CN110577939A - glucose oxidase mutant with improved heat resistance as well as coding gene and application thereof - Google Patents

glucose oxidase mutant with improved heat resistance as well as coding gene and application thereof Download PDF

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CN110577939A
CN110577939A CN201810579016.0A CN201810579016A CN110577939A CN 110577939 A CN110577939 A CN 110577939A CN 201810579016 A CN201810579016 A CN 201810579016A CN 110577939 A CN110577939 A CN 110577939A
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肖志壮
方安然
薛海曌
张稳
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Qingdao Red Cherry Biotechnology Co Ltd
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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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)
    • C12Y101/03004—Glucose oxidase (1.1.3.4)

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Abstract

The invention provides a glucose oxidase mutant with improved heat resistance, a coding gene and application thereof, wherein the glucose oxidase mutant is obtained by performing protein engineering transformation on glucose oxidase from Aspergillus niger by adopting an directed evolution technology, specifically, an error-prone PCR method is used for mutating the glucose oxidase gene, and a high-throughput screening method is used for detecting positive mutation to obtain mutants with improved heat stability. The glucose oxidase mutant provided by the invention has good market application prospect and industrial value.

Description

glucose oxidase mutant with improved heat resistance as well as coding gene and application thereof
Technical Field
The invention belongs to the field of genetic engineering and enzyme engineering, and particularly relates to a glucose oxidase mutant with improved heat resistance, and a coding gene and application thereof.
Background
Glucose Oxidase (GOD) takes molecular oxygen as an electron acceptor and can specifically catalyze beta-D-glucose to generate gluconic acid and hydrogen peroxide. Glucose oxidase is widely applied at present, and GOD is an important tool enzyme of a glucose detection sensor and is more applied in the fields of biomedicine and food processing. The GOD is used as a food additive, and can inhibit bacteria, prolong the shelf life of commodities and prevent food browning. GOD must have a high level of enzyme activity and high enzyme stability if it is to be used industrially on a large scale. The majority of GODs derived from fungi are currently used industrially, and in view of production and application costs, GODs must be stable at higher temperatures and for longer periods of time, e.g. at the high temperatures of bread baking and feed pelleting. The optimization of GOD can utilize the combination of modern molecular technology and biological process engineering technology to realize an economically feasible enzyme production system, combine genetic engineering technology and protein engineering technology, and improve the stability of GOD so as to further improve the industrial value of GOD.
The directed evolution technology belongs to the field of irrational design of proteins, does not need to know the three-dimensional space structure of the proteins, simulates the process of natural evolution (random mutation, recombination and selection) in vitro, makes a large amount of variation of genes, and directionally selects gene sequences with required properties or functions. Directed evolution mimics the natural evolutionary mechanisms of mutation, recombination and selection in vitro, and is the extension and application of the darwinian evolution theory on the molecular level. Error-prone PCR (epPCR) is a technique of asexual evolution, which utilizes the base mismatch occurring during PCR to perform random mutagenesis on a specific gene.
Disclosure of Invention
The invention provides a glucose oxidase mutant with improved heat resistance, a coding gene and application thereof.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
The invention provides a glucose oxidase mutant GOD-T-1 with improved heat resistance, wherein the amino acid sequence of the glucose oxidase mutant GOD-T-1 is shown as SEQ ID NO: 3, the mutant GOD-T-1 has an amino acid sequence of SEQ ID NO: 1 is obtained by changing valine at position 112 of glucose oxidase to isoleucine.
The invention provides a glucose oxidase mutant GOD-T-2 with improved heat resistance, wherein the amino acid sequence of the glucose oxidase mutant GOD-T-2 is shown as SEQ ID NO: 4, the mutant GOD-T-2 has an amino acid sequence of SEQ ID NO: 1, the glucose oxidase is obtained by changing threonine to lysine at amino acid 39 and changing valine to isoleucine at amino acid 112.
The invention provides a glucose oxidase mutant GOD-T-3 with improved heat resistance, wherein the amino acid sequence of the glucose oxidase mutant GOD-T-3 is shown as SEQ ID NO: 5, the mutant GOD-T-3 has an amino acid sequence of SEQ ID NO: 1, the glucose oxidase is obtained by changing threonine to proline at amino acid 93 and changing valine to isoleucine at amino acid 112.
The invention provides a glucose oxidase mutant GOD-T-4 with improved heat resistance, wherein the amino acid sequence of the glucose oxidase mutant GOD-T-4 is shown as SEQ ID NO: 6, the mutant GOD-T-4 has an amino acid sequence of SEQ ID NO: 1, the glucose oxidase is obtained by changing valine at position 112 to isoleucine, amino acid at position 211 from glycine to asparagine, and tyrosine at position 512 to tryptophan.
The invention provides a glucose oxidase mutant GOD-T-5 with improved heat resistance, wherein the amino acid sequence of the glucose oxidase mutant GOD-T-5 is shown as SEQ ID NO: 7, the mutant GOD-T-5 has an amino acid sequence of SEQ ID NO: 1, the glucose oxidase is obtained by changing valine at position 112 to isoleucine, changing lysine to asparagine at position 279, and changing alanine at position 294 to threonine.
The invention provides the coding gene of GOD-T-1, the coding gene of GOD-T-2 or the coding gene of GOD-T-3.
the invention provides the coding gene of GOD-T-4 or the coding gene of GOD-T-5.
The invention provides an expression vector containing the coding gene of GOD-T-1, an expression vector containing the coding gene of GOD-T-2, an expression vector containing the coding gene of GOD-T-3, an expression vector containing the coding gene of GOD-T-4 or an expression vector containing the coding gene of GOD-T-5.
The invention provides application of glucose oxidase mutants GOD-T-1, GOD-T-2, GOD-T-3, GOD-T-4 or GOD-T-5 with improved heat resistance in preparation of animal feed additives.
The animals are fish, prawn, pig, chicken and duck.
