CN115029328B - 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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CN115029328B
CN115029328B CN202210432859.4A CN202210432859A CN115029328B CN 115029328 B CN115029328 B CN 115029328B CN 202210432859 A CN202210432859 A CN 202210432859A CN 115029328 B CN115029328 B CN 115029328B
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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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Abstract

The invention relates to the technical field of genetic engineering, in particular to glucose oxidase mutants GOx-MUT 1-6, and coding genes and application thereof. The present invention provides a glucose oxidase mutant, which is identical to the parent SEQ ID NO: compared with the glucose oxidase shown in 7, the specific activity of the glucose oxidase is remarkably improved under the condition that the thermal stability is not reduced, and the retention rate of the enzyme activity is more than 70% after the glucose oxidase is treated for 5min at 75 ℃. 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 glucose oxidase mutants GOx-MUT 1-6, and coding genes and application thereof.
Background
Glucose oxidase (GOD, EC 1.1.3.4) is an oxidoreductase consisting of two subunits, a dimer, containing 2 Flavin Adenine Dinucleotide (FAD) binding sites. Each monomer contains 2 distinct regions, one non-covalently tightly bound to FAD, predominantly the β -sheet; the other binds to the substrate β -D-glucose, supporting 1 antiparallel β -sheet from 4 α -helices. The physical and chemical properties of GOD from different sources are different, the molecular weight is in the range of 130-175 kDa, and the beta-D-glucose can be specifically catalyzed to produce gluconic acid and hydrogen peroxide under the aerobic condition.
GOD is widely used in the fields of food, chemical industry, biomedical treatment and the like. In the food industry, GOD can be used for catalyzing oxygen in a glucose-depleted vacuum bag, inhibiting growth and reproduction of microorganisms, prolonging the shelf life of food, and improving the taste of flour products. In the chemical industry, GOD is often used not only in bleaching and decolorizing processes, but also as a key enzyme in the production of gluconic acid and derivatives thereof. GOD is a key raw material of a glucose detection kit in the medical field, is also added into toothpaste for reducing the incidence rate of oral diseases, and can be used as an electrode of a biological battery to provide continuous energy sources for biological sensors and artificial organs. Furthermore, the use of GODs is proposed in the production of transgenic plants and other organisms that have reduced sensitivity or increased resistance to pests or diseases. Therefore, the high-efficient production of GOD on a large scale has important economic value. However, GOD often needs to undergo high temperature processes during further processing and production, so that the enzyme activity of GOD is inevitably reduced or even inactivated. For example, the inactivation of glucose oxidase can be caused by a transient high-temperature process in the feed processing process, the application effect of the glucose oxidase is affected, and the temperature resistance of the glucose oxidase is more and more concerned on the premise that the yield of the glucose oxidase is ensured.
GODs are widely distributed in animals, plants and microorganisms. At present, the industrial-level GOD production mainly uses Aspergillus niger and penicillium as production strains, and compared with the penicillium GOD, the GOD expressed by the Aspergillus niger has better thermal stability, but has the problems of low enzyme activity level and complex separation and purification. Pichia pastoris (Pichia pastoris) has clear genetic background, is easy to carry out genetic operation, has moderate glycosylation of secreted protein, is a common host for carrying out exogenous protein expression, particularly utilizes P.pastoris to carry out protein secretion expression, can greatly simplify the separation and purification process, and has important application value.
Disclosure of Invention
The purpose of the present invention is to provide a glucose oxidase mutant with improved specific activity.
It is still another object of the present invention to provide a gene encoding the above 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 still another object of the present invention to provide a method for increasing the specific activity of glucose oxidase.
It is a further object of the present invention to provide the use of the above glucose oxidase mutant.
The glucose oxidase mutant according to the present invention has the following amino acid sequence:
as set forth in SEQ ID NO:7 are substituted at amino acids 243 and 492 of the amino acid sequence shown in figure 7.
The glucose oxidase mutant according to the present invention further comprises a nucleotide sequence as shown in SEQ ID NO:7, at least one of amino acids 11, 12, 14, 15, 16, 43, 51, 88, 163, 241, 343, 360 and 497 of the amino acid sequence shown in fig. 7 is substituted.
