strain is mainly derived from aspergillus niger and mould.Comparatively, Aspergillus Niger
GOD better heat stabilities, and mould production GOD enzyme activity it is higher.GOD as a kind of important industrial enzymes in food service industry,
It is widely used in terms of the deoxidation of the food such as grape wine, beer, fruit juice, milk powder, flour improvement, food brown stain prevention, in medical row
Also have in terms of providing anaerobic environment in the quantitative determination of human body biochemical indicator glucose and feedstuff industry for probiotics in industry
Substantial amounts of application.
In order to meet application demand of the feature industry to GOD, that is, obtain with GOD the features such as stability is good, vigor is high,
Current prevalent means are to excavate novel genetic resources, protein engineering and optimization application environment etc..Utilize protein engineering
Means to GOD carry out molecular improvement with improve its catalytic performance research be even more achieve fast development.For example, prominent by fixed point
The means of change have studied GOD three catalytic residues, i.e., the glutamic acid of the 412nd, the histidine of the 516th and 559.Count mesh
The preceding report paper in terms of enzyme stability Upgrading, on protein surface electrostatic interaction, B-factor values, water delivery
The design and rational strategy such as interaction, hydrogen bond, sat linkage, cation-π interaction, disulfide bond is widely used in protein
In improved stability.But, in above-mentioned strategy, the overwhelming majority is to improve egg to lose enzymatic vigor as cost
White matter stability.Therefore, protein stabilization can be improved by probing into one kind, and not lose the protein Reconstruc-tion policy right and wrong of enzyme activity
It is often significant.The present invention attempts to improve GOD heat endurance on the premise of enzyme activity is not lost, with important theoretical research
Meaning and application value.
The content of the invention
It is an object of the present invention to provide a kind of albumen, with from aspergillus niger (Aspergillus niger) Portugal
Grape carbohydrate oxidase GODA is as female parent, through three point mutation Asn159Asp/Ala160Pro/Val418Glu, and the grape obtained
Carbohydrate oxidase GOD2 mutant, albumen of the present invention be it is following 1), 2) or 3) described in albumen：
1) there is the protein of the amino acid sequence shown in the SEQ ID № .2 in sequence table；
2) by the 159th aspartic acid in the amino acid residue sequence of the SEQ ID № .2 in sequence table, the 160th
Proline, and/or the 418th glutamic acid retain constant and pass through the amino acid residue sequence of the SEQ ID № .2 in sequence table
Cross substitution and/or missing and/or the addition protein as derived from 1) of one or several amino acid residues；
3) the taking by one or several amino acid residues by the amino acid residue sequence of the SEQ ID № .2 in sequence table
1) and/or 2) albumen as derived from that generation and/or missing and/or addition and enzyme activity and heat endurance are enhanced compared with wild-type enzyme
Matter.
Amino acid sequence in sequence table shown in SEQ ID № .2 is made up of 581 amino acid residues.
It is above-mentioned 1), 2) or 3) described in albumen can be artificial synthesized, also can first synthesize its encoding gene, then to carry out restructuring different
Source expression is obtained.It is above-mentioned 1), 2) or 3) described in the encoding gene of albumen can be by by sequence table shown in SEQ ID № .1
DNA sequence dna lacks the codon of one or several coded amino acid residues, and/or the missense of one or several base-pairs of progress is dashed forward
Obtained after change.
The nucleic acid molecules of encoding said proteins fall within protection scope of the present invention.
The nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also be
RNA, such as mRNA, hnRNA or tRNA.
A further object of the present invention is to provide a kind of encoding gene.
The encoding gene has one of following nucleotide sequence：
1) polynucleotide sequence of above-mentioned albumen is encoded；
2) SEQ ID № in sequence table：Nucleotide sequence shown in 1；
3) SEQ ID № in polynucleotide：The polynucleotide sequence of 2 protein sequences；
4) with SEQ ID № in sequence table：In 1 475-480, and/or 1252-1254 nucleotide sequences it is consistent
And can be with SEQ ID № in sequence table under high high stringency conditions：The nucleotide sequence of the 1 DNA sequence dna hybridization limited；
5) can be with SEQ ID № in sequence table under high high stringency conditions：The nucleotide sequence of the 1 DNA sequence dna hybridization limited；
6) there is more than 90% homology, and coding identical function albumen with the DNA sequence dna of 1), 2), 3), 4) or 5) restriction
The DNA sequence dna of matter；Specifically, the homology is more than 95%；Specific again is more than 96%；Again it is specific for 97% with
On；Specific again is more than 98%；Specific again is more than 99%.
