CN115029328A - Glucose oxidase mutant GOx-MUT 1-6 and coding gene and application thereof - Google Patents

Glucose oxidase mutant GOx-MUT 1-6 and coding gene and application thereof Download PDF

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CN115029328A
CN115029328A CN202210432859.4A CN202210432859A CN115029328A CN 115029328 A CN115029328 A CN 115029328A CN 202210432859 A CN202210432859 A CN 202210432859A CN 115029328 A CN115029328 A CN 115029328A
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CN115029328B (en
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江民华
李阳源
黄江
陈琼银
贺金玲
陈丽芝
何小梅
张顺斌
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Guangdong Vtr Bio Tech Co ltd
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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)
    • 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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    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/102—Plasmid DNA for yeast

Abstract

The invention relates to the technical field of genetic engineering, in particular to a glucose oxidase mutant GOx-MUT 1-6 and a coding gene and application thereof. The invention provides a glucose oxidase mutant which has the following advantages compared with a parent SEQ ID NO: compared with glucose oxidase shown in 7, the glucose oxidase has the advantages that the specific activity is remarkably improved under the condition that the thermal stability is not reduced, and the enzyme activity retention rate is over 70 percent after the glucose oxidase is treated at 75 ℃ for 5 min. Is beneficial to the application of the enzyme in industrial production.

Description

Glucose oxidase mutant GOx-MUT 1-6 and coding gene and application thereof
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a glucose oxidase mutant GOx-MUT 1-6 and a coding gene and application thereof.
Background
Glucose oxidase (GOD, EC 1.1.3.4) is an oxidoreductase enzyme, which is a dimer composed of two subunits containing 2 Flavin Adenine Dinucleotide (FAD) binding sites. Each monomer contains 2 distinct regions, one of which binds tightly to FAD non-covalently, predominantly the beta sheet; the other binds to the substrate β -D-glucose, supporting 1 antiparallel β -sheet by 4 α -helices. The GOD from different sources has different physicochemical properties, the molecular weight is 130-175 KDa, and the GOD can specifically catalyze beta-D-glucose to generate gluconic acid and hydrogen peroxide under the aerobic condition.
GOD is widely applied to the fields of food, chemical industry, biomedical treatment and the like. In the food industry, GOD can be used for catalyzing glucose to exhaust oxygen in a vacuum bag, inhibiting the growth and reproduction of microorganisms, prolonging the shelf life of food and improving the mouthfeel of flour products. In the chemical field, GOD is not only often applied to bleaching and decoloring processes, but also is a key enzyme in the production of gluconic acid and derivatives thereof. In the medical field, GOD is a key raw material of a glucose detection kit, is added into toothpaste to reduce the incidence rate of oral diseases, and can also be used as an electrode of a biological battery to provide continuous energy for a biosensor and an artificial organ. Furthermore, the use of GOD is proposed in the production of transgenic plants and other organisms with reduced sensitivity or increased resistance to pests or diseases. Therefore, the large-scale efficient production of GOD has important economic value. However, GOD is often subjected to high temperature during further processing, so that the enzymatic activity of GOD is inevitably reduced or even inactivated. For example, the short-term high-temperature process in the feed processing process can cause the inactivation of glucose oxidase, which affects the application effect of glucose oxidase, and the temperature resistance of glucose oxidase is more and more concerned on the premise that the yield of glucose oxidase is ensured.
GOD is widely distributed in animals, plants and microorganisms. At present, in industrial-level GOD production, Aspergillus niger and Penicillium are mainly used as production strains, and compared with Penicillium GOD, the GOD expressed by Aspergillus niger has good thermal stability, but the problems of low enzyme activity level and complex separation and purification exist. Pichia pastoris (Pichia pastoris) has clear genetic background, is easy to perform genetic operation, has moderate glycosylation of secreted protein, is a common host for expressing foreign protein, particularly utilizes P.pastoris to perform protein secretion expression, can greatly simplify the process of separation and purification, and has important application value.
Disclosure of Invention
The purpose of the present invention is to provide a glucose oxidase mutant having improved specific activity.
It is still another object of the present invention to provide a gene encoding the above-mentioned glucose oxidase mutant.
It is still another object of the present invention to provide a recombinant vector comprising the above glucose oxidase gene.
It is still another object of the present invention to provide a recombinant strain comprising the above glucose oxidase gene.
It is a further object of the present invention to provide a method of increasing the specific activity of glucose oxidase.
The invention further aims to provide application of the glucose oxidase mutant.
The glucose oxidase mutant according to the present invention has the following amino acid sequence:
as shown in SEQ ID NO: 7, the amino acids at positions 243 and 492 of the amino acid sequence shown in figure 7.
The glucose oxidase mutant according to the present invention, further, is represented by SEQ ID NO: 7 at least one of the amino acids at positions 11, 12, 14, 15, 16, 43, 51, 88, 163, 241, 343, 360 and 497 is substituted.
SEQ ID NO：7
SNGIEASLLKDPKLVAGRTYDYIIAGGGLAGLTVAEKLTENPNITVLVIESGSYESDR GPIIEDLNAYGEIFGTSVDHAYETVELATNNRTALIRSGNGLGGSTLINGGTWTRPH KAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNAACHGL NGTVHVGPRDTGDDYSPLMRALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLH EDQVRADAAREWLLPNYQRPNLRVLTGQYVGKVLLSQNATTPRAVGVEFGTHKS NTHNVYAKHEVLLSAGSTVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQT TSTVRSRITSAGAGQGQAAWFATFNETFGKYTEKAHELLNTKLEQWAEEAVARGG FHNTTALLIQYENYRDWIVKDNVAYSELFLDTGGVASFDVWDLLPFTRGYVHILD KDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGNNLA YDADLSAWVEYIPEHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVID GSIPPTQLSSHVMTVFYAMALKIADAVLADYASMQ。
The glucose oxidase mutant of the present invention has the substitution of the 243 amino acid as R243Q and the 492 amino acid as N492D or N492E.
The glucose oxidase mutant according to the present invention, wherein the substitution of the 11 th amino acid is D11N or D11A; a substitution at amino acid position 12 of P12F or P12Q; a substitution at amino acid position 14 of L14G or L14A; the substitution of amino acid at position 15 is V15Q, V15T, or V15S; a substitution of the amino acid at position 16 to a16N or a 16F; a substitution of amino acid 43 with N43D, N43G, N43H, or N43Q; a substitution of amino acid 51 to S51G or S51W; the substitution of amino acid 88 is N88D or N88Q; a substitution at amino acid 163 to a163S or a 163G; the substitution of amino acid 241 to N241H; a substitution at amino acid position 243 with R243Q; a substitution of amino acid 343, 343G, a343D or a343N at position 343; the substitution of amino acid 360 to K360D or K360H; a substitution at amino acid 492 to N492D or N492E; the amino acid substitution at position 497 is D497N, D497M or D497E.