SEQ ID NO：7
SNGIEASLLKDPKLVAGRTYDYIIAGGGLAGLTVAEKLTENPNITVLVIESGSYESDR GPIIEDLNAYGEIFGTSVDHAYETVELATNNRTALIRSGNGLGGSTLINGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNAACHGL NGTVHVGPRDTGDDYSPLMRALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRADAAREWLLPNYQRPNLRVLTGQYVGKVLLSQNATTPRAVGVEFGTHKS NTHNVYAKHEVLLSAGSTVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGKYTEKAHELLNTKLEQWAEEAVARGG FHNTTALLIQYENYRDWIVKDNVAYSELFLDTGGVASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGNNLA YDADLSAWVEYIPEHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQLSSHVMTVFYAMALKIADAVLADYASMQ。
The glucose oxidase mutant according to the present invention, wherein the substitution of amino acid 243 is R243Q and the substitution of amino acid 492 is N492D or N492E.
The glucose oxidase mutant according to the present invention, wherein the substitution of amino acid 11 is D11N or D11A; substitution of amino acid 12 to P12F or P12Q; substitution of amino acid 14 is L14G or L14A; the substitution of amino acid 15 is V15Q, V T or V15S; substitution of amino acid 16 is a16N or a16F; the substitution of amino acid 43 is N43D, N43G, N H or N43Q; the substitution of amino acid 51 is S51G or S51W; the substitution of amino acid 88 is N88D or N88Q; the substitution of amino acid 163 is a163S or a163G; substitution of amino acid 241 is N241H; the substitution of amino acid 243 is R243Q; the substitution at amino acid 343 is a343G, A343D or a343N; the substitution of amino acid 360 is K360D or K360H; the substitution at amino acid 492 is N492D or N492E; the substitution at amino acid 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-SEQ ID NO: shown at 6.
Wherein, the mutant GOx-MUT1 comprises mutation sites: P12F, A16N, N88D, A163S, R243Q, K360D, N492D (SEQ ID NO: 1);
the mutant GOx-MUT2 comprises mutation sites: N43D, N88Q, A163S, R243Q, K360D, N492D (SEQ ID NO: 2);
the mutant GOx-MUT3 comprises mutation sites: L14G, N88Q, A163S, R243Q, A343N, K360D, N492D (SEQ ID NO: 3);
the mutant GOx-MUT4 comprises mutation sites: d11N, S51G, A163S, R243Q, K360H, N492D, D497E (SEQ ID NO: 4);
the mutant GOx-MUT5 comprises mutation sites: V15Q, A163S, N241H, R243Q, N492E (SEQ ID NO: 5);
the mutant GOx-MUT6 comprises mutation sites: S51W, R243Q, A343N, K360D, N492D, D497M (SEQ ID NO: 6).
The glucose oxidase gene according to the present invention encodes any one of the above-mentioned glucose oxidase mutants.
The nucleotide sequence of the parent glucose oxidase gene is shown in SEQ ID NO: shown at 8.