The invention has the advantages and beneficial technical effects that: the invention aims to perform protein engineering modification on glucose oxidase derived from Aspergillus niger by using an directed evolution technology, and in order to achieve the aim, the invention uses an error-prone PCR method to mutate the glucose oxidase gene, and then detects positive mutation by using a high-throughput screening method to obtain mutants with improved thermal stability, compared with wild-type glucose oxidase GOD-1, the 5 mutants obtained by the invention, namely GOD-T-1, GOD-T-2, GOD-T-3, GOD-T-4 and GOD-T-5, have obviously improved thermal stability, and after the glucose oxidase is treated for 3min at 70 ℃, the residual enzyme activity is respectively improved by 1.2, 1.9, 2.9, 2.2 and 1.5 times. The glucose oxidase mutant provided by the invention has good market application prospect and industrial value.
Drawings
FIG. 1 is a diagram of alignment of glucose oxidase mutant GOD-T-1 with wild-type amino acid sequence;
FIG. 2 is a diagram of alignment of glucose oxidase mutant GOD-T-2 with wild-type amino acid sequence;
FIG. 3 is a diagram of alignment of glucose oxidase mutant GOD-T-3 with wild-type amino acid sequence;
FIG. 4 is a diagram of alignment of glucose oxidase mutant GOD-T-4 with wild-type amino acid sequence;
FIG. 5 is a diagram of alignment of the glucose oxidase mutant GOD-T-5 with the wild type amino acid sequence;
FIG. 6 shows the residual enzyme activities of glucose oxidase mutants GOD-T-1, GOD-T-2, GOD-T-3, GOD-T-4 and GOD-T-5 at different temperatures.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the accompanying drawings and specific embodiments.
The present invention uses conventional techniques and methods used in the field of molecular biology. The examples are only for illustrating the present invention and do not limit the scope of the present invention.
Example 1: construction of glucose oxidase GOD-1 mutation library by error-prone PCR (error-prone PCR) method
Glucose oxidase gene GOD-1 derived from Aspergillus niger is composed of 589 amino acids (shown as SEQ ID NO: 1), and the glucose oxidase gene GOD-1 is synthesized by a whole-gene synthesis method (shown as SEQ ID NO: 2). The synthesized gene has the signal peptide sequence removed from the original gene and has EcoR I and Not I enzyme cutting sites at two ends. The glucose oxidase GOD-1 gene was amplified using the synthesized gene as a template, and mutations were randomly introduced using GeneMorpt II random mutation PCR kit (Stratagene).
The primers used were:
5′-GCGCGAATTCTTGCCACATTACATTAGATCCAAC-3′(SEQ ID No：8)，
5′-TAAAGCGGCCGCTTATTGCATAGAAGCGTAATCTTCC-3' (SEQ ID No: 9). The EcoR I and Not I cleavage sites are underlined, respectively.
The reaction conditions are as follows: pre-denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 60s, annealing at 58 ℃ for 60s and extension at 72 ℃ for 2min for 30 cycles, and recovering the target gene fragment.
The target fragment was digested with EcoR I and Not I, and ligated with the similarly digested pET 21a (+) vector (ampicillin resistance gene) using Ligase. And (2) transforming the connected fragments into escherichia coli BL21-DE3, coating an LB plate containing ampicillin, performing inverted culture at 37 ℃, picking a monoclonal to a 96-well plate after a transformant appears on the plate, performing shake culture at 30 ℃ and 220rpm for 12h with each well containing 150uL of LB culture medium (containing 1mM IPTG and 50 mu g/mL ampicillin), placing the well plate at-20 ℃, and repeatedly freezing and thawing to break the wall to obtain a crude enzyme solution containing glucose oxidase. Respectively taking out 5uL of lysate to two new 96-well plates, treating one of the two new 96-well plates at 70 ℃ for 3min, adding color development solution containing o-dianisidine methanol buffer solution, glucose buffer solution and horseradish peroxidase solution into the two 96-well plates, reacting for 3min at 37 ℃, adding 100uL2M sulfuric acid to terminate the reaction, and determining the residual enzyme activity according to the color development reaction.
And taking a strain with higher residual activity than the wild GOD-1 into a new 96-well culture plate, and repeatedly screening. 1 mutant was screened and numbered GOD-T-1, which had a residual activity 1.2 times that of the wild-type control, and was subjected to DNA sequencing.
the sequencing result shows that, as shown in FIG. 1, the error-prone PCR of the round obtained a mutant GOD-T-1 containing a single-point mutation of V112I, and the amino acid sequence of the mutant GOD-T-1 is SEQ ID NO: 3.
GOD-T-1: valine V at position 112 to isoleucine I (GTT to ATT).
Example 2: second round error-prone PCR construction of mutant libraries and screening of mutant library constructions
The process for constructing the mutant GOD-T-1 with improved heat resistance, which is selected by the first round of error-prone PCR method, is used as a template for the second round of error-prone PCR, and the process, primers and PCR reaction conditions for constructing the mutant library are the same as those in example 1.
Through a second round of error-prone PCR, a large number of mutant gene fragments were also obtained. The constructed mutant was transferred to Escherichia coli expression strain BL21-DE3, GOD-T-1 was used as a control in screening for heat resistance positive mutation, the rest of the procedures were the same as in example 2, and a strain having a higher residual activity than that of the mutant GOD-T-1 was taken out and placed in a new 96-well culture plate, and repeated screening was carried out.
4 mutants are obtained in the round of screening, named as GOD-T-2, GOD-T-3, GOD-T-4 and GOD-T-5 respectively, the thermal stability of the mutants is higher than that of GOD-T-1, and the mutant strains are picked and sent to a sequencing company for sequencing.
The sequencing results showed that, as shown in fig. 2, fig. 3, fig. 4 and fig. 5, the error-prone PCR of this round yielded a mutant GOD-T-2 containing two point mutations of T39K and V112I, the amino acid sequence of which is SEQ ID NO: 4. a mutant GOD-T-3 containing two-point mutation of T93P and V112I, wherein the amino acid sequence of the mutant GOD-T-3 is SEQ ID NO: 5. a mutant GOD-T-4 containing three point mutations of V112I, G211D and Y512W, and the amino acid sequence of the mutant GOD-T-4 is SEQ ID NO: 6. a mutant GOD-T-5 containing three point mutations of V112I, K279N and A294T, and the amino acid sequence of the mutant GOD-T-5 is SEQ ID NO: 7.