Above-mentioned high high stringency conditions can be that with 6 × SSC, 0.5%SDS solution hybridizes at 65 DEG C, then with 2 × SSC,
0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
Wherein, the SEQ ID № in sequence table：1 is made up of 1743 nucleotides, and its open reading frame (ORF) is certainly
SEQ ID № in the 5 ' nucleotides of end 1-1743, polynucleotide：Protein shown in 2, i.e., GOD2 of the present invention
Albumen.
Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing above-mentioned nucleic acid molecules fall within the present invention's
Protection domain.
The recombinant vector can be recombinant expression carrier, or recombinant cloning vector.
The starting strain of the recombinant bacterium includes Escherichia coli, saccharomycete, bacillus, and/or Bacillus acidi lactici.
The saccharomycete includes Pichia pastoris, beer yeast cells, and/or many types of inferior yeast cells.
The recombinant expression carrier can use existing expression vector establishment.The expression vector can also include foreign gene
3 ' ends untranslated region, i.e., the DNA fragmentation comprising polyadenylation signals and any other participation mRNA processing or gene expression.Institute
State the 3 ' ends that the bootable polyadenylic acid of polyadenylation signals is added to mRNA precursor.Use the gene constructed recombinant expression carrier
When, any enhanced, composing type, organizing specific type or inducible promoter can be added before its transcription initiation nucleotides,
They can be used alone or are used in combination with other promoters；In addition, using the gene constructed recombinant expression carrier of the present invention
When, it is also possible to use enhancer, including translational enhancer or transcriptional enhancer.For the ease of being identified recombinant bacterium and being screened,
Expression carrier used thereof can be processed, such as addition, expression can produce enzyme or the luminophor of color change in Host Strains
Gene (gus gene, GFP genes, luciferase genes etc.), resistant antibiotic marker (gentamicin label, card
That mycin label etc.).Also any selected marker can be not added with, directly with adverse circumstance screening conversion bacterial strain.
The carrier specially pPIC9 that sets out of the recombinant expression carrier；The starting strain of the recombinant bacterium is specially
GS115；
The recombinant expression carrier is specially pPIC9-GOD2；The recombinant bacterium is specially GS115/GOD2.
The primer pair for expanding encoding gene total length of the present invention or its any fragment falls within the scope of protection of the invention.
It is a further object to provide a kind of preparation method of glucose oxidase mutant, 1) methods described is
Or 2) methods described：
1) methods described include by the 159th asparagine of wild type glucose oxidase amino acid sequence, the 160th
Alanine, and/or the 418th valine are changed into aspartic acid, proline, and/or glutamic acid respectively；
2) methods described is included the with the amino acid sequence of wild type glucose oxidase amino acid sequence homologous
159th, 160, and/or 418 amino acids are changed into aspartic acid, proline, and/or glutamic acid respectively；
In methods described, the amino acid sequence of the wild type glucose oxidase is by the SEQ ID № in sequence table
.2 the amino acids of the 159th, 160, and/or 418 of shown amino acid sequence are changed into asparagine, alanine, valine respectively
Amino acid sequence afterwards；The amino acid sequence with wild type glucose oxidase amino acid sequence homologous be specially with it is described
There is more than 90% homology, and the amino acid sequence of function identical protein with wild type glucose oxidase amino acid sequence
Row；Specifically, the homology is more than 95%；Specific again is more than 96%；Specific again is more than 97%；It is specific again
For more than 98%；Specific again is more than 99%.
The glucose oxidase mutant is compared with the wild type glucose oxidase, with following at least one
Shape：1) glucose oxidase enzymatic activity strengthens；2) glucose oxidase heat endurance strengthens.
A further object is for the present invention provides a kind of preparation method of glucose oxidase mutant, methods described bag
Include：
1) encoding gene described above is prepared；
2) prepare comprising step 1) recombinant expression carrier of the encoding gene；
3) step 2 is made) expression vector expression to be to obtain destination protein glucose oxidase mutant.
Specifically, encoding gene described above has SEQ ID № in sequence table：Nucleotide sequence shown in 1；Have again
Body, SEQ ID № in the sequence table that encoding gene described above has：1 nucleotide sequence of 1-1743.
