Heat-resistant glucose oxidase mutant, preparation method therefor and use thereof

Abstract

The present invention belongs to the technical field of genetic engineering. Provided are a heat-resistant glucose oxidase mutant, a preparation method therefor and the use thereof. Compared with a parent glucose oxidase having the amino acid sequence shown as SEQ ID NO: 1, the glucose oxidase mutant comprises the following mutations: K37C and A572C. Compared with the parent glucose oxidase, the glucose oxidase mutant has significantly improved thermal stability, exhibits better heat resistance, and can maintain higher enzyme activity at high temperatures, and therefore is beneficial to industrial application.

Description

Heat-resistant glucose oxidase mutant and preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a heat-resistant glucose oxidase mutant and a preparation method and application thereof. BACKGROUND

[0002] Glucose oxidase (E.C.1.1.3.4, GOD) can specifically catalyze β-D-glucose to generate gluconic acid and hydrogen peroxide under aerobic conditions. Glucose oxidase is a homodimeric molecule containing two flavin adenine dinucleotide (FAD) binding sites. Each monomer contains two completely different regions: one region mainly consists of β-sheets and is tightly bound to the flavin adenine dinucleotide (FAD) coenzyme molecule by non-covalent bonds; the other region consists of four α-helices supporting a β-sheet in an anti-parallel arrangement and can bind to the substrate β-D-glucose. The enzyme can catalyze β-D-glucose to be oxidized into gluconolactone and hydrogen peroxide, and the gluconolactone can be converted into gluconic acid under non-enzymatic reactions.

[0003] The addition of glucose oxidase to feed can specifically oxidize β-D-glucose into gluconic acid and hydrogen peroxide. The reaction product gluconic acid can reduce the pH value in the gastrointestinal tract to create an acidic environment for the growth of lactic acid bacteria. The generated hydrogen peroxide has a sterilization effect, and when the hydrogen peroxide accumulates to a certain concentration, it directly inhibits the growth and reproduction of Escherichia coli, Salmonella, Pasteurella, Staphylococcus, and Vibrio. In terms of improving animal digestion and absorption, promoting growth, preventing and treating diseases, replacing part of the drugs and antibiotics, and saving costs, it has outstanding performance. As a new type of growth-promoting feed additive, it will be widely used in green breeding production due to its non-pollution, high safety, and strong health care properties, and has a broad market application prospect. In addition, glucose oxidase also has important applications in the fields of food and medical diagnosis.

[0004] Currently, the main sources of microbial GOD are Aspergillus niger and Penicillium. Compared with Penicillium, the GOD produced by Aspergillus niger has the advantages of good thermal stability and strong substrate specificity. CN105002147A discloses a mutant glucose oxidase with improved expression, an encoding gene thereof, and an application. By site-directed mutagenesis, the amino acid sequence Y76C and Q279K sites of the glucose oxidase are mutated, the expression is improved, and the stability is good. C101348794A discloses a coding gene of high-activity glucose oxidase, a preparation method and an application. By introducing two mutations at positions 1752 and 2272 of the Aspergillus niger GOD structural gene through overlap PCR, the mutant enzyme activity is improved.

[0005] However, since the heat resistance of conventional glucose oxidase is still poor, it is difficult to adapt to the industrial temperature generated when used in feed and food production, resulting in that conventional glucose oxidase is difficult to meet the actual production demand. Based on this, the present application hopes to provide a glucose oxidase with good heat resistance.

[0006] SUMMARY

[0007] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a heat-resistant glucose oxidase mutant and its preparation method and application. Compared with the parent glucose oxidase, the heat stability of the glucose oxidase mutant is obviously improved, which shows good heat resistance and can maintain higher enzyme activity at high temperature, which is beneficial to industrial application.

[0008] The present application provides a glucose oxidase mutant, which, relative to the parent glucose oxidase of the amino acid sequence shown in SEQ ID NO: 1, comprises the following mutations: K37C, A572C.

[0009] Experiments show that, compared with the parent glucose oxidase, the residual enzyme activity of the glucose oxidase mutant with K37C and A572C mutations is obviously higher after heat treatment, indicating that the occurrence of the above mutations effectively improves the heat resistance of the glucose oxidase and has better thermal stability, which is more beneficial to industrial application.

[0010] Preferably, the glucose oxidase mutant, relative to the parent glucose oxidase of the amino acid sequence shown in SEQ ID NO: 1, further comprises at least one of the following mutations: H277F, M305L, W350Y, Q453K, Y80V, Q248V, K252V, P443C, A292L, T296V.