GOD-T-2: threonine 39 of the enzyme was changed to lysine K (DNA sequence ACT to AAA) and valine V112 to isoleucine I (GTT to ATT).
GOD-T-3: threonine T at position 93 of the enzyme is changed to proline P (ACT to CCA), valine V at position 112 to isoleucine I (GTT to ATT).
GOD-T-4: valine V at position 112 to isoleucine I (GTT to ATT), glycine G at position 211 to asparagine D (GGA to GAC), tyrosine Y at position 512 to tryptophan W (TAC to TGG).
GOD-T-5: valine V at position 112 was changed to isoleucine I (GTT to ATT), lysine K at position 279 was changed to asparagine N (AAG to AAC), and alanine A at position 294 was changed to threonine T (GCT to ACT).
Example 3: construction of Pichia pastoris engineering strain
PCR amplification was performed using the primers described in example 1, using the mutants obtained in examples 1 and 2 as templates, and the PCR reaction conditions were the same as in example 1.
The amplified glucose oxidase mutant gene fragments obtained in example 1 and example 2, and wild-type gene fragments were linked to expression vector pPIC9K via EcoR I and Not I sites to construct expression vectors pPIC9K-GOD-1, pPIC9K-GOD-T-1, pPIC9K-GOD-T-2, pPIC9K-GOD-T-3, pPIC9K-GOD-T-4, and pPIC 9K-GOD-T-5. The expression vector is transferred into escherichia coli DH5 alpha competence, and a large amount of plasmids are extracted after transformants are picked.
The expression plasmid was linearized with SalI, and the linearized Fragment was purified and collected with a Fragment purification Kit (TaKaRa MiniBEST DNA Fragment purification Kit), transformed into Pichia pastoris GS115 by the electrotransformation method, and coated on MD plates. And (3) coating colonies growing on the MD plate on YPD plates of geneticin with gradually increasing concentrations (1mg/mL, 2mg/mL, 4mg/mL and 8mg/mL) to screen positive transformants with multiple copies, so as to obtain the pichia pastoris recombinant strain.
Transformants of 6 genes are respectively named as Pichia pastoris GOD-1(Pichia pastoris GOD-1), Pichia pastoris GOD-T-1(Pichia pastoris GOD-T-1), Pichia pastoris GOD-T-2(Pichia pastoris GOD-T-2), Pichia pastoris GOD-T-3(Pichia pastoris GOD-T-3), Pichia pastoris GOD-T-4 (Pichia pastoris GOD-T-4) and Pichia pastoris GOD-T-5(Pichia pastoris GOD-T-5), transformants of each gene are respectively selected and transferred into BMGY culture medium, after shaking culture for 18h at 30 ℃, 220rpm, thalli are obtained by centrifugation, a proper amount of thalli are transferred into BMMY culture medium, the thalli concentration is enabled to reach OD600 ═ 1, the oscillating culture is continued at 30 ℃, 220rpm, methanol was added at 1% culture volume every 24 h. After the induction expression for 4d, the culture solution is centrifuged to obtain a supernatant, and the supernatant is subjected to glucose oxidase activity determination and thermal stability determination.
Example 4: determination of enzyme activity and thermal stability of mutant and wild type expression product
The enzyme activity determination method comprises the following steps:
Taking 2.5mL of o-dianisidine buffer solution (prepared by adding 0.1mL of 1% o-dianisidine methanol stock solution into 12mL of 0.1M phosphate buffer solution with pH 6.0), adding 0.3mL of 18% glucose solution and 0.1mL of 0.03% peroxidase solution into a colorimetric tube, preserving the temperature at 37 ℃ for 5min, adding 0.1mL of glucose oxidase enzyme solution (adding 0.1mL of distilled water into a blank tube), reacting for 3min, adding 2mL of 2M sulfuric acid, and uniformly mixing to terminate the reaction. The blank is determined at a wavelength of 540nm using a standard blank as a blank control (A)0) And a sample solution (A)1) The absorbance of (a). Result in Δ A ═ A1-A0
Calculating the enzyme activity of the sample:
X＝(ΔA×n×3)/(11.3×t×0.1)
T-time of measurement, min
0.1-sample volume, mL
11.3-extinction coefficient
N-dilution multiple
3-volume of reaction solution, mL
enzyme activity units and definitions: under the conditions of pH5.5 and 37 deg.C, 1.0 μmol/min of beta-D-glucose can be oxidized into gluconic acid and H2O2The amount of enzyme (c) is one unit.
The fermentation supernatant described in example 3 was diluted to about 20U/mL with a phosphate buffer solution of pH6.0, and after treatment at 70 ℃ for 3min, the residual enzyme activity was determined, and the relative enzyme activity was calculated with the enzyme activity of the untreated sample as 100%. As shown in FIG. 6, the wild-type glucose oxidase was treated at 70 ℃ for 3min, and only 23% of the enzyme activity remained, while the mutants, GOD-T-1, GOD-T-2, GOD-T-3, GOD-T-4 and GOD-T-5, were also treated at 70 ℃ for 3min and still maintained 30-60% of the enzyme activity. Compared with wild genes, the heat resistance of the gene is respectively improved by 1.2, 1.9, 2.9, 2.2, 1.5 and 1.3 times.
therefore, compared with the wild type, the heat resistance of the mutated glucose oxidase is greatly improved, and the application of the mutated glucose oxidase in industry and agriculture is more facilitated.