It is also another object of the present invention to provide the encoding gene, the recombinant vector, expression cassette, transgenic cell line
Or recombinant bacterium, the primer pair, and/or the preparation method are following 1) -4) it is at least one in application：
1) glucose oxidase is prepared and/or containing glucose oxidase Related product；
2) glucose oxidase mutant and/or the Related product containing glucose oxidase mutant are prepared；
3) prepare enzymatic activity glucose oxidase mutant enhanced compared with wild type and/or increase containing enzymatic activity compared with wild type
The Related product of strong glucose oxidase mutant；
4) heat endurance glucose oxidase mutant enhanced compared with wild type is prepared and/or wilder containing heat endurance
The Related product of the enhanced glucose oxidase mutant of type.
It is also another object of the present invention to provide described encoding gene, described recombinant vector, expression cassette, transgenosis are thin
Born of the same parents system or recombinant bacterium, the primer pair, and/or the preparation method are in application and preparation in including food, medicine, animal feed
And/or the application in the additive in textile industry field.
It is also another object of the present invention to provide described albumen, described recombinant vector, expression cassette, transgenic cell line
Or the glucose oxidase mutant for preparing of recombinant bacterium, and/or the preparation method is following 1) -4) it is at least one in
Using：
1) prepare and contain and/or in itself directly as glucose oxidase Related product；
2) prepare and contain and/or in itself directly as glucose oxidase mutant Related product；
3) prepare and contain and/or related directly as enzymatic activity glucose oxidase mutant enhanced compared with wild type in itself
Product；
4) prepare and contain and/or be mutated body phase directly as heat endurance glucose oxidase enhanced compared with wild type in itself
Close product.
It is also another object of the present invention to provide described albumen, described recombinant vector, expression cassette, transgenic cell line
Or the glucose oxidase mutant for preparing of recombinant bacterium, and/or the preparation method in application and preparation in including food, doctor
The application in additive in medicine, animal feed and/or textile industry field.
Instant invention overcomes the deficiencies in the prior art there is provided a kind of high enzymatic activity and excellent heat endurance, be suitable for
The glucose oxidase applied in the fields such as food, medicine, feed and textile industry.
The mutant enzyme enzyme activity that the present invention is provided has brought up to 273.7U/mg, increase rate by the 228.0U/mg of wild type
For 20%；Wild type GODA is handled at 60 DEG C after 120min, and enzyme activity falls below 89.2U/mg, and remaining enzyme activity is equivalent to processing
Preceding 37.6%；And after mutant GOD2 handles 120min at 60 DEG C, remaining enzyme activity is 105.3U/mg, compared with wild type
Improve 1.18 times.Handled at 60 DEG C after 30min, GODA remaining enzyme activity is 130.8U/mg, and GOD2 remaining enzyme activity is
180.5U/mg, increase rate is 40%；Wild type GODA is handled at 70 DEG C after 2min, and enzyme activity falls below 116.9U/mg,
50% of remaining enzyme activity equivalent to before processing；And after mutant GOD2 handles 2min at 70 DEG C, remaining enzyme activity is
186.4U/mg, 1.59 times are improved compared with wild type.Therefore, the glucose oxidase mutant GOD2 that the present invention is provided can be well
The demand applied in the fields such as food, medicine, feed and textile industry is met, there is boundless application prospect.
Brief description of the drawings
Fig. 1 relatively schemes for wild type GODA and mutant GOD2 activity ratio.
Fig. 2 is that the stability of wild type GODA and mutant GOD2 at 60 DEG C compares figure.
Fig. 3 is that the stability of wild type GODA and mutant GOD2 at 70 DEG C compares figure.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Test material and reagent
Test material and reagent
1st, bacterial strain and carrier：Expressive host Pichia pastoris GS115, expression plasmid carrier pPIC9
(Invitrogen) preserved for this laboratory.
2nd, enzyme and other biochemical reagents：Restriction endonuclease is purchased from Fermentas companies, and ligase is purchased from Promaga companies.Its
It is all domestic AR (can be commercially available from common biochemical Reagent Company).