[0011] On the basis of the K37C and A572C mutations of the parent glucose oxidase, one or more of the above mutation sites are introduced, and the results show that the obtained glucose oxidase mutant not only has good heat resistance, but also can further improve its basic enzyme activity.

[0012] More preferably, relative to the parent glucose oxidase of the amino acid sequence shown in SEQ ID NO: 1, the glucose oxidase mutant has any one of the following mutations (1)-(12):

[0013] (1) K37C+A572C+H277F; (2) K37C+A572C+M305L; (3) K37C+A572C+W350Y; (4) K37C+A572C+Q453K; (5) K37C+A572C+Q453K+W350Y; (6) K37C+A572C+Q453K+M305L; (7) K37C+A572C+Y80V; (8) K37C+A572C+Q248V; (9) K37C+A572C+K252V; (10) K37C+A572C+P443C; (11) K37C+A572C+A292L; (12) K37C+A572C+T296V.

[0014] The present application also provides a nucleic acid molecule comprising the nucleotide fragments shown in (a) and / or (b):

[0015] (a) a nucleotide fragment encoding the above-mentioned glucose oxidase mutant;

[0016] (b) a nucleotide fragment reverse complementary to (a).

[0017] The present application also provides a recombinant expression vector comprising the above-mentioned nucleic acid molecule.

[0018] The present application also provides a recombinant cell comprising the above-mentioned nucleic acid molecule or the above-mentioned recombinant expression vector.

[0019] Preferably, the recombinant cell comprises a bacterial or fungal cell.

[0020] More preferably, the fungal cell is a Pichia cell.

[0021] The present application also provides a method for preparing the above-mentioned glucose oxidase mutant, comprising the following steps:

[0022] (1) culturing the recombinant cell;

[0023] (2) inducing the recombinant cell to express the above-mentioned glucose oxidase mutant.

[0024] The present application also provides the use of the above-mentioned glucose oxidase mutant in the field of food, medical diagnosis or feed.

[0025] Glucose oxidase is known to have many uses in industrial production, in the food industry, can prevent food oxidation, sugar preservation. And refined glucose oxidase, can be used for medical diagnosis, such as colorimetric blood glucose test paper and blood glucose meter based on biosensor. In the feed industry, as a feed additive, glucose oxidase can relieve intestinal mycotoxin poisoning, reduce the harm of excessive feed mold; at the same time, glucose oxidase catalyzes the glucose in the animal intestine to produce gluconic acid, which can reduce the pH value of gastric chyme, effectively inhibit the reproduction of harmful bacteria, promote the growth of beneficial bacteria, also can activate pepsin activity, which is beneficial to the digestion and absorption of protein, vitamin, mineral and other nutrients, thereby improving the feed conversion efficiency; the livestock and poultry feed added with glucose oxidase can remove free radicals produced by animals due to stress reaction, protect the integrity of intestinal epithelial cells, and block the invasion of pathogens.

[0026] Compared with the prior art, the beneficial effects of the present application are:

[0027] The glucose oxidase mutant provided by the present application has significantly improved heat resistance compared with the parent glucose oxidase of the amino acid sequence shown in SEQ ID NO: 1, and the enzyme activity of part of the glucose oxidase mutant is also obviously improved, which is conducive to the actual industrial application of the enzyme. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is the relative enzyme activity of wild-type glucose oxidase and glucose oxidase mutant at different reaction temperatures;

[0029] Figure 2 is the relative enzyme activity of wild-type glucose oxidase and glucose oxidase mutant at different pH values. DETAILED DESCRIPTION

[0030] In order to make those skilled in the art more clearly understand the technical solutions described in the present application, the following examples are used for illustration. It should be pointed out that the following examples do not constitute a limitation on the scope of protection required by the present application.

[0031] The biological materials, reagents or devices used in the following examples can be obtained from conventional commercial channels, or can be obtained by existing known methods, unless otherwise specified. The molecular biology experimental methods not specifically described in the following examples are carried out according to the specific methods listed in the book of "Molecular Cloning Experiment Guide" (third edition) J. Sambrook, or according to the reagent kit and product instruction.

[0032] Variant, Mutant: The terms "variant" "mutant" mean a polypeptide having glucose oxidase activity that comprises a mutation (i.e., a substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to the parent glucose oxidase set forth in SEQ ID NO: 1. A substitution means replacing the amino acid occupying a position with a different amino acid; a deletion means removing the amino acid occupying a position; and an insertion means adding an amino acid after abutting and immediately following the amino acid occupying a position. Mutants of the present application have at least 20%, e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the glucose oxidase activity of the mature polypeptide of SEQ ID NO: 1.