Example 5: culture application experiment of glucose oxidase
5.1 Experimental animals
The carps used in the experiment are all individuals which are incubated in the same batch, have regular specifications and good growth states, the carps are temporarily cultured in a circular temporary culture pond with the diameter of 5m and the height of 1.6m before the experiment, and are separately cultured in a glass fish tank with the length of 80cm multiplied by 60cm multiplied by 40cm after being domesticated for 10 days by using a carp compound feed without any enzyme preparation. The initial average body weight (12.33 +/-0.60) g of the carps in each treatment group has no significant difference.
5.2 materials of the experiment
the carp feed formula comprises: 12% of fish meal, 25% of soybean meal, 20% of rapeseed meal, 7% of cottonseed meal, 15% of corn germ cake, 16% of wheat bran and 5% of additive;
A glucose oxidase preparation (produced by the glucose oxidase strain provided by the invention, i.e. Pichia pastoris GOD-T-3);
Sieving feed raw materials and enzyme preparation raw materials with 40 mesh sieve, stirring, extruding with small granulator (maximum extrusion temperature is 70 deg.C) to obtain hard granule feed with diameter of 2mm, oven drying, and storing in ventilated and cool dry place.
Water vat, water pipe, water pump, air pump, gauze.
5.3 design of the experiment
After the experimental carp is temporarily raised for 10 days, the 350 tails of healthy and disease-free carps with the weight of 11-13g are selected and randomly divided into 5 groups, each group is 7 in number, each group is 8 in number, and the grouping condition is shown in table 1.
TABLE 1 Experimental grouping
Numbering Grouping Treatment of
1 Control group Carp basic ration
2 experimental group 1 carp basic ration + GOD0.5g/kg
3 Experimental group 2 Carp basic ration + GOD1g/kg
5.4 Breeding management
The experiment is carried out in a glass jar indoors, dissolved oxygen is kept at 5-7mg/L, water temperature is controlled at 25-28 ℃, pH value is 7.6-8.2, 8: feeding 2 times at a ratio of 00: 16:00 regularly, wherein the feeding amount is 2-5% of the body weight, the feeding amount is adjusted along with the growth and the ingestion of the carps, the feeding is stopped 12 hours before the test is finished, and the body weight of each group is weighed. Changing water every two days, wherein the water changing amount is not more than 1/3, disinfecting the water with norweck every 10 days, and keeping the whole experiment for 45 days.
5.5 results of the experiment
TABLE 245 Tian carp culture experiment results
As can be seen from table 2, in the 45-day growth cycle, the weight gain rates of the experimental group 1 and the experimental group 2 are both significantly higher than that of the blank control group (P <0.01), and are respectively increased by 10.27% and 25.73% compared with that of the blank control group, wherein the weight gain rate of the experimental group 2 is the highest; the specific growth rate of the carps can be obviously improved by the experimental group 1 and the experimental group 2, both are obviously higher than that of a blank control group (P <0.01), and are respectively improved by 6.47% and 15.42% compared with the blank control group, wherein the specific growth rate of the carps in the experimental group 2 is the highest; the bait coefficient of the carp can be obviously reduced by the experimental group 1 and the experimental group 2, the bait coefficient is obviously lower than that of a blank control group (P <0.01), the bait coefficient is respectively reduced by 15.57% and 20.42% compared with that of the blank control group, and the bait coefficient of the carp in the experimental group 2 is the lowest. According to the data, the growth performance of the carps is greatly influenced by adding the glucose oxidase, the growth of the carps can be obviously improved, and the production efficiency of cultivation is increased. In the production practice, the GOD is mixed with aquatic feed for use, so that the weight gain of a human body can be improved, the feed coefficient can be improved, the death rate can be reduced, the excretion of organic matters in excrement can be reduced, and the aquaculture water environment can be improved.
The application of the glucose oxidase of the present invention is not limited to carp application, as the glucose oxidase can be added to basal diet, and can be used for feeding other livestock and poultry and aquatic animals. Can be added into compound feed of pig, chicken and prawn in the course of breeding.
the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> Qingdao red cherry Biotechnology Co., Ltd
<120> glucose oxidase mutant with improved heat resistance, and coding gene and application thereof
<160> 9
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Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val
530 535 540
Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr
545 550 555 560
Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys
565 570 575
Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln
580 585
<210> 2
<211> 1770
<212> DNA
<213> Aspergillus niger (Aspergillus niger)
<400> 2
ttgccacatt acattagatc caacggtatt gaagcatcct tgttgactga tcctaaggat 60
gtttcaggta gaactgttga ttacattatt gctggaggtg gattgactgg tttgactact 120
gctgctagat tgactgaaaa cccaaacatt tcagttttgg ttattgaatc aggttcttac 180
gaatccgata gaggtcctat tattgaagat ttgaacgctt acggggacat ttttggttct 240
tccgttgatc atgcttacga aactgttgaa ttggctacta acaaccaaac tgctttgatt 300
agatccggta acggattggg tggttccact ttggttaacg gaggtacttg gactagacct 360
cataaggctc aagttgattc ctgggaaact gtttttggta acgaaggatg gaactgggat 420
aacgttgctg cttactcctt gcaagctgaa cgagcgcggg cccctaacgc taagcaaatt 480