3rd, culture medium：
LB culture mediums：0.5% yeast extract, 1% peptone, 1%NaCl, pH 7.0
YPD culture mediums：1% yeast extract, 2% peptone, 2% glucose
MD solid mediums：2% glucose, 1.5% agarose, 1.34%YNB, 0.00004%Biotin
MM solid mediums：1.5% agarose, 1.34%YNB, 0.00004%Biotin, 0.5% methanol
BMGY culture mediums：1% yeast extract, 2% peptone, 1% glycerine (V/V), 1.34%YNB, 0.00004%
Biotin
BMMY culture mediums：1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 0.5% first
Alcohol (V/V)
4th, the experimental methods of molecular biology illustrated in detail, equal reference are not done in the present embodiment《Molecular Cloning: A Laboratory
Guide》Listed specific method is carried out in the book of (third edition) J. Pehanorm Brookers one, or according to kit and product description
Carry out.
The rite-directed mutagenesis of embodiment 1, glucose oxidase encoding gene
Homologous modeling is carried out to glucose oxidase GODA, and mutational site is designed as the 159th asparagine, 160
Position alanine and 418 valines sport aspartic acid, proline and glutamic acid respectively.Pass through Over-lap PCR method
Mutational site is introduced, and sequence verification is carried out to it, mutator GOD2 is obtained.The Over-lap PCR the primers such as institute of table 1
Show：
Table 1
Sequencing result shows that above-mentioned Over-lap PCR amplifications are obtained with SEQ ID № in sequence table：1 nucleotides
Sequence, common 1743bp, wherein SEQ ID № in coding head of district 1743bp, the coding region sequence such as sequence table：1-1743 in 1
Shown in the nucleotides of position, SEQ ID № in polynucleotide：Amino acid sequence shown in 2, totally 581 amino acid residues.This is had
SEQ ID № in ordered list：The fragment of nucleotide sequence shown in 1 is named as mutant gene GOD2；There to be sequence table
Middle SEQ ID №：The albumen of amino acid sequence shown in 2 is named as glucose oxidase mutant GOD2.
The glucose oxidase mutant GOD2 of embodiment 2 preparation
(1) recombinant plasmid pPIC9-GOD2 preparation
Expression vector pPIC9 is subjected to double digestion (Eco RI+Not I)；The tool that above-described embodiment 1 is prepared simultaneously
SEQ ID № in ordered list：The nucleic acid fragment of 1 nucleotide sequence carries out double digestion (Eco RI+Not I)；Cut above-mentioned
Two good nucleic acid fragments are attached, and obtain the recombinant plasmid containing the mutant gene GOD2.
Above-mentioned gained recombinant plasmid is sent into sequencing, the correctness of sequence is verified.By the external source base inserted in gained plasmid
The sequence of cause is SEQ ID №：The recombinant plasmid of nucleotides shown in 1, is named as pPIC9-GOD2.
(2) recombinant bacterium GS115/GOD2 preparation
Recombinant plasmid pPIC9-GOD2 is converted into Pichia pastoris GS115 cell, restructuring yeast strains GS115/GOD2 is obtained.
The plasmid for extracting recombinant bacterium sends to sequencing, and the correct recombinant bacterium containing plasmid pPIC9-GOD2 of sequencing is named as into GS115/
GOD2。
(3) glucose oxidase mutant GOD2 preparation
Above-mentioned restructuring yeast strains GS115/GOD2 is taken, in the 1L triangular flasks for being inoculated in 300mL BMGY culture mediums, is placed in
30 DEG C, 220rpm shaking table cultures 48h；Nutrient solution 3000g is centrifuged into 5min afterwards, supernatant is abandoned, precipitation contains 0.5% with 100mL
The BMMY culture mediums of (0.5ml/100ml culture mediums) methanol are resuspended, and are again placed in 30 DEG C, Fiber differentiation under the conditions of 220rpm.
0.5mL methanol is added every 12h, the methanol volumetric concentration in bacterium solution is maintained at 0.5%, while taking supernatant to reclaim and affine layer
Analysis purifying glucose oxidase mutant GOD2, for Enzyme assay.
The glucose oxidase mutant GOD2 of embodiment 3 and the property analysis of wild type compare
(1) enzyme activity com-parison and analysis
GOD enzyme activity is measured using ultraviolet spectrophotometer method.Specific method is as follows：Enzyme is carried out under prescribed conditions
Promote reaction, enzymatic reaction system is：200 μ L reaction system, including 50 μ L dilution enzyme liquid suitably, 20 μ L 10mM ABTS
Solution, 20 μ L 50U/mL HRP solution, 90 μ L disodium hydrogen phosphates-citric acid Buffer, and 20 μ L 1M glucose solution.