The glucose oxidase mutant provided by the invention has an amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: and 6.
Wherein, the mutant GOx-MUT1 comprises the following mutation sites: P12F, A16N, N88D, A163S, R243Q, K360D, N492D (SEQ ID NO: 1);
mutant GOx-MUT2 contains the following mutation sites: N43D, N88Q, A163S, R243Q, K360D, N492D (SEQ ID NO: 2);
mutant GOx-MUT3 contains the following mutation sites: L14G, N88Q, A163S, R243Q, A343N, K360D, N492D (SEQ ID NO: 3);
mutant GOx-MUT4 contains the following mutation sites: D11N, S51G, A163S, R243Q, K360H, N492D, D497E (SEQ ID NO: 4);
mutant GOx-MUT5 contains the following mutation sites: V15Q, A163S, N241H, R243Q, N492E (SEQ ID NO: 5);
mutant GOx-MUT6 contains the following mutation sites: S51W, R243Q, A343N, K360D, N492D, D497M (SEQ ID NO: 6).
The glucose oxidase gene of the present invention encodes any one of the glucose oxidase mutants described above.
The nucleotide sequence of the parent glucose oxidase gene is shown as SEQ ID NO: shown in fig. 8.
SEQ ID NO：8：
tctaatggtattgaggcttccttgttgaaagacccaaaacttgtcgccggtagaacctacgactacatcattgccggtggtggtttgg ctggtttgaccgttgctgagaagttgaccgagaatcctaacatcactgttttggttattgagtccggttcctacgagtctgaccgtggt ccaattattgaggatttgaatgcctacggtgaaatcttcggaacttctgtcgaccacgcctatgagaccgttgagttggctactaaca atagaactgctttgatccgttccggtaacggtttgggaggatccactttgattaacggtggaacctggactagaccacataaagccc aagtcgactcctgggagactgtcttcggaaacgaaggttggaactgggactctgttgctgcttactcccttcaggctgaaagagctc gtgccccaaatgctaagcagatcgccgctggtcactactttaacgccgcatgccacggtttgaacggtactgttcacgttggacca cgtgatactggtgatgactactctccattgatgagagccttgatgtctgctgtcgaagatcgtggagtccctaccaagaaggacttg ggttgcggagaccctcatggtgtctccatgttcccaaacaccttgcacgaggaccaagttcgtgctgacgctgccagagaatggtt gcttcctaactaccagagaccaaacttgagggtcttgactggtcagtacgtcggtaaggtcttgttgtctcagaacgctaccacccc aagagctgttggtgtcgagttcggtactcacaagtctaacacccacaacgtctacgctaagcatgaggtccttttgtccgccggttct actgtttccccaaccatcttggagtattctggaattggtatgaaatctattttggagcctttgggaatcgacaccgttgttgaccttccag ttggtttgaacttgcaggaccagaccacctccactgtccgttctcgtattacttccgctggtgctggacaaggtcaagctgcctggtt cgctaccttcaatgagacctttggtaagtacaccgagaaggcccacgagttgttgaacaccaagttggagcaatgggctgaagag gctgtcgctagaggtggattccataataccaccgccttgttgatccaatacgaaaattatagagattggattgttaaggacaatgttgc ttactccgagttgtttttggataccggtggagtcgcttcctttgacgtctgggacttgttgcctttcacccgtggttacgttcacattttgg acaaagatccttacttgcgtcacttcgcctacgacccacagtacttcttgaacgagttggacttgttgggtcaagctgctgctactca gttggcccgtaacatttctaactctggtgccatgcaaacctacttcgctggagagaccattccaggaaacaacttggcctacgatgc cgacttgtctgcctgggtcgagtacatccctgaacatttccgtccaaactatcacggtgtcggaacctgctccatgatgccaaagga aatgggtggagtcgtcgacaatgccgctcgtgtttacggagtccagggtttgagagtcatcgacggttctatcccaccaacccaatt gtcctcccacgtcatgactgtcttctacgctatggccttgaagatcgctgacgctgttcttgctgactacgcttctatgcagtaa。
The nucleotide sequence of the glucose oxidase mutant coding gene is shown as SEQ ID NO: 9 to SEQ ID NO: as shown at 14.
Wherein, the mutant GOx-MUT1 comprises the following mutation sites: P12F, A16N, N88D, A163S, R243Q, K360D and N492D, and the nucleotide sequence of the coding gene is shown as SEQ ID NO: 9;
mutant GOx-MUT2 contains the following mutation sites: N43D, N88Q, A163S, R243Q, K360D and N492D, wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO: 10;
mutant GOx-MUT3 contains the following mutation sites: L14G, N88Q, A163S, R243Q, A343N, K360D and N492D, wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO: 11;
mutant GOx-MUT4 contains the following mutation sites: D11N, S51G, A163S, R243Q, K360H, N492D and D497E, wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO: 12;
mutant GOx-MUT5 contains the following mutation sites: V15Q, A163S, N241H, R243Q and N492E, wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO: 13;
mutant GOx-MUT6 contains the mutation sites: S51W, R243Q, A343N, K360D, N492D and D497M, wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO: 14.
the invention provides a recombinant vector containing the glucose oxidase mutant gene.
The invention provides a recombinant strain containing the glucose oxidase mutant gene.
The method for improving the specific activity of the glucose oxidase comprises the following steps of: 7, wherein the amino acids at positions 243 and 492 of the glucose oxidase are substituted.
The method for improving the specific activity of glucose oxidase, provided by the invention, comprises the following steps of: 7 is subjected to one or more of the following substitutions:
a substitution of the amino acid at position 11 with D11N or D11A;
a substitution at amino acid position 12 of P12F or P12Q;
a substitution at amino acid position 14 of L14G or L14A;
the substitution of amino acid at position 15 is V15Q, V15T, or V15S;
a substitution of the amino acid at position 16 to a16N or a 16F;
a substitution of amino acid 43 with N43D, N43G, N43H, or N43Q;
a substitution of amino acid 51 to S51G or S51W;
the substitution of amino acid 88 is N88D or N88Q;
a substitution at amino acid 163 to a163S or a 163G;
the substitution of amino acid 241 to N241H;
the substitution of the amino acid at position 243 is R243Q;
the substitution of the amino acid at position 343 with A343G, A343D or A343N;
the substitution of amino acid 360 to K360D or K360H;
a substitution at amino acid 492 to N492D or N492E;
the amino acid substitution at position 497 is D497N, D497M or D497E.