gctgctggtc attactttaa cgcttcctgt catggagtta acggaactgt tcatgctggt 540
ccaagagata ctggagacga ttactcccca attgttaagg ctttgatgtc cgctgttgaa 600
gataggggtg ttccaactaa gaaggatttt ggatgtggcg acccacatgg agtttctatg 660
tttccaaaca ctttgcatga agatcaagtt agatccgatg ctgctagaga atggttgttg 720
cctaactacc aaagacctaa cttgcaagtt ttgactggtc aatacgttgg taaagttttg 780
ttgtcacaaa acggaactac tcctagagct gttggagttg aatttggtac tcataagggt 840
aacactcata acgtttacgc taagcatgaa gttttgttgg ctgctggttc cgctgtttca 900
ccaactattt tggaatactc cggtattggt atgaagtcta ttttggaacc attgggtatt 960
gatactgttg ttgatttgcc agttggattg aacttgcaag atcaaactac tgctactgtt 1020
agatccagaa ttacttccgc tggagctgga caaggacaag ctgcttggtt tgctactttt 1080
aacgaaactt ttggagatta ctccgaaaag gctcatgaat tgttgaacac taagttggaa 1140
caatgggctg aagaagctgt tgctagaggt ggttttcata acactactgc tttgttgatt 1200
caatacgaaa actacagaga ttggattgtt aaccataacg ttgcttactc agaattgttt 1260
ttggatactg ctggagttgc ttcctttgat gtttgggatt tgttgccctt cactagagga 1320
tacgttcata ttttggataa agacccatac ttgcatcatt ttgcttacga tccacaatac 1380
tttttgaacg aattggattt gttgggacaa gctgctgcta ctcaattggc tagaaacatt 1440
tctaactcgg gtgctatgca aacttacttt gctggagaaa ctattccagg tgacaacttg 1500
gcttacgatg ctgatttgtc cgcttggact gaatacattc cataccattt tagacctaac 1560
taccacggag ttggtacttg ttctatgatg ccaaaggaaa tgggaggtgt tgttgataac 1620
gctgctagag tttacggagt tcaaggattg agagttattg atggttcaat tccaccaact 1680
caaatgtctt cacatgttat gactgtgttt tacgctatgg ctttgaagat ttcagatgct 1740
attttggaag attacgcttc tatgcaataa 1770
<210> 3
<211> 589
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Leu Pro His Tyr Ile Arg Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr
1 5 10 15
Asp Pro Lys Asp Val Ser Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly
20 25 30
Gly Gly Leu Thr Gly Leu Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro
35 40 45
Asn Ile Ser Val Leu Val Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg
50 55 60
Gly Pro Ile Ile Glu Asp Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser
65 70 75 80
Ser Val Asp His Ala Tyr Glu Thr Val Glu Leu Ala Thr Asn Asn Gln
85 90 95
Thr Ala Leu Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile
100 105 110
Asn Gly Gly Thr Trp Thr Arg Pro His Lys Ala Gln Val Asp Ser Trp
115 120 125
Glu Thr Val Phe Gly Asn Glu Gly Trp Asn Trp Asp Asn Val Ala Ala
130 135 140
Tyr Ser Leu Gln Ala Glu Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile
145 150 155 160
Ala Ala Gly His Tyr Phe Asn Ala Ser Cys His Gly Val Asn Gly Thr
165 170 175
Val His Ala Gly Pro Arg Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val
180 185 190
Lys Ala Leu Met Ser Ala Val Glu Asp Arg Gly Val Pro Thr Lys Lys
195 200 205
Asp Phe Gly Cys Gly Asp Pro His Gly Val Ser Met Phe Pro Asn Thr
210 215 220
Leu His Glu Asp Gln Val Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu
225 230 235 240
Pro Asn Tyr Gln Arg Pro Asn Leu Gln Val Leu Thr Gly Gln Tyr Val
245 250 255
Gly Lys Val Leu Leu Ser Gln Asn Gly Thr Thr Pro Arg Ala Val Gly
260 265 270
Val Glu Phe Gly Thr His Lys Gly Asn Thr His Asn Val Tyr Ala Lys
275 280 285
His Glu Val Leu Leu Ala Ala Gly Ser Ala Val Ser Pro Thr Ile Leu
290 295 300
Glu Tyr Ser Gly Ile Gly Met Lys Ser Ile Leu Glu Pro Leu Gly Ile
305 310 315 320
Asp Thr Val Val Asp Leu Pro Val Gly Leu Asn Leu Gln Asp Gln Thr
325 330 335
Thr Ala Thr Val Arg Ser Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly
340 345 350
Gln Ala Ala Trp Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser
355 360 365
Glu Lys Ala His Glu Leu Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu
370 375 380
Glu Ala Val Ala Arg Gly Gly Phe His Asn Thr Thr Ala Leu Leu Ile
385 390 395 400
Gln Tyr Glu Asn Tyr Arg Asp Trp Ile Val Asn His Asn Val Ala Tyr
405 410 415
Ser Glu Leu Phe Leu Asp Thr Ala Gly Val Ala Ser Phe Asp Val Trp
420 425 430
Asp Leu Leu Pro Phe Thr Arg Gly Tyr Val His Ile Leu Asp Lys Asp
435 440 445
Pro Tyr Leu His His Phe Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu
450 455 460
Leu Asp Leu Leu Gly Gln Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile
465 470 475 480
Ser Asn Ser Gly Ala Met Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro
485 490 495
Gly Asp Asn Leu Ala Tyr Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr
500 505 510
Ile Pro Tyr His Phe Arg Pro Asn Tyr His Gly Val Gly Thr Cys Ser
515 520 525
Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val
530 535 540
Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr
545 550 555 560
Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys
565 570 575
Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln
580 585
<210> 4
<211> 589
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Leu Pro His Tyr Ile Arg Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr
1 5 10 15
Asp Pro Lys Asp Val Ser Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly
20 25 30
Gly Gly Leu Thr Gly Leu Lys Thr Ala Ala Arg Leu Thr Glu Asn Pro
35 40 45
Asn Ile Ser Val Leu Val Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg
50 55 60
Gly Pro Ile Ile Glu Asp Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser
65 70 75 80
Ser Val Asp His Ala Tyr Glu Thr Val Glu Leu Ala Thr Asn Asn Gln
85 90 95
Thr Ala Leu Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile
100 105 110
Asn Gly Gly Thr Trp Thr Arg Pro His Lys Ala Gln Val Asp Ser Trp
115 120 125
Glu Thr Val Phe Gly Asn Glu Gly Trp Asn Trp Asp Asn Val Ala Ala
130 135 140
Tyr Ser Leu Gln Ala Glu Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile
145 150 155 160
Ala Ala Gly His Tyr Phe Asn Ala Ser Cys His Gly Val Asn Gly Thr
165 170 175
Val His Ala Gly Pro Arg Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val
180 185 190
Lys Ala Leu Met Ser Ala Val Glu Asp Arg Gly Val Pro Thr Lys Lys
195 200 205
Asp Phe Gly Cys Gly Asp Pro His Gly Val Ser Met Phe Pro Asn Thr