Growth curve of the 3min internal optical densities with the time is determined at 420nm wavelength, according to the slope of straight line every 30s records once
Try to achieve enzyme activity.1 enzyme-activity unit (U) is defined as under given conditions, 1 μm of ol of generation oxidized form ABTS in the unit interval
Required enzyme amount.
Glucose oxidase mutant GOD2 prepared by above-described embodiment 2 after purification, with wild type glucose oxidase
Enzymatic reaction is carried out at pH 6.5,30 DEG C to determine its enzymatic activity.
Enzyme activity determination result is as shown in figure 1, the enzyme GODA vigor of wild type is 228.0U/mg, and glucose oxidase is mutated
Body GOD2 enzyme activity is 273.7U/mg, and wilder enzyme improves 20%.
(2) thermal stability analysis compares
Glucose oxidase mutant GOD2 prepared by above-described embodiment 2 after purification, with wild type glucose oxidase
Together, the heat endurance at 60 DEG C or 70 DEG C is determined, assay method is as follows:
The mutant and the thermal stability determination of wild type are in 0.1mol/L citrate-phosphate disodium hydrogen buffer solutions
Handled under (pH 6.5) buffer solution system different temperatures (60 DEG C or 70 DEG C) different time (handle 2 at 60 DEG C respectively, 5,10,20,
30th, 60,90 and 120min；2,5,10,15 and 20min are handled at 70 DEG C respectively), then the remaining enzymatic activity of progress is surveyed at 30 DEG C
It is fixed.
As shown in Fig. 2 after wild type GODA handles 120min at 60 DEG C, enzyme activity falls below 89.2U/mg, residual enzyme
Live equivalent to the 37.6% of before processing；And after mutant GOD2 handles 120min at 60 DEG C, remaining enzyme activity is 105.3U/
Mg, 1.18 times are improved compared with wild type.Handled at 60 DEG C after 30min, GODA remaining enzyme activity is 130.8U/mg, and GOD2's is surplus
Remaining enzyme activity is 180.5U/mg, and increase rate is 40%.It is well positioned to meet the fields such as food, medicine, feed and textile industry
The demand of middle application, there is boundless application prospect.
As shown in figure 3, after wild type GODA handles 2min at 70 DEG C, enzyme activity falls below 116.9U/mg, remaining enzyme activity
Equivalent to the 50% of before processing；And after mutant GOD2 handles 2min at 70 DEG C, remaining enzyme activity is 186.4U/mg, compared with
Wild type improves 1.59 times.
Sequence table
<110>Feed institute of the Chinese Academy of Agricultural Sciences
<120>A kind of glucose oxidase mutant and its encoding gene and application
<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
tctcctatcg tcaaggcctt gatgtccgct gttgaagaca gaggtgtccc aactaagaag 600
gacttgggtt gcggagaccc acatggtgtt tctatgttcc ctaacacctt gcacgaggac 660
caagtcagat ccgatgctgc ccgtgaatgg ttgcttccaa actaccaaag acctaacttg 720
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
tgggttgaat acattccata caacttcaga cctaactacc atggtgtcgg aacctgttct 1560
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
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Thr
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
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
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His
195 200 205
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
290 295 300
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
450 455 460
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 (10)
1. a kind of albumen, it is characterised in that the albumen for it is following 1), 2) or 3) described in albumen：
1) there is the protein of the amino acid sequence shown in the SEQ ID № .2 in sequence table；
2) by the 159th aspartic acid in the amino acid residue sequence of the SEQ ID № .2 in sequence table, the 160th dried meat ammonia
Acid, and/or the 418th glutamic acid retain constant and the amino acid residue sequence of the SEQ ID № .2 in sequence table are passed through into one
Or the substitution of several amino acid residues and/or missing and/or the addition protein as derived from 1)；
3) by the amino acid residue sequence of the SEQ ID № .2 in sequence table by one or several amino acid residues substitution and/
1) and/or 2) or the protein as derived from that missing and/or addition and enzyme activity and heat endurance are enhanced compared with wild-type enzyme.
2. a kind of encoding gene, it is characterised in that the encoding gene has one of following nucleotide sequence：
1) polynucleotide sequence of albumen described in coding claim 1；
2) SEQ ID № in sequence table：Nucleotide sequence shown in 1；
3) SEQ ID № in polynucleotide：The polynucleotide sequence of 2 protein sequences；
4) with SEQ ID № in sequence table：In 1 475-480, and/or 1252-1254 nucleotide sequences it is consistent and
Can be with SEQ ID № in sequence table under high high stringency conditions：The nucleotide sequence of the 1 DNA sequence dna hybridization limited；
5) can be with SEQ ID № in sequence table under high high stringency conditions：The nucleotide sequence of the 1 DNA sequence dna hybridization limited；
6) there is more than 90% homology with the DNA sequence dna of 1), 2), 3), 4) or 5) restriction, and encodes identical function protein
DNA sequence dna.