SEQ ID NO：8：
tctaatggtattgaggcttccttgttgaaagacccaaaacttgtcgccggtagaacctacgactacatcattgccggtggtggtttgg ctggtttgaccgttgctgagaagttgaccgagaatcctaacatcactgttttggttattgagtccggttcctacgagtctgaccgtggtccaattattgaggatttgaatgcctacggtgaaatcttcggaacttctgtcgaccacgcctatgagaccgttgagttggctactaaca atagaactgctttgatccgttccggtaacggtttgggaggatccactttgattaacggtggaacctggactagaccacataaagcccaagtcgactcctgggagactgtcttcggaaacgaaggttggaactgggactctgttgctgcttactcccttcaggctgaaagagctc gtgccccaaatgctaagcagatcgccgctggtcactactttaacgccgcatgccacggtttgaacggtactgttcacgttggaccacgtgatactggtgatgactactctccattgatgagagccttgatgtctgctgtcgaagatcgtggagtccctaccaagaaggacttg ggttgcggagaccctcatggtgtctccatgttcccaaacaccttgcacgaggaccaagttcgtgctgacgctgccagagaatggttgcttcctaactaccagagaccaaacttgagggtcttgactggtcagtacgtcggtaaggtcttgttgtctcagaacgctaccacccc aagagctgttggtgtcgagttcggtactcacaagtctaacacccacaacgtctacgctaagcatgaggtccttttgtccgccggttctactgtttccccaaccatcttggagtattctggaattggtatgaaatctattttggagcctttgggaatcgacaccgttgttgaccttccag ttggtttgaacttgcaggaccagaccacctccactgtccgttctcgtattacttccgctggtgctggacaaggtcaagctgcctggttcgctaccttcaatgagacctttggtaagtacaccgagaaggcccacgagttgttgaacaccaagttggagcaatgggctgaagaggctgtcgctagaggtggattccataataccaccgccttgttgatccaatacgaaaattatagagattggattgttaaggacaatgttgc ttactccgagttgtttttggataccggtggagtcgcttcctttgacgtctgggacttgttgcctttcacccgtggttacgttcacattttggacaaagatccttacttgcgtcacttcgcctacgacccacagtacttcttgaacgagttggacttgttgggtcaagctgctgctactca gttggcccgtaacatttctaactctggtgccatgcaaacctacttcgctggagagaccattccaggaaacaacttggcctacgatgccgacttgtctgcctgggtcgagtacatccctgaacatttccgtccaaactatcacggtgtcggaacctgctccatgatgccaaagga aatgggtggagtcgtcgacaatgccgctcgtgtttacggagtccagggtttgagagtcatcgacggttctatcccaccaacccaattgtcctcccacgtcatgactgtcttctacgctatggccttgaagatcgctgacgctgttcttgctgactacgcttctatgcagtaa。
The nucleotide sequence of the coding gene of the glucose oxidase mutant is shown as SEQ ID NO: 9-SEQ ID NO:14.
Wherein, the mutant GOx-MUT1 comprises mutation sites: P12F, A16N, N88D, A163S, R243Q, K360D, N492D, and the nucleotide sequence of the coding gene is shown in SEQ ID NO:9, a step of performing the process;
the mutant GOx-MUT2 comprises mutation sites: N43D, N88Q, A163S, R243Q, K360D, N492D, the nucleotide sequence of the coding gene of which is shown in SEQ ID NO:10;
the mutant GOx-MUT3 comprises mutation sites: L14G, N88Q, A163S, R243Q, A343N, K360D, N492D, and the nucleotide sequence of the coding gene is shown in SEQ ID NO:11;
the mutant GOx-MUT4 comprises mutation sites: D11N, S51G, A163S, R243Q, K360H, N492D, D497E, the nucleotide sequence of the encoding gene thereof is as shown in SEQ ID NO:12;
the mutant GOx-MUT5 comprises mutation sites: V15Q, A163S, N241H, R243Q, N492E, the nucleotide sequence of the encoding gene of which is set forth in SEQ ID NO:13;
the mutant GOx-MUT6 comprises mutation sites: S51W, R243Q, A343N, K360D, N492D, D497M, and 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 comprising the glucose oxidase mutant gene.
The method for improving the specific activity of glucose oxidase comprises the following steps of: 7, amino acid substitutions at positions 243 and 492 of the glucose oxidase.
The method for improving the specific activity of glucose oxidase, provided by the invention, comprises the following steps of: 7, performing one or more of the following substitutions:
substitution of amino acid 11 is D11N or D11A;
substitution of amino acid 12 to P12F or P12Q;
substitution of amino acid 14 is L14G or L14A;
the substitution of amino acid 15 is V15Q, V T or V15S;
substitution of amino acid 16 is a16N or a16F;
the substitution of amino acid 43 is N43D, N43G, N H or N43Q;
the substitution of amino acid 51 is S51G or S51W;
the substitution of amino acid 88 is N88D or N88Q;
the substitution of amino acid 163 is a163S or a163G;
substitution of amino acid 241 is N241H;
the substitution of amino acid 243 is R243Q;
the substitution at amino acid 343 is a343G, A343D or a343N;
the substitution of amino acid 360 is K360D or K360H;
the substitution at amino acid 492 is N492D or N492E;
the substitution at amino acid 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 production of feed additives, food additives, sodium gluconate or calcium gluconate.