210 215 220
Leu His Glu Asp Gln Val Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu
225 230 235 240
Pro Asn Tyr Gln Arg Pro Asn Leu Gln Val Leu Thr Gly Gln Tyr Val
245 250 255
Gly Lys Val Leu Leu Ser Gln Asn Gly Thr Thr Pro Arg Ala Val Gly
260 265 270
Val Glu Phe Gly Thr His Lys Gly Asn Thr His Asn Val Tyr Ala Lys
275 280 285
His Glu Val Leu Leu Ala Ala Gly Ser Ala Val Ser Pro Thr Ile Leu
290 295 300
Glu Tyr Ser Gly Ile Gly Met Lys Ser Ile Leu Glu Pro Leu Gly Ile
305 310 315 320
Asp Thr Val Val Asp Leu Pro Val Gly Leu Asn Leu Gln Asp Gln Thr
325 330 335
Thr Ala Thr Val Arg Ser Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly
340 345 350
Gln Ala Ala Trp Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser
355 360 365
Glu Lys Ala His Glu Leu Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu
370 375 380
Glu Ala Val Ala Arg Gly Gly Phe His Asn Thr Thr Ala Leu Leu Ile
385 390 395 400
Gln Tyr Glu Asn Tyr Arg Asp Trp Ile Val Asn His Asn Val Ala Tyr
405 410 415
Ser Glu Leu Phe Leu Asp Thr Ala Gly Val Ala Ser Phe Asp Val Trp
420 425 430
Asp Leu Leu Pro Phe Thr Arg Gly Tyr Val His Ile Leu Asp Lys Asp
435 440 445
Pro Tyr Leu His His Phe Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu
450 455 460
Leu Asp Leu Leu Gly Gln Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile
465 470 475 480
Ser Asn Ser Gly Ala Met Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro
485 490 495
Gly Asp Asn Leu Ala Tyr Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr
500 505 510
Ile Pro Tyr His Phe Arg Pro Asn Tyr His Gly Val Gly Thr Cys Ser
515 520 525
Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val
530 535 540
Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr
545 550 555 560
Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys
565 570 575
Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln
580 585
<210> 5
<211> 589
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Leu Pro His Tyr Ile Arg Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr
1 5 10 15
Asp Pro Lys Asp Val Ser Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly
20 25 30
Gly Gly Leu Thr Gly Leu Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro
35 40 45
Asn Ile Ser Val Leu Val Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg
50 55 60
Gly Pro Ile Ile Glu Asp Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser
65 70 75 80
Ser Val Asp His Ala Tyr Glu Thr Val Glu Leu Ala Pro Asn Asn Gln
85 90 95
Thr Ala Leu Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile
100 105 110
Asn Gly Gly Thr Trp Thr Arg Pro His Lys Ala Gln Val Asp Ser Trp
115 120 125
Glu Thr Val Phe Gly Asn Glu Gly Trp Asn Trp Asp Asn Val Ala Ala
130 135 140
Tyr Ser Leu Gln Ala Glu Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile
145 150 155 160
Ala Ala Gly His Tyr Phe Asn Ala Ser Cys His Gly Val Asn Gly Thr
165 170 175
Val His Ala Gly Pro Arg Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val
180 185 190
Lys Ala Leu Met Ser Ala Val Glu Asp Arg Gly Val Pro Thr Lys Lys
195 200 205
Asp Phe Gly Cys Gly Asp Pro His Gly Val Ser Met Phe Pro Asn Thr
210 215 220
Leu His Glu Asp Gln Val Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu
225 230 235 240
Pro Asn Tyr Gln Arg Pro Asn Leu Gln Val Leu Thr Gly Gln Tyr Val
245 250 255
Gly Lys Val Leu Leu Ser Gln Asn Gly Thr Thr Pro Arg Ala Val Gly
260 265 270
Val Glu Phe Gly Thr His Lys Gly Asn Thr His Asn Val Tyr Ala Lys
275 280 285
His Glu Val Leu Leu Ala Ala Gly Ser Ala Val Ser Pro Thr Ile Leu
290 295 300
Glu Tyr Ser Gly Ile Gly Met Lys Ser Ile Leu Glu Pro Leu Gly Ile
305 310 315 320
Asp Thr Val Val Asp Leu Pro Val Gly Leu Asn Leu Gln Asp Gln Thr
325 330 335
Thr Ala Thr Val Arg Ser Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly
340 345 350
Gln Ala Ala Trp Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser
355 360 365
Glu Lys Ala His Glu Leu Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu
370 375 380
Glu Ala Val Ala Arg Gly Gly Phe His Asn Thr Thr Ala Leu Leu Ile
385 390 395 400
Gln Tyr Glu Asn Tyr Arg Asp Trp Ile Val Asn His Asn Val Ala Tyr
405 410 415
Ser Glu Leu Phe Leu Asp Thr Ala Gly Val Ala Ser Phe Asp Val Trp
420 425 430
Asp Leu Leu Pro Phe Thr Arg Gly Tyr Val His Ile Leu Asp Lys Asp
435 440 445
Pro Tyr Leu His His Phe Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu
450 455 460
Leu Asp Leu Leu Gly Gln Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile
465 470 475 480
Ser Asn Ser Gly Ala Met Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro
485 490 495
Gly Asp Asn Leu Ala Tyr Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr
500 505 510
Ile Pro Tyr His Phe Arg Pro Asn Tyr His Gly Val Gly Thr Cys Ser
515 520 525
Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val
530 535 540
Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr
545 550 555 560
Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys
565 570 575
Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln
580 585
<210> 6
<211> 589
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Leu Pro His Tyr Ile Arg Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr
1 5 10 15
Asp Pro Lys Asp Val Ser Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly
20 25 30
Gly Gly Leu Thr Gly Leu Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro
35 40 45
Asn Ile Ser Val Leu Val Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg
50 55 60
Gly Pro Ile Ile Glu Asp Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser
65 70 75 80
Ser Val Asp His Ala Tyr Glu Thr Val Glu Leu Ala Thr Asn Asn Gln
85 90 95
Thr Ala Leu Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile
100 105 110
Asn Gly Gly Thr Trp Thr Arg Pro His Lys Ala Gln Val Asp Ser Trp
115 120 125
Glu Thr Val Phe Gly Asn Glu Gly Trp Asn Trp Asp Asn Val Ala Ala
130 135 140
Tyr Ser Leu Gln Ala Glu Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile
145 150 155 160
Ala Ala Gly His Tyr Phe Asn Ala Ser Cys His Gly Val Asn Gly Thr
165 170 175
Val His Ala Gly Pro Arg Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val
180 185 190
Lys Ala Leu Met Ser Ala Val Glu Asp Arg Gly Val Pro Thr Lys Lys
195 200 205
Asp Phe Asp Cys Gly Asp Pro His Gly Val Ser Met Phe Pro Asn Thr
210 215 220
Leu His Glu Asp Gln Val Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu
225 230 235 240
Pro Asn Tyr Gln Arg Pro Asn Leu Gln Val Leu Thr Gly Gln Tyr Val
245 250 255
Gly Lys Val Leu Leu Ser Gln Asn Gly Thr Thr Pro Arg Ala Val Gly
260 265 270
Val Glu Phe Gly Thr His Lys Gly Asn Thr His Asn Val Tyr Ala Lys
275 280 285
His Glu Val Leu Leu Ala Ala Gly Ser Ala Val Ser Pro Thr Ile Leu
290 295 300
Glu Tyr Ser Gly Ile Gly Met Lys Ser Ile Leu Glu Pro Leu Gly Ile
305 310 315 320
Asp Thr Val Val Asp Leu Pro Val Gly Leu Asn Leu Gln Asp Gln Thr
325 330 335
Thr Ala Thr Val Arg Ser Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly
340 345 350
Gln Ala Ala Trp Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser
355 360 365
Glu Lys Ala His Glu Leu Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu
370 375 380
Glu Ala Val Ala Arg Gly Gly Phe His Asn Thr Thr Ala Leu Leu Ile
385 390 395 400
Gln Tyr Glu Asn Tyr Arg Asp Trp Ile Val Asn His Asn Val Ala Tyr
405 410 415
Ser Glu Leu Phe Leu Asp Thr Ala Gly Val Ala Ser Phe Asp Val Trp
420 425 430
Asp Leu Leu Pro Phe Thr Arg Gly Tyr Val His Ile Leu Asp Lys Asp
435 440 445
Pro Tyr Leu His His Phe Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu
450 455 460
Leu Asp Leu Leu Gly Gln Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile
465 470 475 480
Ser Asn Ser Gly Ala Met Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro
485 490 495
Gly Asp Asn Leu Ala Tyr Asp Ala Asp Leu Ser Ala Trp Thr Glu Trp
500 505 510
Ile Pro Tyr His Phe Arg Pro Asn Tyr His Gly Val Gly Thr Cys Ser
515 520 525
Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val
530 535 540
Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr
545 550 555 560
Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys
565 570 575
Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln
580 585
<210> 7
<211> 589
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Leu Pro His Tyr Ile Arg Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr
1 5 10 15
Asp Pro Lys Asp Val Ser Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly
20 25 30
Gly Gly Leu Thr Gly Leu Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro
35 40 45
Asn Ile Ser Val Leu Val Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg
50 55 60
Gly Pro Ile Ile Glu Asp Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser
65 70 75 80
Ser Val Asp His Ala Tyr Glu Thr Val Glu Leu Ala Thr Asn Asn Gln
85 90 95
Thr Ala Leu Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile
100 105 110
Asn Gly Gly Thr Trp Thr Arg Pro His Lys Ala Gln Val Asp Ser Trp
115 120 125
Glu Thr Val Phe Gly Asn Glu Gly Trp Asn Trp Asp Asn Val Ala Ala
130 135 140
Tyr Ser Leu Gln Ala Glu Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile
145 150 155 160
Ala Ala Gly His Tyr Phe Asn Ala Ser Cys His Gly Val Asn Gly Thr
165 170 175
Val His Ala Gly Pro Arg Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val
180 185 190
Lys Ala Leu Met Ser Ala Val Glu Asp Arg Gly Val Pro Thr Lys Lys
195 200 205
Asp Phe Gly Cys Gly Asp Pro His Gly Val Ser Met Phe Pro Asn Thr
210 215 220
Leu His Glu Asp Gln Val Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu
225 230 235 240
Pro Asn Tyr Gln Arg Pro Asn Leu Gln Val Leu Thr Gly Gln Tyr Val
245 250 255
Gly Lys Val Leu Leu Ser Gln Asn Gly Thr Thr Pro Arg Ala Val Gly
260 265 270
Val Glu Phe Gly Thr His Asn Gly Asn Thr His Asn Val Tyr Ala Lys
275 280 285
His Glu Val Leu Leu Thr Ala Gly Ser Ala Val Ser Pro Thr Ile Leu
290 295 300
Glu Tyr Ser Gly Ile Gly Met Lys Ser Ile Leu Glu Pro Leu Gly Ile
305 310 315 320
Asp Thr Val Val Asp Leu Pro Val Gly Leu Asn Leu Gln Asp Gln Thr
325 330 335
Thr Ala Thr Val Arg Ser Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly
340 345 350
Gln Ala Ala Trp Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser
355 360 365
Glu Lys Ala His Glu Leu Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu
370 375 380
Glu Ala Val Ala Arg Gly Gly Phe His Asn Thr Thr Ala Leu Leu Ile
385 390 395 400
Gln Tyr Glu Asn Tyr Arg Asp Trp Ile Val Asn His Asn Val Ala Tyr
405 410 415
Ser Glu Leu Phe Leu Asp Thr Ala Gly Val Ala Ser Phe Asp Val Trp
420 425 430
Asp Leu Leu Pro Phe Thr Arg Gly Tyr Val His Ile Leu Asp Lys Asp
435 440 445
Pro Tyr Leu His His Phe Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu
450 455 460
Leu Asp Leu Leu Gly Gln Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile
465 470 475 480
Ser Asn Ser Gly Ala Met Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro
485 490 495
Gly Asp Asn Leu Ala Tyr Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr
500 505 510
Ile Pro Tyr His Phe Arg Pro Asn Tyr His Gly Val Gly Thr Cys Ser
515 520 525
Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val
530 535 540
Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr
545 550 555 560
Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys
565 570 575
Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln
580 585
<210> 8
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gcgcgaattc ttgccacatt acattagatc caac 34
<210> 9
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
taaagcggcc gcttattgca tagaagcgta atcttcc 37

Claims (10)

1. The glucose oxidase mutant GOD-T-1 with improved heat resistance is characterized in that: the amino acid sequence of the glucose oxidase mutant GOD-T-1 is shown as SEQ ID NO: 3, the mutant GOD-T-1 has an amino acid sequence of SEQ ID NO: 1 is obtained by changing valine at position 112 of glucose oxidase to isoleucine.