[0033] The present application provides a glucose oxidase mutant comprising the following mutations relative to the parent glucose oxidase set forth in the amino acid sequence of SEQ ID NO: 1: K37C, A572C. In a preferred embodiment, the variant has at least 98%, e.g., at least 98% or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.

[0034] Mutants of the present application include substitutions with cysteine at positions 37 and 572 and form disulfide bonds. In addition, the variant includes a substitution at one or more positions corresponding to positions 80, 248, 252, 277, 292, 296, 305, 350, 443, and 453 of SEQ ID NO: 1. In one embodiment, the variant includes a substitution at one or two positions corresponding to positions 80, 248, 252, 277, 292, 296, 305, 350, 443, and 453 of SEQ ID NO: 1.

[0035] In a preferred embodiment, the glucose oxidase mutant of the present application includes substitutions with cysteine at positions 37 and 572 and at least one of the following mutations relative to the parent glucose oxidase of SEQ ID NO: 1: H277F, M305L, W350Y, Q453K, Y80V, Q248V, K252V, P443C, A292L, T296V.

[0036] In a preferred embodiment, the glucose oxidase mutant of the present application has any one of the following mutations:

[0037] K37C+A572C; K37C+A572C+H277F; K37C+A572C+M305L; K37C+A572C+W350Y; K37C+A572C+Q453K; K37C+A572C+Q453K+W350Y; K37C+A572C+Q453K+M305L; K37C+A572C+Y80V; K37C+A572C+Q248V; K37C+A572C+K252V; K37C+A572C+P443C; K37C+A572C+A292L; K37C+A572C+T296V.

[0038] Expression: The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be measured, e.g., to detect increased expression, by techniques known in the art, such as measuring the level of mRNA and / or translated polypeptide.

[0039] Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences providing for its expression.

[0040] Fermentable medium: The term "fermentable medium" or "fermentation medium" refers to a medium comprising one or more (e.g., two, several) sugars, such as glucose, fructose, sucrose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and / or soluble oligosaccharides, wherein the medium is capable of being partially converted (fermented) by a host cell into a desired product, such as ethanol. In some cases, the fermentation medium is derived from a natural source, such as sugar cane, starch, or cellulose; and can be pre-treated for enzymatic hydrolysis (saccharification) from such a source. The term fermentation medium is understood herein to refer to the medium prior to the addition of the fermenting organism, e.g., the medium resulting from a saccharification process, as well as the medium used in a simultaneous saccharification and fermentation process (SSF).

[0041] Recombinant production of the glucose oxidase variants according to the present application can be performed in hosts known in the art. Suitable hosts can be selected from filamentous fungal strains, such as Aspergillus niger, Aspergillus sojae, and Aspergillus oryyzae. Suitable hosts can be selected from yeast strains, such as, for example, Pichia pastoris, Saccharomyces cerevisiae, and Hansenula polymorpha.

[0042] The cells expressing the glucose oxidase mutant used in the present application can be expressed in a bacterial host, or the protein secreted into the periplasm or extracellular space, in some embodiments of the present application, by any method known to those skilled in the art. When the glucose oxidase mutant is secreted into the nutrient medium, it can be recovered directly from the medium. If the glucose oxidase mutant is not secreted, it can be recovered from cell lysates.

[0043] The culture of the expression organism is prepared in an appropriate volume according to standard fermentation procedures. In preferred embodiments, the cells are grown in a fermenter, and the growth conditions are optionally controlled, such as pH, temperature, oxygen, and / or nutrient supply. The first step in purification involves separation of the cells from the supernatant using one or more of several techniques such as sedimentation, microfiltration, centrifugation, or flocculation. In preferred embodiments, the method of choice is microfiltration. If expressed intracellularly, the cells are treated to release the protein from the intracellular space. These treatments can include, for example, pressure, enzymatic, osmotic shock, freezing, sonication, or other treatments, resulting in a cell extract that can or can not be further purified.

[0044] In some embodiments of the present application, the glucose oxidase mutant is secreted into the supernatant after induction of the culture, and further purification from the supernatant or concentrated supernatant can be performed using one or more of several methods including extraction or fractionation methods such as ammonium sulfate or ethanol or acid precipitation, or chromatographic 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 chelate affinity chromatography, resulting in a high purity target protein. In other preferred embodiments, the target protein is purified to high purity by HPLC.