3. a kind of recombinant vector, expression cassette, transgenic cell line or recombinant bacterium, it is characterised in that the recombinant vector, expression
Box, transgenic cell line or recombinant bacterium contain the encoding gene described in claim 2.
4. a kind of primer pair, it is characterised in that encoding gene total length described in the amplifiable claim 2 of primer pair or its is any
Fragment.
1) or 2) 5. a kind of preparation method of glucose oxidase mutant, it is characterised in that methods described is methods described：
1) methods described is included the 159th asparagine of wild type glucose oxidase amino acid sequence, the 160th the third ammonia
Acid, and/or the 418th valine are changed into aspartic acid, proline, and/or glutamic acid respectively；
2) methods described include by with the 159th of the amino acid sequence of wild type glucose oxidase amino acid sequence homologous the,
160, and/or 418 amino acids are changed into aspartic acid, proline, and/or glutamic acid respectively；
In methods described, the amino acid sequence of the wild type glucose oxidase is by shown in the SEQ ID № .2 in sequence table
The amino acids of the 159th, 160, and/or 418 of amino acid sequence be changed into the ammonia after asparagine, alanine, valine respectively
Base acid sequence；The amino acid sequence with wild type glucose oxidase amino acid sequence homologous be specially with it is described with it is wild
Type glucose oxidase amino acid sequence has more than 90% homology, and the amino acid sequence of function identical protein.
6. a kind of preparation method of glucose oxidase mutant, it is characterised in that methods described includes：
1) encoding gene described in claim 2 is prepared；
2) prepare comprising step 1) recombinant expression carrier of the encoding gene；
3) step 2 is made) expression vector expression to be to obtain destination protein glucose oxidase mutant.
7. the recombinant vector described in encoding gene, claim 3, expression cassette, transgenic cell line described in claim 2 or again
Group bacterium, primer pair described in claim 4, and/or preparation method described in claim 5,6 are following 1) -4) it is at least one in
Using：
1) glucose oxidase is prepared and/or containing glucose oxidase Related product；
2) glucose oxidase mutant and/or the Related product containing glucose oxidase mutant are prepared；
3) enzymatic activity glucose oxidase mutant enhanced compared with wild type is prepared and/or enhanced compared with wild type containing enzymatic activity
The Related product of glucose oxidase mutant；
4) prepare heat endurance glucose oxidase mutant enhanced compared with wild type and/or increase containing heat endurance compared with wild type
The Related product of strong glucose oxidase mutant.
8. the recombinant vector described in encoding gene, claim 3, expression cassette, transgenic cell line described in claim 2 or again
Preparation method described in primer pair described in group bacterium, claim 4, and/or claim 5,6 is in application and preparation in including food, doctor
The application in additive in medicine, animal feed and/or textile industry field.
9. recombinant vector, expression cassette, transgenic cell line or the restructuring described in albumen, claim 3 described in claim 1
The glucose oxidase mutant that preparation method described in bacterium, and/or claim 5,6 is prepared is following 1) -4) at least one
Application in kind：
1) prepare and contain and/or in itself directly as glucose oxidase Related product；
2) prepare and contain and/or in itself directly as glucose oxidase mutant Related product；
3) prepare and contain and/or produced in itself directly as enzymatic activity glucose oxidase mutant correlation enhanced compared with wild type
Product；
4) prepare and contain and/or produced in itself directly as heat endurance glucose oxidase mutant correlation enhanced compared with wild type
Product.
10. recombinant vector, expression cassette, transgenic cell line or the restructuring described in albumen, claim 3 described in claim 1
The glucose oxidase mutant that preparation method described in bacterium, and/or claim 5,6 is prepared is in application and preparation in including food
The application in additive in product, medicine, animal feed and/or textile industry field.
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| 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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| 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 |
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| WO2023236638A1 (en) * | 2022-06-09 | 2023-12-14 | 中国农业科学院北京畜牧兽医研究所 | Thermal stability-improved glucose oxidase goxm10 mutant e361p, derivative mutant thereof, and use thereof |
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