The glucose oxidase mutant can be applied to feed production, food processing, medicine, chemical industry and agriculture, such as the production of feed additives, food additives, sodium gluconate or calcium gluconate.
The invention provides a glucose oxidase mutant which has the following advantages compared with a parent SEQ ID NO: compared with glucose oxidase shown in 7, the glucose oxidase has the advantages that the specific activity is remarkably improved under the condition that the thermal stability is not reduced, and the enzyme activity retention rate is over 70 percent after the glucose oxidase is treated at 75 ℃ for 5 min. Is beneficial to the application of the enzyme in industrial production.
Drawings
FIG. 1 shows the optimum reaction pH for the glucose oxidase mutants of the present application;
FIG. 2 shows the optimal reaction temperature for the glucose oxidase mutants of the present application;
FIG. 3 shows the thermostability of the glucose oxidase mutants of the present application.
Detailed Description
The following examples are provided to better illustrate the present invention and should not be construed as limiting the invention. The molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions. The reagents and biomaterials, if not specifically indicated, are commercially available.
The glucose oxidase mutant provided by the invention is based on an amino acid sequence SEQ ID NO: 7 (the nucleic acid sequence is SEQ ID NO.8) is obtained by multiple times of mutation and high-throughput screening. Increased specific activity, meaning relative to the amino acid sequence SEQ ID NO: 7 (hereinafter referred to as GOx), the specific activity is remarkably improved under the condition of not reducing the thermal stability of the glucose oxidase shown in the specification, and the enzyme activity retention rate is over 70 percent after the glucose oxidase is treated at 75 ℃ for 5 min.
The invention also provides a method for preparing glucose oxidase with improved specific activity, which comprises the following steps:
a) constructing a recombinant expression vector containing a gene encoding the glucose oxidase mutant of the present invention;
b) introducing the recombinant expression vector into a host cell;
c) inducing the host cell to express the glucose oxidase mutant.
The glucose oxidase mutant is secreted into the nutrient medium and can be directly recovered from the medium. If the glucose oxidase mutant is not secreted, it can be recovered from the cell lysate.
Glucose oxidase proteins can be expressed in a variety of expression systems, and accordingly appropriate downstream processing and purification steps must be selected. Cells expressing the glucose oxidase variant are by any method known to those skilled in the art, and in some embodiments of the invention, the glucose oxidase variant may be expressed in a bacterial host and the protein secreted into the periplasm or extracellular space. The culture of the expression organism is prepared in appropriate volumes according to standard fermentation methods. In a preferred embodiment, the cells are grown in a fermentor, and optionally growth conditions such as pH, temperature, oxygen and/or nutrient supply are controlled. The first step of purification involves separating the cells from the supernatant using one or more of several techniques such as sedimentation, microfiltration, centrifugation or flocculation. In a preferred embodiment, the applicable method is microfiltration. If expressed intracellularly, the cells are treated to release the protein from the intracellular space. These treatments may include, for example, pressurization, enzymatic, osmotic shock, freezing, sonication, or other treatments, thereby producing a cell extract, which may or may not be further purified.
In some embodiments of the invention, the glucose oxidase is secreted into the supernatant after culturing by induction, and further protein purification from the supernatant or concentrated supernatant may be carried out using one or more of several methods including: extraction or fractionation methods such as ammonium sulfate or ethanol or acid precipitation, or chromatography methods including, but not limited to, ion exchange, hydrophobic interaction, hydroxyapatite, size fractionation by gel filtration, phosphocellulose or lectin chromatography, and affinity chromatography, or any combination thereof. In some preferred methods, the affinity tagged protein is purified by metal chelator affinity chromatography to obtain a high purity protein of interest. In other preferred embodiments, the target protein is obtained in high purity by HPLC purification.
In other embodiments of the invention, the supernatant, or the supernatant partially purified by ultrafiltration, or the supernatant concentrated and/or twice filtered (diafiltered), continues to be dried by any of several technical methods: such as, but not limited to, spray drying, freeze drying, reflux evaporation, thin layer evaporation, centrifugal evaporation, conveyor drying, or any combination thereof.
In a further embodiment of the invention, the fermented cell suspension comprising the expressed glucose oxidase is dried as a whole using a method such as, but not limited to, fluid bed drying, conveyor drying, spray drying or drum drying or any combination thereof.
The term "activity" or "catalytic activity" as used herein quantitatively describes the conversion of a given substrate under specified reaction conditions. The term "specific activity" quantitatively describes the catalytic activity relative to the amount of enzyme under the specified reaction conditions.
The invention provides a glucose oxidase mutant with improved specific activity relative to a parent glucose oxidase GOx. The glucose oxidase mutant coding gene is similar to the gene of SEQ ID NO: 1 has a sequence identity of at least 97% and less than 100%, and wherein the glucose oxidase variant has glucose oxidase activity.
In some embodiments, the glucose oxidase mutant is as set forth in SEQ ID NO: 7, at least one of amino acids at positions 11, 12, 14, 15, 16, 43, 51, 88, 163, 241, 243, 343, 360, 492, and 497 is substituted, and the glucose oxidase variant has glucose oxidase activity.
In some embodiments, the glucose oxidase mutant comprises the amino acid sequence set forth in SEQ ID NO: 7, and at least one of positions 11, 12, 14, 15, 16, 43, 51, 88, 163, 241, 343, 360 and 497 is substituted, said glucose oxidase variant having glucose oxidase activity.
In other preferred embodiments, the glucose oxidase mutant comprises the amino acid sequence set forth in SEQ ID NO: 1, at least one of the amino acids at positions 243 and 492, 360 and 163, and at least one of the amino acids at positions 11, 12, 14, 15, 16, 43, 51, 88, 241, 343 and 497, wherein the glucose oxidase variant has glucose oxidase activity.
In an embodiment of the invention, the glucose oxidase mutant is selected from one or more of the following group of substitutions: a substitution of the amino acid at position 11 with D11N or D11A; a substitution at amino acid position 12 of P12F or P12Q; a substitution at amino acid position 14 of L14G or L14A; the substitution of amino acid at position 15 is V15Q, V15T, or V15S; a substitution of the amino acid at position 16 to a16N or a 16F; a substitution of amino acid 43 to N43D, N43G, N43H, or N43Q; a substitution of amino acid 51 to S51G or S51W; the substitution of amino acid 88 is N88D or N88Q; a substitution at amino acid 163 to a163S or a 163G; the substitution of amino acid 241 to N241H; the substitution of the amino acid at position 243 is R243Q; a substitution of amino acid 343, 343G, a343D or a343N at position 343; the substitution of amino acid 360 to K360D or K360H; a substitution at amino acid 492 to N492D or N492E; and the substitution of the amino acid at position 497 is D497N, D497M or D497E.