The present invention provides a glucose oxidase mutant, which is identical to the parent SEQ ID NO: compared with the glucose oxidase shown in 7, the specific activity of the glucose oxidase is remarkably improved under the condition that the thermal stability is not reduced, and the retention rate of the enzyme activity is more than 70% after the glucose oxidase is treated for 5min at 75 ℃. Is beneficial to the application of the enzyme in industrial production.
Drawings
FIG. 1 shows the optimal reaction pH of the glucose oxidase mutants of the present application;
FIG. 2 shows the optimal reaction temperatures 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 given for better illustration of the invention and should not be construed as limiting the invention. The molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j. The reagents and biological materials, unless otherwise specified, are commercially available.
The glucose oxidase mutant provided by the invention is based on an amino acid sequence SEQ ID NO:7 (nucleic acid sequence is SEQ ID NO. 8) is obtained through multiple mutation and high-throughput screening. Increased specific activity means an increased specific activity relative to the amino acid sequence of SEQ ID NO:7 (hereinafter referred to as GOx) the specific activity is significantly improved without decreasing the thermal stability of the glucose oxidase, and the retention rate of the enzyme activity is above 70% after 5min of treatment at 75 ℃.
The invention also provides a method for preparing the glucose oxidase with improved specific activity, which comprises the following steps:
a) Constructing a recombinant expression vector comprising 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 recovered directly from the medium. If the glucose oxidase mutant is not secreted, it can be recovered from the cell lysate.
The glucose oxidase protein can be expressed in a variety of expression systems and appropriate downstream processing and purification steps must be selected accordingly. Cells expressing the glucose oxidase variants in some embodiments of the invention, the glucose oxidase variants can be expressed in a bacterial host and the protein secreted into the periplasm or extracellular space by any method known to those of skill in the art. Culture of the expression organisms was prepared in appropriate volumes according to standard fermentation methods. In preferred embodiments, the cells are grown in a fermenter, and optionally growth conditions such as pH, temperature, oxygen, and/or nutrient supply are controlled. The first step of purification involves separating cells from the supernatant using one or more of several techniques such as sedimentation, microfiltration, centrifugation or flocculation. In a preferred embodiment, a suitable method is microfiltration. If expressed in cells, 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 to produce a cell extract, which may or may not be subjected to further purification.
In some embodiments of the invention, after the glucose oxidase is cultured by induction, secretion into the supernatant, and further protein purification from the supernatant or concentrated supernatant may be performed 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 interactions, hydroxyapatite, particle size fractionation by gel filtration, phosphocellulose or lectin chromatography and affinity chromatography, or any combination thereof. In some preferred methods, the affinity tag protein is purified by metal chelator affinity chromatography to obtain the target protein in high purity. 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 concentrated and/or secondary filtered (diafiltrated) supernatant, is further dried by any of several techniques: such as, but not limited to, spray drying, freeze drying, reflux evaporation (down-draught evaporation), thin layer evaporation, centrifugal evaporation, conveyor drying, or any combination thereof.
In a further embodiment of the invention, the fermentation cell suspension comprising 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 defined 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 codes for a gene, and the glucose oxidase mutant has the nucleotide sequence shown in SEQ ID NO: 1. the amino acid sequence has at least 97% and less than 100% sequence identity, and wherein the glucose oxidase variant has glucose oxidase activity.
In some embodiments, the glucose oxidase mutant is described as set forth in SEQ ID NO:7, at least one amino acid of amino acid sequence 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 amino acids 243 and 492, 11, 12, 14, 15, 16, 43, 51, 88, 163, 241, 343, 360, and 497 of the amino acid sequence, and a variant of the glucose oxidase 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 amino acids at positions 243 and 492, at least one of amino acids at positions 360 and 163, and at least one of amino acids at positions 11, 12, 14, 15, 16, 43, 51, 88, 241, 343, and 497 of the amino acid sequence shown in fig. 1, and a variant of the glucose oxidase having glucose oxidase activity.