[0045] In other embodiments of the present application, the supernatant, or the supernatant partially purified by ultrafiltration, or the supernatant concentrated and / or diafiltered, is further dried by any of several techniques including, but not limited to, spray drying, freeze drying, down-draught evaporation, thin layer evaporation, centrifugal evaporation, conveyor dryer, or any combination thereof.

[0046] In further embodiments of the present application, the fermentation cell suspension including the expressed glucose oxidase is dried as a whole using methods including, but not limited to, fluidized bed drying, conveyor drying, spray drying, or drum drying, or any combination thereof.

[0047] The term "activity" or "catalytic activity" quantitatively describes the conversion of a given substrate under defined reaction conditions. The term "specific activity" quantitatively describes the catalytic activity relative to the enzyme amount under defined reaction conditions.

[0048] Example 1: Design screening of disulfide bond pairs of glucose oxidase

[0049] Using the published three-dimensional structure of wild-type Aspergillus niger glucose oxidase (the amino acid sequence of which is shown as SEQ ID NO: 1), and referring to the three-dimensional structure file PDB ID 1CF3, the disulfide bond pair site K37C+A572C was finally designed and screened after creative labor.

[0050] Example 2: Construction and expression of glucose oxidase mutant (GOX3)

[0051] Experimental materials and reagents:

[0052] 1. Strains and vectors

[0053] The E. coli strain Top10, Pichia pastoris GS115, the vector pPIC9K, and the antibiotic G418 were purchased from Invitrogen Company.

[0054] 2. Enzymes and kits

[0055] PCR enzymes, plasmid extraction kits, and gel purification kits were purchased from Shanghai Biotechnology Company, and restriction endonucleases were purchased from NEB Company.

[0056] 3. Culture medium

[0057] The E. coli culture medium was LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). LB-Amp was LB medium plus 100 μg / mL ampicillin. LB-Zeocin was LB medium plus 25 μg / mL Zeocin. The yeast culture medium was YPD (1% yeast extract, 2% peptone, 2% glucose). The yeast screening medium was YPDZ (YPD + 100 μg / mL Zeocin). The yeast induction medium was BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (v / v)) and BMMY (0.5% methanol instead of glycerol, the rest of the components were the same as BMGY). The recombinant yeast fermentation basic salt medium: diammonium phosphate 5%, potassium dihydrogen phosphate 0.5%, magnesium sulfate heptahydrate 1.5%, potassium sulfate 1.95%, calcium sulfate 0.1%, antifoam 0.03%. After high pressure, 4.35 mL PTM1 was added per liter. PTM1 (trace salt solution): copper sulfate 0.6%, potassium iodide 0.018%. Manganese sulfate monohydrate 0.3%, sodium molybdate dihydrate 0.02%, boric acid 0.002%, flowing water cobalt chloride 0.05%, zinc chloride 2%, ferric sulfate heptahydrate 6.5%, concentrated sulfuric acid 0.5%, biotin 0.02%

[0058] 4. Chemical reagents:

[0059] The glucose oxidase standard, o-dianisidine hydrochloride and horseradish peroxide were purchased from Sigma Company, glucose was purchased from OXIOD Company, and other reagents were purchased from Guangzhou Chemical Reagent Factory.

[0060] 5. Glucose oxidase determination method

[0061] The glucose oxidase activity was determined by o-dianisidine spectrophotometry (determination conditions: 37°C, pH 5.5). Under the action of glucose oxidase, glucose and oxygen reacted to generate gluconic acid and hydrogen peroxide. Hydrogen peroxide and colorless reduced o-dianisidine generated water and red oxidized o-dianisidine under the action of peroxidase. The absorbance of the reaction solution was determined at 540 nm, and the enzyme activity of glucose oxidase was calculated according to the standard curve.

[0062] Table 1: disulfide bond mutant primers

[0063] The amino acid sequence of the parent GOX2 of the glucose oxidase mutant in the application is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2. The related disulfide bond mutant GOX3 obtained by screening in Example 1 was amplified by PCR using the parent recombinant vector pPIC9K-GOX2 as a template. The related amplification primers are shown in Table 1.