More specifically, glucose oxidase mutants having an increased specific activity relative to the parent glucose oxidase preferably comprise the group of:
P12F, a16N, N88D, a163S, R243Q, K360D, N492D; or
N43D, N88Q, a163S, R243Q, K360D, N492D; or
L14G, N88Q, a163S, R243Q, a343N, K360D, N492D; or
D11N, S51G, a163S, R243Q, K360H, N492D, D497E; or
V15Q, a163S, N241H, R243Q, N492E; or
S51W、R243Q、A343N、K360D、N492D、D497M。
The invention provides application of the glucose oxidase mutant in feed production, food processing and medicines. Including, but not limited to, as a poultry feed additive, mold, in feed productionThe application of the toxin antidote and the like; the method is used for deoxidizing, improving flour, removing glucose, measuring glucose content, prolonging the quality guarantee period and the preservation period of food and the like in the food industry, and can be used for producing sodium gluconate or calcium gluconate; in the pharmaceutical industry, it can be used for removing or alleviating dental plaque, tartar and caries, and can be used for treating H 2 O 2 The sensitive lymphoma is used as a kit, an enzyme electrode and the like for in vitro quantitative analysis of glucose in serum (plasma), urine and cerebrospinal fluid.
Experimental materials and reagents:
1. bacterial strains and vectors
A strain containing a parent glucose oxidase GOx gene (SEQ ID NO:8) and an expression plasmid, an escherichia coli strain Top10, pichia pastoris X33, a vector pPICZ alpha A, a vector pGAPz alpha A and an antibiotic Zeocin.
2. Enzyme and kit
Super fidelity 2 × Master Mix PCR polymerase, restriction enzyme, plasmid extraction kit and purification kit.
3. Culture medium
The E.coli medium was LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). LB + Amp Medium LB medium was supplemented with ampicillin to a final concentration of 100 ug/mL. LB + Zeo Medium LB medium to a final concentration of 25ug/mL Zeocin was added.
The yeast medium was YPD medium (1% yeast extract, 2% peptone, 2% glucose). The yeast screening culture medium is YPD + Zeo culture medium (the YPD + Zeo culture medium is YPD culture medium added with Zeocin with final concentration of 100 ug/mL). Yeast induction medium BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (v/v)) and BMMY (the remainder was identical to BMGY except that glycerol was replaced with 0.5% methanol).
Recombinant yeast fermentation basic salt culture medium: 5% of diammonium hydrogen phosphate, 0.5% of potassium dihydrogen phosphate, 1.5% of magnesium sulfate heptahydrate, 1.95% of potassium sulfate, 0.1% of calcium sulfate and 0.03% of defoaming agent. 4.35ml PTM1 per liter after the high pressure. PTM1 (trace salt solution): copper sulfate 0.6% and potassium iodide 0.018%. 0.3 percent of manganese sulfate monohydrate, 0.02 percent of sodium molybdate dihydrate, 0.002 percent of boric acid, 0.05 percent of flowing cobalt chloride, 2 percent of zinc chloride, 6.5 percent of ferric sulfate heptahydrate, 0.5 percent of concentrated sulfuric acid and 0.02 percent of biotin.
4. Chemical reagents:
glucose oxidase standard, o-dianisidine hydrochloride and horseradish peroxide, glucose.
5. Glucose oxidase determination method
The activity of the glucose oxidase is measured by o-dianisidine spectrophotometry. Under the action of glucose oxidase, glucose and oxygen react to generate gluconic acid and hydrogen peroxide, and the hydrogen peroxide and colorless reduced o-dianisidine generate water and red oxidized o-dianisidine under the action of peroxidase. And measuring the light absorption value of the reaction liquid at the temperature of 37 ℃ and the pH value of 5.5 and under the condition of 540nm, and calculating the enzyme activity of the glucose oxidase according to a standard curve.
Example 1 site-directed mutagenesis
Taking a parent recombinant vector pPICz alpha A-GOx as a template, designing primers to construct corresponding GOx mutants D11N, L14A, V15Q, A16N, S51G, K360H and D497N, and carrying out PCR amplification.
And detecting the PCR amplification result by agarose electrophoresis, and purifying and recovering the target product of the PCR amplification. Digesting the template by using restriction endonuclease DpnI, transferring the decomposed product into escherichia coli Top10 competent cells by adopting a chemical conversion heat shock method, verifying recombinant transformants by bacterial liquid PCR, extracting plasmids of the transformants which are verified to be correct, and sequencing to determine corresponding mutants. And (3) linearizing the correctly sequenced mutant plasmid by using PmeI, purifying a linear plasmid fragment, transferring the linear plasmid fragment into a pichia pastoris X33 competent cell by adopting an electrical transformation method, and screening by adopting a YPD + Zeo culture medium.
The resulting yeast recombinant transformants were picked up one by one with toothpicks into 24-well plates, 1mL of BMGY-containing medium was added to each well, cultured at 30 ℃ and 220rpm for about 24 hours, and centrifuged to remove the supernatant. Then respectively adding 1.6mLBMMY culture medium to carry out induction culture. After 24h of culture, the supernatant is taken out by centrifugation, 200 mu L of the supernatant is respectively taken out to a 96-well plate, and the enzyme activity of the glucose oxidase is measured.
Example 2 site-directed saturation mutagenesis and screening
Taking pPICz alpha A-GOx as a template, respectively carrying out saturation mutation on 7 sites in the embodiment 1, specifically carrying out mutant construction by referring to the construction method in the embodiment 1, obtaining 16 effective mutation sites by a high-throughput screening method, respectively measuring corresponding enzyme activity and calculating specific activity. The relative specific activities of the mutants were calculated by dividing the specific activities of the mutants by the specific activities of the original parent glucose oxidase GOx. The calculation results show that the relative specific activities of the 16 mutants are improved in different amplitudes according to D11A, D11N, L14G, L14A, V15Q, V15T, V15S, A16F, A16N, S51G, S51W, K360D, K360H, D497N, D497E and D497M.