2. The glucose oxidase mutant GOD-T-2 with improved heat resistance is characterized in that: the amino acid sequence of the glucose oxidase mutant GOD-T-2 is shown as SEQ ID NO: 4, the mutant GOD-T-2 has an amino acid sequence of SEQ ID NO: 1, the glucose oxidase is obtained by changing threonine to lysine at amino acid 39 and changing valine to isoleucine at amino acid 112.
3. The glucose oxidase mutant GOD-T-3 with improved heat resistance is characterized in that: the amino acid sequence of the glucose oxidase mutant GOD-T-3 is shown as SEQ ID NO: 5, the mutant GOD-T-3 has an amino acid sequence of SEQ ID NO: 1, the glucose oxidase is obtained by changing threonine to proline at amino acid 93 and changing valine to isoleucine at amino acid 112.
4. The glucose oxidase mutant GOD-T-4 with improved heat resistance is characterized in that: the amino acid sequence of the glucose oxidase mutant GOD-T-4 is shown as SEQ ID NO: 6, the mutant GOD-T-4 has an amino acid sequence of SEQ ID NO: 1, the glucose oxidase is obtained by changing valine at position 112 to isoleucine, amino acid at position 211 from glycine to asparagine, and tyrosine at position 512 to tryptophan.
5. The glucose oxidase mutant GOD-T-5 with improved heat resistance is characterized in that: the amino acid sequence of the glucose oxidase mutant GOD-T-5 is shown as SEQ ID NO: 7, the mutant GOD-T-5 has an amino acid sequence of SEQ ID NO: 1, the glucose oxidase is obtained by changing valine at position 112 to isoleucine, changing lysine to asparagine at position 279, and changing alanine at position 294 to threonine.
6. GOD-T-1 encoding gene according to claim 1, GOD-T-2 encoding gene according to claim 2, or GOD-T-3 encoding gene according to claim 3.
7. GOD-T-4 encoding gene according to claim 4 or GOD-T-5 encoding gene according to claim 5.
8. An expression vector comprising the gene encoding GOD-T-1 according to claim 1, an expression vector comprising the gene encoding GOD-T-2 according to claim 2, an expression vector comprising the gene encoding GOD-T-3 according to claim 3, an expression vector comprising the gene encoding GOD-T-4 according to claim 4, or an expression vector comprising the gene encoding GOD-T-5 according to claim 5.
9. Application of glucose oxidase mutant GOD-T-1, GOD-T-2, GOD-T-3, GOD-T-4 or GOD-T-5 with improved heat resistance in preparation of animal feed additive.
10. Use of a glucose oxidase mutant, GOD-T-1, GOD-T-2, GOD-T-3, GOD-T-4 or GOD-T-5, according to claim 9 for the preparation of an animal feed additive, characterized in that: the animals are fish, prawn, pig, chicken and duck.
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Cited By (7)

* Cited by examiner, † Cited by third party
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CN111004786A (en) * 2019-12-25 2020-04-14 广东溢多利生物科技股份有限公司 Glucose oxidase and carrier and application thereof
CN112877306A (en) * 2021-04-28 2021-06-01 中国农业科学院北京畜牧兽医研究所 Super heat-resistant glucose oxidase AtGOD, gene and application thereof
CN114517192A (en) * 2020-04-27 2022-05-20 青岛尚德生物技术有限公司 Protease mutant BLAPR1 with improved heat stability and coding gene and application thereof
CN114736879A (en) * 2022-06-09 2022-07-12 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
CN114736880A (en) * 2022-06-09 2022-07-12 中国农业科学院北京畜牧兽医研究所 Mutant D497N of glucose oxidase GoxM10 with improved acid stability as well as derivative mutant and application thereof
CN114736881A (en) * 2022-06-09 2022-07-12 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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CN111004786B (en) * 2019-12-25 2021-12-07 广东溢多利生物科技股份有限公司 Glucose oxidase and carrier and application thereof
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CN112877306A (en) * 2021-04-28 2021-06-01 中国农业科学院北京畜牧兽医研究所 Super heat-resistant glucose oxidase AtGOD, gene and application thereof
CN112877306B (en) * 2021-04-28 2021-07-20 中国农业科学院北京畜牧兽医研究所 Super-heat-resistant glucose oxidase AtGOD and gene and application thereof
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections
CN114736880A (en) * 2022-06-09 2022-07-12 中国农业科学院北京畜牧兽医研究所 Mutant D497N of glucose oxidase GoxM10 with improved acid stability as well as derivative mutant and application thereof
CN114736880B (en) * 2022-06-09 2022-09-27 中国农业科学院北京畜牧兽医研究所 Mutant D497N of glucose oxidase GoxM10 with improved acid stability as well as 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
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
CN114736881A (en) * 2022-06-09 2022-07-12 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
CN114736879A (en) * 2022-06-09 2022-07-12 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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