In an embodiment of the invention, the glucose oxidase mutant is selected from one or more of the following substitutions: substitution of amino acid 11 is D11N or D11A; substitution of amino acid 12 to P12F or P12Q; substitution of amino acid 14 is L14G or L14A; the substitution of amino acid 15 is V15Q, V T or V15S; substitution of amino acid 16 is a16N or a16F; the substitution of amino acid 43 is N43D, N43G, N H or N43Q; the substitution of amino acid 51 is S51G or S51W; the substitution of amino acid 88 is N88D or N88Q; the substitution of amino acid 163 is a163S or a163G; substitution of amino acid 241 is N241H; the substitution of amino acid 243 is R243Q; the substitution at amino acid 343 is a343G, A343D or a343N; the substitution of amino acid 360 is K360D or K360H; the substitution at amino acid 492 is N492D or N492E; and the substitution of amino acid 497 is D497N, D497M or D497E.
More specifically, the 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 (b)
N43D, N88Q, A163S, R243Q, K360D, N492D; or (b)
L14G, N88Q, A163S, R243Q, A343N, K360D, N492D; or (b)
d11N, S51G, A163S, R243Q, K360H, N492D, D497E; or (b)
V15Q, A163S, N241H, R243Q, N492E; or (b)
S51W、R243Q、A343N、K360D、N492D、D497M。
The invention provides application of the glucose oxidase mutant in feed production, food processing and medicine. Including, but not limited to, use in feed production as poultry feed additives, mycotoxin antidotes and the like; in the food industry for deoxidizing, improving flour, removing glucose, measuring glucose content, prolonging the quality and fresh-keeping period of food, etc., for example, can be used in the production of sodium gluconate or calcium gluconate; in the pharmaceutical industry, can be used for removing or relieving dental plaque, tartar and caries formation, and can be used for treating H 2 O 2 Sensitive lymphoma is treated, and the sensitive lymphoma is used as a kit, an enzyme electrode and the like for in vitro quantitative analysis of glucose in serum (pulp), urine and cerebrospinal fluid and the like.
Experimental materials and reagents:
1. strain and vector
The strain containing the parent glucose oxidase GOx gene (SEQ ID NO: 8) and the expression plasmid, E.coli strain Top10, pichia X33, vector pPICZ alpha A, vector pGAPz alpha A, antibiotic Zeocin.
2. Enzyme and kit
Super fidelity 2X Master Mix PCR polymerase, restriction enzyme, plasmid extraction kit, 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 ampicillin was added to LB medium at a final concentration of 100 ug/mL. LB+Zeo medium Zeocin was added to LB medium at a final concentration of 25 ug/mL.
The yeast medium was YPD medium (1% yeast extract, 2% peptone, 2% glucose). Yeast selection medium was YPD+Zeo medium (YPD+Zeo medium was YPD medium supplemented with Zeocin at a 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 (divided by 0.5% methanol instead of glycerol, the rest of the components are identical to BMGY).
Recombinant yeast fermentation basal salt culture medium: 5% of diammonium phosphate, 0.5% of monopotassium phosphate, 1.5% of magnesium sulfate heptahydrate, 1.95% of potassium sulfate, 0.1% of calcium sulfate and 0.03% of defoamer. 4.35ml of PTM1 per liter after high pressure. PTM1 (trace salt solution): copper sulfate 0.6% and potassium iodide 0.018%. Manganese sulfate monohydrate 0.3%, sodium molybdate dihydrate 0.02%, boric acid 0.002%, cobalt chloride running water 0.05%, zinc chloride 2%, ferric sulfate heptahydrate 6.5%, concentrated sulfuric acid 0.5% and biotin 0.02%.
4. Chemical reagent:
glucose oxidase standard, o-dianisidine hydrochloride and horseradish peroxide were purchased for glucose.