TABLE 1 comparison of relative specific Activity of GOx and GOx mutants
Figure BDA0003611612060000101
Example 3 semi-saturation mutagenesis and screening
The method comprises the following steps of designing a semi-saturated mutation primer by taking pPICz alpha A-GOx as a template, carrying out mutation on 8 sites of 12 th site, 43 th site, 88 th site, 163 th site, 241 th site, 243 th site, 343 rd site and 492 th site respectively, constructing the mutant by referring to the construction method in the embodiment 1, obtaining 17 effective mutation sites by a high-throughput screening method, measuring corresponding enzyme activity and calculating specific activity respectively. The relative specific activities of the mutants were calculated by dividing the specific activities of the mutants by the specific activities of the original parent glucose oxidase GOx. The calculation results show that the relative specific activities of the 17 mutants are also improved in P12F, P12Q, N43D, N43G, N43H, N43Q, N88Q, N88D, A163S, A163G, N241H, R243Q, A343G, A343D, A343N, N492E and N492D.
TABLE 2 GOx and GOx mutants relative specific Activity comparison
Figure BDA0003611612060000102
Figure BDA0003611612060000111
Example 4 combining mutations and screening
On the basis of the forward mutation sites with improved relative specific activity in example 2 and example 3, double-site or multi-site combination mutation is carried out. Purifying original parent glucose oxidase GOx and glucose oxidase GOx mutant by nickel affinity chromatography purification method, screening by high-throughput method, respectively measuring corresponding enzyme activity and calculating specific activity. The relative specific activities of the mutants were calculated by dividing the specific activities of the mutants by the specific activities of the original parent glucose oxidase GOx. Through multiple rounds of combination mutation and screening, 6 combination mutations are finally screened in the experiment, and are respectively:
GOx-MUT1 contains mutation sites as follows: P12F, a16N, N88D, a163S, R243Q, K360D, N492D;
GOx-MUT2 contains mutation sites as follows: N43D, N88Q, a163S, R243Q, K360D, N492D;
GOx-MUT3 contains mutation sites of: L14G, N88Q, a163S, R243Q, a343N, K360D, N492D;
GOx-MUT4 contains mutation sites as follows: D11N, S51G, a163S, R243Q, K360H, N492D, D497E;
GOx-MUT5 contains mutation sites as follows: V15Q, a163S, N241H, R243Q, N492E;
GOx-MUT6 contains mutation sites as follows: S51W, R243Q, A343N, K360D, N492D and D497M.
TABLE 3 comparison of the specific Activity of the original glucose oxidase GOx and the combination mutants
Figure BDA0003611612060000112
Example 5 optimal reaction pH for the original glucose oxidase GOx and the mutants GOx-MUT1, GOx-MUT2, GOx-MUT3, GOx-MUT4, GOx-MUT5 and GOx-MUT6
The enzyme activity of glucose oxidase was measured at 37 ℃ under the conditions of pH3, pH3.5, pH4, pH4.5, pH5, pH5.5, pH6, pH6.5, pH7 and pH7.5, respectively, and the results are shown in FIG. 1. As can be seen from FIG. 1, the relative enzyme activity trends of GOx-MUT1, GOx-MUT2, GOx-MUT3, GOx-MUT4, GOx-MUT5 and GOx-MUT6 under different pH conditions are substantially consistent with that of the original glucose oxidase GOx, the optimum pH of the original glucose oxidase is 5.5, and the optimum reaction pH range of the mutant is pH5.0-pH 6.0.
Example 6 optimal reaction temperature and Heat resistance of the original glucose oxidase GOx and the mutants GOx-MUT1, GOx-MUT2, GOx-MUT3, GOx-MUT4, GOx-MUT5 and GOx-MUT6
Measuring the enzyme activity of the glucose oxidase at 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ and 70 ℃ respectively under the condition of pH5.5, wherein the enzyme activity of the glucose oxidase measured at 37 ℃ is used as a reference, and the relative enzyme activities of the enzymes under different temperature conditions are calculated. The results are shown in FIG. 2, where the optimum reaction temperature range for the glucose oxidase mutant was 25 ℃ to 45 ℃. Wherein, the optimal reaction temperature range of the parent glucose oxidase GOx is 25-50 ℃.
To investigate the stability of the glucose oxidases GOx and the mutants GOx-MUT1 to GOx-MUT6 at different temperatures, the supernatants were respectively left to stand at 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃ for 5min, and then the enzyme activities were measured at 37 ℃ and pH5.5, with the relative enzyme activity of the samples without heat treatment being 100%, the results are shown in FIG. 3. As can be seen from FIG. 3, after the parent glucose oxidase GOx is treated at 75 ℃ for 5min, the enzyme activity retention rate is 70%, and similarly, after the mutants GOx-MUT1 to GOx-MUT6 are treated at 75 ℃ for 5min, the enzyme activity retention rates are all above 70%, wherein the enzyme activity retention rate of GOx-MUT6 can also be 81%.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Sequence listing
<110> Guangdong Yiduoli Biotechnology corporation
<120> glucose oxidase mutant GOx-MUT 1-6 and coding gene and application thereof
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 583
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Ser Asn Gly Ile Glu Ala Ser Leu Leu Lys Asp Phe Lys Leu Val Asn
1 5 10 15
Gly Arg Thr Tyr Asp Tyr Ile Ile Ala Gly Gly Gly Leu Ala Gly Leu
20 25 30
Thr Val Ala Glu Lys Leu Thr Glu Asn Pro Asn Ile Thr Val Leu Val
35 40 45
Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Glu Ile Phe Gly Thr Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Thr Asp Asn Arg Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ser Cys His Gly Leu Asn Gly Thr Val His Val Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Leu Met Arg Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ala Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
Asn Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Ser Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ser
275 280 285
Ala Gly Ser Thr Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Gly Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr
485 490 495
Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Glu His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Leu Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu
565 570 575
Ala Asp Tyr Ala Ser Met Gln
580
<210> 2
<211> 583
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Ser Asn Gly Ile Glu Ala Ser Leu Leu Lys Asp Pro Lys Leu Val Ala
1 5 10 15
Gly Arg Thr Tyr Asp Tyr Ile Ile Ala Gly Gly Gly Leu Ala Gly Leu
20 25 30
Thr Val Ala Glu Lys Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val
35 40 45
Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Glu Ile Phe Gly Thr Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Thr Gln Asn Arg Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ser Cys His Gly Leu Asn Gly Thr Val His Val Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Leu Met Arg Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ala Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
Asn Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Ser Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ser
275 280 285
Ala Gly Ser Thr Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Gly Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr
485 490 495
Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Glu His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Leu Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu
565 570 575
Ala Asp Tyr Ala Ser Met Gln
580
<210> 3
<211> 583
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Ser Asn Gly Ile Glu Ala Ser Leu Leu Lys Asp Pro Lys Gly Val Ala
1 5 10 15
Gly Arg Thr Tyr Asp Tyr Ile Ile Ala Gly Gly Gly Leu Ala Gly Leu
20 25 30
Thr Val Ala Glu Lys Leu Thr Glu Asn Pro Asn Ile Thr Val Leu Val
35 40 45
Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Glu Ile Phe Gly Thr Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Thr Gln Asn Arg Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ser Cys His Gly Leu Asn Gly Thr Val His Val Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Leu Met Arg Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ala Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