5. Glucose oxidase assay method
The glucose oxidase activity was determined by o-dianisidine spectrophotometry. Under the action of glucose oxidase, glucose reacts with oxygen to generate gluconic acid and hydrogen peroxide, and the hydrogen peroxide and colorless reduced o-dianisidine react with peroxidase to generate water and red oxidized o-dianisidine. The absorbance of the reaction solution was measured at 37℃and pH5.5 at 540nm, and the enzyme activity of glucose oxidase was calculated from the standard curve.
Example 1 site-directed mutagenesis
The parent recombinant vector pPICzalpha A-GOx is used as a template, and primers are designed to construct corresponding GOx mutants D11N, L14A, V15Q, A16N, S51G, K360H and D497N for PCR amplification.
And detecting the PCR amplification result by agarose electrophoresis, and purifying and recovering the target product of PCR amplification. And (3) digesting the template by using restriction endonuclease DpnI, transferring the decomposed product into competent cells of escherichia coli Top10 by adopting a chemical conversion heat shock method, verifying the recombinant transformant by bacterial liquid PCR, extracting plasmids of the transformant with correct verification, and sequencing to determine the corresponding mutant. The mutant plasmid with correct sequence was linearized with PmeI, the linear plasmid fragment was purified, transformed into Pichia X33 competent cells by electrotransformation and screened using YPD+Zeo medium.
The resulting yeast recombinant transformants were picked up one by one with toothpick into 24 well plates, 1mL of BMGY-containing medium was added to each well, cultured at 30℃and 220rpm for about 24 hours, and the supernatant was removed by centrifugation. Then 1.6mLBMMY culture medium was added for induction culture. After culturing for 24 hours, the supernatant was collected by centrifugation, and 200. Mu.L to 96-well plates were taken out of the supernatant, respectively, to perform enzyme activity measurement of glucose oxidase.
Example 2 site-directed saturation mutagenesis and screening
The pPICz alpha A-GOx is used as a template, 7 sites in the embodiment 1 are subjected to saturation mutation respectively, the specific construction method refers to the construction method in the embodiment 1, the mutant construction is carried out, 16 effective mutation sites are obtained through a high-throughput screening method, and the corresponding enzyme activities are measured respectively and the specific activities are calculated. The relative specific activity of the mutant was calculated as the specific activity of the mutant divided by the specific activity of the original parent glucose oxidase GOx. The calculation results show that the relative specific activities of the 16 mutants are improved in different magnitudes by D11A, D11N, L14G, L A, V15Q, V15T, V S, A16F, A N, S51G, S51W, K360D, K H, D497N, D497E and D497M.
TABLE 1 comparative relative activities of GOx and GOx mutations
Example 3, half-saturation mutagenesis and screening
The specific construction method refers to the construction method in embodiment 1, mutant construction is carried out by taking pPICz alpha A-GOx as a template, designing a half-saturation mutation primer, respectively mutating 8 sites of the 12 th site, the 43 rd site, the 88 th site, the 163 th site, the 241 th site, the 243 rd site, the 343 rd site and the 492 rd site, 17 effective mutation sites are obtained by a high-throughput screening method, and corresponding enzyme activities are respectively measured and specific activities are calculated. The relative specific activity of the mutant was calculated as the specific activity of the mutant divided by the specific activity of the original parent glucose oxidase GOx. The calculation results show that the relative specific activities of the 17 mutants are also improved by P12F, P12Q, N43D, N43G, N43H, N43Q, N88Q, N88D, A163S, A163G, N241H, R243Q, A343G, A343D, A343N, N492E and N492D.
TABLE 2 comparative relative activities of GOx and GOx mutations
Example 4, combinatorial mutagenesis and screening
Double-site or multi-site combination mutation was performed on the basis of the forward mutation sites of the relative live promotion of example 2 and example 3. And (3) respectively purifying the original parent glucose oxidase GOx and the mutant of the glucose oxidase GOx by adopting a nickel affinity chromatography purification method, screening by adopting a high-flux method, respectively measuring the corresponding enzyme activities and calculating the specific activities. The relative specific activity of the mutant was calculated as the specific activity of the mutant divided by the specific activity of the original parent glucose oxidase GOx. Through multiple rounds of combination mutation and screening, the experiment finally screens 6 combination mutations, respectively:
the GOx-MUT1 comprises the following mutation sites: P12F, A16N, N88D, A163S, R243Q, K360D, N492D;
the GOx-MUT2 contains the following mutation sites: N43D, N88Q, A163S, R243Q, K360D, N492D;
GOx-MUT3 contains the following mutation sites: L14G, N88Q, A163S, R243Q, A343N, K360D, N492D;
GOx-MUT4 contains the following mutation sites: d11N, S51G, A163S, R243Q, K360H, N492D, D497E;
GOx-MUT5 contains the following mutation sites: V15Q, A163S, N241H, R243Q, N492E;
GOx-MUT6 contains the following mutation sites: S51W, R243Q, A343N, K360D, N492D, D497M.
TABLE 3 comparison of specific Activity of the original glucose oxidase GOx and the combination mutant
Example 5 optimal reaction pH for the original glucose oxidase GOx and the mutant GOx-MUT1, GOx-MUT2, GOx-MUT3, GOx-MUT4, GOx-MUT5 and GOx-MUT6
The enzyme activities of glucose oxidase were measured at 37℃under 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 tendencies 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 optimal pH of the original glucose oxidase is 5.5, and the optimal reaction pH range of the mutant is pH5.0-pH6.0.
EXAMPLE 6 optimal reaction temperatures and thermostabilities of the original glucose oxidase GOx and the mutants GOx-MUT1, GOx-MUT2, GOx-MUT3, GOx-MUT4, GOx-MUT5 and GOx-MUT6
The enzyme activities of glucose oxidase were measured at 25℃at 30℃at 35℃at 40℃at 45℃at 50℃at 55℃at 60℃at 65℃at 70℃under pH5.5, wherein the relative enzyme activities of the enzymes at different temperatures were calculated by using the enzyme activities of glucose oxidase measured at 37℃as a control. As shown in FIG. 2, the optimal 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 ℃ to 50 ℃.
To investigate the stability of glucose oxidase GOx and mutant GOx-MUT1 to GOx-MUT6 at different temperatures, the supernatants were allowed to stand at 35℃at 40℃at 45℃at 50℃at 55℃at 60℃at 65℃at 70℃at 75℃for 5min, and then the enzyme activities were measured at 37℃at pH5.5, with the relative enzyme activities of the non-heat-treated samples being 100%, and the results are shown in FIG. 3. As can be seen from FIG. 3, the retention of the enzyme activity of the parent glucose oxidase GOx was 70% after 5min at 75℃and, similarly, the retention of the enzyme activity of the mutants GOx-MUT1 to GOx-MUT6 was 70% or more after 5min at 75℃and, among them, the retention of the enzyme activity of GOx-MUT6 was 81%.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Sequence listing
<110> Guangdong Yiduoli Biotech stock Co., ltd
<120> glucose oxidase mutant GOx-MUT 1-6, encoding 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 (5)

1. The glucose oxidase mutant is characterized in that the amino acid sequence of the glucose oxidase mutant is shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. or SEQ ID NO: 6. as shown.
2. A glucose oxidase gene encoding the glucose oxidase mutant according to claim 1.
3. A recombinant vector comprising the glucose oxidase gene of claim 2.
4. A recombinant strain comprising the glucose oxidase gene of claim 2.
5. A method for increasing the specific activity of glucose oxidase, said method comprising the steps of: 7. the procedure shown for the glucose oxidase was composed of the following mutations:
P12F, A16N, N88D, A163S, R243Q, K360D and N492D; or alternatively
N43D, N88Q, A163S, R243Q, K360D and N492D; or alternatively
L14G, N88Q, A163S, R243Q, A343N, K360D and N492D; or alternatively
d11N, S51G, A163S, R243Q, K360H, N492D and D497E; or alternatively
V15Q, A163S, N241H, R243Q and N492E; or alternatively
S51W, R243Q, A343N, K360D, N492D and D497M.
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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

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