Asn Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Ser Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ser
275 280 285
Ala Gly Ser Thr Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Asn Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Gly Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr
485 490 495
Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Glu His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Leu Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu
565 570 575
Ala Asp Tyr Ala Ser Met Gln
580
<210> 4
<211> 583
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Ser Asn Gly Ile Glu Ala Ser Leu Leu Lys Asn Pro Lys Leu Val Ala
1 5 10 15
Gly Arg Thr Tyr Asp Tyr Ile Ile Ala Gly Gly Gly Leu Ala Gly Leu
20 25 30
Thr Val Ala Glu Lys Leu Thr Glu Asn Pro Asn Ile Thr Val Leu Val
35 40 45
Ile Glu Gly Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Glu Ile Phe Gly Thr Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ser Cys His Gly Leu Asn Gly Thr Val His Val Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Leu Met Arg Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ala Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
Asn Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Ser Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ser
275 280 285
Ala Gly Ser Thr Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly His Tyr Thr Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Gly Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr
485 490 495
Glu Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Glu His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Leu Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu
565 570 575
Ala Asp Tyr Ala Ser Met Gln
580
<210> 5
<211> 583
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Ser Asn Gly Ile Glu Ala Ser Leu Leu Lys Asp Pro Lys Leu Gln Ala
1 5 10 15
Gly Arg Thr Tyr Asp Tyr Ile Ile Ala Gly Gly Gly Leu Ala Gly Leu
20 25 30
Thr Val Ala Glu Lys Leu Thr Glu Asn Pro Asn Ile Thr Val Leu Val
35 40 45
Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Glu Ile Phe Gly Thr Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ser Cys His Gly Leu Asn Gly Thr Val His Val Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Leu Met Arg Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ala Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
His Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Ser Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ser
275 280 285
Ala Gly Ser Thr Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Lys Tyr Thr Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Gly Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Glu Asn Leu Ala Tyr
485 490 495
Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Glu His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Leu Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu
565 570 575
Ala Asp Tyr Ala Ser Met Gln
580
<210> 6
<211> 583
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Ser Asn Gly Ile Glu Ala Ser Leu Leu Lys Asp Pro Lys Leu Val Ala
1 5 10 15
Gly Arg Thr Tyr Asp Tyr Ile Ile Ala Gly Gly Gly Leu Ala Gly Leu
20 25 30
Thr Val Ala Glu Lys Leu Thr Glu Asn Pro Asn Ile Thr Val Leu Val
35 40 45
Ile Glu Trp Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Glu Ile Phe Gly Thr Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ala Cys His Gly Leu Asn Gly Thr Val His Val Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Leu Met Arg Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ala Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
Asn Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Ser Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ser
275 280 285
Ala Gly Ser Thr Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Asn Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Gly Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr
485 490 495
Met Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Glu His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Leu Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu
565 570 575
Ala Asp Tyr Ala Ser Met Gln
580
<210> 7
<211> 583
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Ser Asn Gly Ile Glu Ala Ser Leu Leu Lys Asp Pro Lys Leu Val Ala
1 5 10 15
Gly Arg Thr Tyr Asp Tyr Ile Ile Ala Gly Gly Gly Leu Ala Gly Leu
20 25 30
Thr Val Ala Glu Lys Leu Thr Glu Asn Pro Asn Ile Thr Val Leu Val
35 40 45
Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Glu Ile Phe Gly Thr Ser Val Asp His Ala Tyr
65 70 75 80
Glu Thr Val Glu Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ser
85 90 95
Gly Asn Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr
100 105 110
Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
130 135 140
Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ala Cys His Gly Leu Asn Gly Thr Val His Val Gly Pro Arg
165 170 175
Asp Thr Gly Asp Asp Tyr Ser Pro Leu Met Arg Ala Leu Met Ser Ala
180 185 190
Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp
195 200 205
Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
210 215 220
Arg Ala Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
225 230 235 240
Asn Leu Arg Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
245 250 255
Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Ser Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ser
275 280 285
Ala Gly Ser Thr Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
290 295 300
Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
305 310 315 320
Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
325 330 335
Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala
340 345 350
Thr Phe Asn Glu Thr Phe Gly Lys Tyr Thr Glu Lys Ala His Glu Leu
355 360 365
Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
370 375 380
Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
385 390 395 400
Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp
405 410 415
Thr Gly Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr
420 425 430
Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe
435 440 445
Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln
450 455 460
Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met
465 470 475 480
Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asn Asn Leu Ala Tyr
485 490 495
Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Glu His Phe Arg
500 505 510
Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met
515 520 525
Gly Gly Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu
530 535 540
Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Leu Ser Ser His Val
545 550 555 560
Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu
565 570 575
Ala Asp Tyr Ala Ser Met Gln
580
<210> 8
<211> 1752
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tctaatggta ttgaggcttc cttgttgaaa gacccaaaac ttgtcgccgg tagaacctac 60
gactacatca ttgccggtgg tggtttggct ggtttgaccg ttgctgagaa gttgaccgag 120
aatcctaaca tcactgtttt ggttattgag tccggttcct acgagtctga ccgtggtcca 180
attattgagg atttgaatgc ctacggtgaa atcttcggaa cttctgtcga ccacgcctat 240
gagaccgttg agttggctac taacaataga actgctttga tccgttccgg taacggtttg 300
ggaggatcca ctttgattaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360
tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420
cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480
aacgccgcat gccacggttt gaacggtact gttcacgttg gaccacgtga tactggtgat 540
gactactctc cattgatgag agccttgatg tctgctgtcg aagatcgtgg agtccctacc 600
aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660
gaggaccaag ttcgtgctga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720
aacttgaggg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780
accccaagag ctgttggtgt cgagttcggt actcacaagt ctaacaccca caacgtctac 840
gctaagcatg aggtcctttt gtccgccggt tctactgttt ccccaaccat cttggagtat 900
tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960
ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020
gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtaag 1080
tacaccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140
gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200
gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cggtggagtc 1260
gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320
aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380
ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440
caaacctact tcgctggaga gaccattcca ggaaacaact tggcctacga tgccgacttg 1500
tctgcctggg tcgagtacat ccctgaacat ttccgtccaa actatcacgg tgtcggaacc 1560
tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620
gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaattgtc ctcccacgtc 1680
atgactgtct tctacgctat ggccttgaag atcgctgacg ctgttcttgc tgactacgct 1740
tctatgcagt aa 1752
<210> 9
<211> 1752
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
tctaatggta ttgaggcttc cttgttgaaa gacttcaaac ttgtcaacgg tagaacctac 60
gactacatca ttgccggtgg tggtttggct ggtttgaccg ttgctgagaa gttgaccgag 120
aatcctaaca tcactgtttt ggttattgag tccggttcct acgagtctga ccgtggtcca 180
attattgagg atttgaatgc ctacggtgaa atcttcggaa cttctgtcga ccacgcctat 240
gagaccgttg agttggctac tgacaataga actgctttga tccgttccgg taacggtttg 300
ggaggatcca ctttgattaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360
tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420
cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480
aacgcctctt gccacggttt gaacggtact gttcacgttg gaccacgtga tactggtgat 540
gactactctc cattgatgag agccttgatg tctgctgtcg aagatcgtgg agtccctacc 600
aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660
gaggaccaag ttcgtgctga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720
aacttgcagg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780
accccaagag ctgttggtgt cgagttcggt actcacaagt ctaacaccca caacgtctac 840
gctaagcatg aggtcctttt gtccgccggt tctactgttt ccccaaccat cttggagtat 900
tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960
ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020
gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080
tacaccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140
gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200
gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cggtggagtc 1260
gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320
aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380
ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440
caaacctact tcgctggaga gaccattcca ggagacaact tggcctacga tgccgacttg 1500
tctgcctggg tcgagtacat ccctgaacat ttccgtccaa actatcacgg tgtcggaacc 1560
tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620
gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaattgtc ctcccacgtc 1680
atgactgtct tctacgctat ggccttgaag atcgctgacg ctgttcttgc tgactacgct 1740
tctatgcagt aa 1752
<210> 10
<211> 1752
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tctaatggta ttgaggcttc cttgttgaaa gacccaaaac ttgtcgccgg tagaacctac 60
gactacatca ttgccggtgg tggtttggct ggtttgaccg ttgctgagaa gttgaccgag 120
aatcctgata tcactgtttt ggttattgag tccggttcct acgagtctga ccgtggtcca 180
attattgagg atttgaatgc ctacggtgaa atcttcggaa cttctgtcga ccacgcctat 240
gagaccgttg agttggctac tcagaataga actgctttga tccgttccgg taacggtttg 300
ggaggatcca ctttgattaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360
tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420
cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480
aacgcctctt gccacggttt gaacggtact gttcacgttg gaccacgtga tactggtgat 540
gactactctc cattgatgag agccttgatg tctgctgtcg aagatcgtgg agtccctacc 600
aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660
gaggaccaag ttcgtgctga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720
aacttgcagg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780
accccaagag ctgttggtgt cgagttcggt actcacaagt ctaacaccca caacgtctac 840
gctaagcatg aggtcctttt gtccgccggt tctactgttt ccccaaccat cttggagtat 900
tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960
ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020
gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080
tacaccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140
gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200
gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cggtggagtc 1260
gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320
aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380
ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440
caaacctact tcgctggaga gaccattcca ggagacaact tggcctacga tgccgacttg 1500
tctgcctggg tcgagtacat ccctgaacat ttccgtccaa actatcacgg tgtcggaacc 1560
tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620
gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaattgtc ctcccacgtc 1680
atgactgtct tctacgctat ggccttgaag atcgctgacg ctgttcttgc tgactacgct 1740
tctatgcagt aa 1752
<210> 11
<211> 1752
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tctaatggta ttgaggcttc cttgttgaaa gacccaaaag gagtcgccgg tagaacctac 60
gactacatca ttgccggtgg tggtttggct ggtttgaccg ttgctgagaa gttgaccgag 120
aatcctaaca tcactgtttt ggttattgag tccggttcct acgagtctga ccgtggtcca 180
attattgagg atttgaatgc ctacggtgaa atcttcggaa cttctgtcga ccacgcctat 240
gagaccgttg agttggctac tcagaataga actgctttga tccgttccgg taacggtttg 300
ggaggatcca ctttgattaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360
tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420
cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480
aacgcctctt gccacggttt gaacggtact gttcacgttg gaccacgtga tactggtgat 540
gactactctc cattgatgag agccttgatg tctgctgtcg aagatcgtgg agtccctacc 600
aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660
gaggaccaag ttcgtgctga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720
aacttgcagg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780
accccaagag ctgttggtgt cgagttcggt actcacaagt ctaacaccca caacgtctac 840
gctaagcatg aggtcctttt gtccgccggt tctactgttt ccccaaccat cttggagtat 900
tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960
ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020
gctggtaacg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080
tacaccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140
gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200
gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cggtggagtc 1260
gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320
aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380
ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440
caaacctact tcgctggaga gaccattcca ggagacaact tggcctacga tgccgacttg 1500
tctgcctggg tcgagtacat ccctgaacat ttccgtccaa actatcacgg tgtcggaacc 1560
tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620
gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaattgtc ctcccacgtc 1680
atgactgtct tctacgctat ggccttgaag atcgctgacg ctgttcttgc tgactacgct 1740
tctatgcagt aa 1752
<210> 12
<211> 1752
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tctaatggta ttgaggcttc cttgttgaaa aacccaaaac ttgtcgccgg tagaacctac 60
gactacatca ttgccggtgg tggtttggct ggtttgaccg ttgctgagaa gttgaccgag 120
aatcctaaca tcactgtttt ggttattgag ggaggttcct acgagtctga ccgtggtcca 180
attattgagg atttgaatgc ctacggtgaa atcttcggaa cttctgtcga ccacgcctat 240
gagaccgttg agttggctac taacaataga actgctttga tccgttccgg taacggtttg 300
ggaggatcca ctttgattaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360
tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420
cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480
aacgcctctt gccacggttt gaacggtact gttcacgttg gaccacgtga tactggtgat 540
gactactctc cattgatgag agccttgatg tctgctgtcg aagatcgtgg agtccctacc 600
aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660
gaggaccaag ttcgtgctga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720
aacttgcagg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780
accccaagag ctgttggtgt cgagttcggt actcacaagt ctaacaccca caacgtctac 840
gctaagcatg aggtcctttt gtccgccggt tctactgttt ccccaaccat cttggagtat 900
tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960
ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020
gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtcac 1080
tacaccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140
gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200
gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cggtggagtc 1260
gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320
aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380
ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440
caaacctact tcgctggaga gaccattcca ggagacaact tggcctacca ggccgacttg 1500
tctgcctggg tcgagtacat ccctgaacat ttccgtccaa actatcacgg tgtcggaacc 1560
tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620
gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaattgtc ctcccacgtc 1680
atgactgtct tctacgctat ggccttgaag atcgctgacg ctgttcttgc tgactacgct 1740
tctatgcagt aa 1752
<210> 13
<211> 1752
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
tctaatggta ttgaggcttc cttgttgaaa gacccaaaac ttcaggccgg tagaacctac 60
gactacatca ttgccggtgg tggtttggct ggtttgaccg ttgctgagaa gttgaccgag 120
aatcctaaca tcactgtttt ggttattgag tccggttcct acgagtctga ccgtggtcca 180
attattgagg atttgaatgc ctacggtgaa atcttcggaa cttctgtcga ccacgcctat 240
gagaccgttg agttggctac taacaataga actgctttga tccgttccgg taacggtttg 300
ggaggatcca ctttgattaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360
tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420
cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480
aacgcctctt gccacggttt gaacggtact gttcacgttg gaccacgtga tactggtgat 540
gactactctc cattgatgag agccttgatg tctgctgtcg aagatcgtgg agtccctacc 600
aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660
gaggaccaag ttcgtgctga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720
cacttgcagg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780
accccaagag ctgttggtgt cgagttcggt actcacaagt ctaacaccca caacgtctac 840
gctaagcatg aggtcctttt gtccgccggt tctactgttt ccccaaccat cttggagtat 900
tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960
ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020
gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtaag 1080
tacaccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140
gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200
gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cggtggagtc 1260
gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320
aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380
ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440
caaacctact tcgctggaga gaccattcca ggagaaaact tggcctacga tgccgacttg 1500
tctgcctggg tcgagtacat ccctgaacat ttccgtccaa actatcacgg tgtcggaacc 1560
tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620
gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaattgtc ctcccacgtc 1680
atgactgtct tctacgctat ggccttgaag atcgctgacg ctgttcttgc tgactacgct 1740
tctatgcagt aa 1752
<210> 14
<211> 1752
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tctaatggta ttgaggcttc cttgttgaaa gacccaaaac ttgtcgccgg tagaacctac 60
gactacatca ttgccggtgg tggtttggct ggtttgaccg ttgctgagaa gttgaccgag 120
aatcctaaca tcactgtttt ggttattgag tggggttcct acgagtctga ccgtggtcca 180
attattgagg atttgaatgc ctacggtgaa atcttcggaa cttctgtcga ccacgcctat 240
gagaccgttg agttggctac taacaataga actgctttga tccgttccgg taacggtttg 300
ggaggatcca ctttgattaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360
tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420
cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480
aacgccgcat gccacggttt gaacggtact gttcacgttg gaccacgtga tactggtgat 540
gactactctc cattgatgag agccttgatg tctgctgtcg aagatcgtgg agtccctacc 600
aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660
gaggaccaag ttcgtgctga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720
aacttgcagg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780
accccaagag ctgttggtgt cgagttcggt actcacaagt ctaacaccca caacgtctac 840
gctaagcatg aggtcctttt gtccgccggt tctactgttt ccccaaccat cttggagtat 900
tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960
ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020
gctggtaacg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080
tacaccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140
gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200
gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cggtggagtc 1260
gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320
aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380
ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440
caaacctact tcgctggaga gaccattcca ggagacaact tggcctacat ggccgacttg 1500
tctgcctggg tcgagtacat ccctgaacat ttccgtccaa actatcacgg tgtcggaacc 1560
tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620
gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaattgtc ctcccacgtc 1680
atgactgtct tctacgctat ggccttgaag atcgctgacg ctgttcttgc tgactacgct 1740
tctatgcagt aa 1752

Claims (10)

1. A glucose oxidase mutant, characterized in that the glucose oxidase mutant has the following amino acid sequence:
as shown in SEQ ID NO: 7, the amino acids at positions 243 and 492 of the amino acid sequence shown in figure 7.
2. The glucose oxidase mutant according to claim 1, further comprising the amino acid sequence as set forth in SEQ ID NO: 7 at least one of the amino acids at positions 11, 12, 14, 15, 16, 43, 51, 88, 163, 241, 343, 360 and 497 is substituted.
3. The glucose oxidase mutant according to claim 1, wherein the substitution of the amino acid at position 243 is R243Q, and the substitution of the amino acid at position 492 is N492D or N492E.
4. The glucose oxidase mutant according to claim 2, wherein the substitution of the 11 th amino acid is D11N or D11A; a substitution at amino acid position 12 of P12F or P12Q; a substitution at amino acid position 14 of L14G or L14A; the substitution of amino acid 15 to V15Q, V15T, or V15S; a substitution of the amino acid at position 16 to a16N or a 16F; a substitution of amino acid 43 to N43D, N43G, N43H, or N43Q; a substitution of amino acid 51 to S51G or S51W; the substitution of amino acid 88 is N88D or N88Q; a substitution at amino acid 163 to a163S or a 163G; the substitution of amino acid 241 to N241H; a substitution at amino acid position 243 with R243Q; the substitution of the amino acid at position 343 with A343G, A343D or A343N; the substitution of amino acid 360 to K360D or K360H; a substitution at amino acid 492 to N492D or N492E; the amino acid substitution at position 497 is D497N, D497M or D497E.
5. The glucose oxidase mutant according to claim 1, wherein the amino acid sequence of the glucose oxidase mutant is as shown in SEQ ID NO: 1 to SEQ ID NO: and 6.
6. A recombinant vector comprising a gene encoding the glucose oxidase mutant according to any one of claims 1 to 5.
7. A recombinant strain, wherein the recombinant vector comprises a gene encoding the glucose oxidase mutant according to any one of claims 1 to 5.
8. A method of increasing the specific activity of glucose oxidase comprising administering to a subject in need thereof an effective amount of an enzyme having the amino acid sequence of SEQ ID NO: 7, wherein the amino acids at positions 243 and 492 of the glucose oxidase are substituted.
9. The method of increasing the specific activity of glucose oxidase according to claim 8, wherein the amino acid sequence of SEQ ID NO: 7 is subjected to one or more of the following substitutions:
a substitution of the amino acid at position 11 with D11N or D11A;
a substitution at amino acid position 12 of P12F or P12Q;
a substitution at amino acid position 14 of L14G or L14A;
the substitution of amino acid 15 to V15Q, V15T, or V15S;
a substitution of the amino acid at position 16 to a16N or a 16F;
a substitution of amino acid 43 with N43D, N43G, N43H, or N43Q;
a substitution of amino acid 51 to S51G or S51W;
a substitution at amino acid position 88 with N88D or N88Q;
a substitution at amino acid 163 to a163S or a 163G;
a substitution of amino acid 241 to N241H;
a substitution at amino acid position 243 with R243Q;
the substitution of the amino acid at position 343 with A343G, A343D or A343N;
the substitution of amino acid 360 to K360D or K360H;
a substitution at amino acid 492 to N492D or N492E;
the amino acid substitution at position 497 is D497N, D497M or D497E.
10. The glucose oxidase mutant of claim 1, for use in feed production, food additives and medicine.
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