[0064] PCR amplification detection by agarose gel electrophoresis: the target product amplified by PCR was purified and recovered. The template was digested with restriction endonuclease Dpnl, and the digested product was transformed into E. coli Top 10 competent cells by chemical transformation heat shock method. The recombinant transformants were verified by bacterial liquid PCR, and the plasmid of the correct transformants was extracted for sequencing to determine the corresponding mutant. The mutant plasmid with correct sequencing was linearized with Pmel, the linear plasmid fragment was purified, and the electric transformation method was used to transform into Pichia pastoris GS115 competent cells, and YPD+G418 medium was used for screening. The toothpick was used to pick the obtained yeast recombinant transformants one by one into a 24-well plate, 1 mL of BMGY medium was added to each well, and the culture was incubated at 30°C, 220 rpm for about 24 h, and the supernatant was centrifuged. Then 1.6 mL of BMMY medium was added for induction culture. After 24 h of culture, the supernatant was centrifuged, and 200 μL of the supernatant was taken out and added to a 24-well plate for glucose oxidase heat-resistant property analysis.

[0065] Example 3: Determination of heat stability of glucose oxidase mutant (GOX3)

[0066] The 24-well plate fermentation supernatant was diluted to about 10 U / mL with distilled water, and the residual enzyme activity was determined after treatment at 80°C for 3 min. The enzyme activity of the untreated sample was taken as 100%, and the relative enzyme activity was calculated. The results showed that the introduction of disulfide bond had a significant effect on the mutant. The results showed that after heat treatment, the residual enzyme activity of wild-type glucose oxidase GOX2 was only 41%, while the heat stability of glucose oxidase GOX3 mutant was significantly improved, and the residual enzyme activity after heat treatment was 76%, which was more than 50% higher than that of the wild type. The above results showed that the introduction of disulfide bond could obtain a mutant with higher heat stability.

[0067] Example 4: Semi-saturation mutation and combination mutation of glucose oxidase

[0068] In order to further improve the heat resistance of glucose oxidase, semi-saturation mutation was carried out to screen mutants with better heat resistance. The semi-saturation mutation primer (the sequence information is shown in Table 2) was designed using pPIC9K-GOX3 as the template, and the semi-saturation mutation was carried out. The mutant was constructed according to the construction method in Example 2, and finally, the mutant with improved properties was obtained by high-throughput screening method (the mutation site is shown in Table 3).

[0069] Table 2: Semi-saturation mutation primer

[0070] Table 3: Glucose oxidase mutation site

[0071] The enzyme activity of each glucose oxidase mutant screened by the present application was determined according to the glucose oxidase enzyme activity determination method of Example 2, and the results are shown in Table 4 below, with the enzyme activity of the parent glucose oxidase GOX2 as the benchmark.

[0072] Table 4: Relative enzyme activity of glucose oxidase mutants of the present application in fermentation

[0073] As can be seen from Table 4, the enzyme activity of most of the glucose oxidase mutants obtained by mutation based on GOX2 is improved, among which GOX15 has the highest improvement rate of relative enzyme activity compared to the parent enzyme, which can reach 29.6%.

[0074] Example 5: Heat resistance of glucose oxidase mutants

[0075] The fermentation supernatant containing each glucose oxidase mutant was diluted with distilled water to about 10 U / mL of glucose oxidase enzyme activity, and then treated at 80°C for 3 minutes, after which the residual enzyme activity was determined. The corresponding enzyme activity of the sample without heat treatment was taken as 100%, and the relative enzyme activity was calculated.

[0076] Table 5: Heat resistance of glucose oxidase mutants

[0077] The results in Table 5 show that the heat resistance of the glucose oxidase mutants of the present application is improved after heat treatment compared to the parent. Among them, the residual enzyme activity of GOX6, 7, 8, and 9 after heat treatment can still maintain above 81%, which is significantly better than the residual enzyme activity of the wild-type glucose oxidase GOX2 (only 41.5%). The above results show that by introducing appropriate disulfide bonds and new site combination mutations, mutants with higher thermal stability can be obtained.

[0078] Example 5: Optimal reaction temperature of glucose oxidase mutants

[0079] The enzyme activity of each glucose oxidase was taken as 100% at pH 5.5 and 37°C. Then, the relative enzyme activity of each glucose oxidase was determined at pH 5.5 and at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, and 75°C, respectively, and the results are shown in Figure 1 and Table 6. The optimal reaction temperature range of each glucose oxidase is 35°C-50°C, and the enzyme activity is basically unchanged. Above 55°C, the relative enzyme activity of GOX2 is 78.7%, but the relative enzyme activity of mutants GOX3, GOX5-GOX9 is all above 90%.

[0080] Table 6: Optimal reaction temperature of glucose oxidase mutants

[0081] Example 6: Optimum reaction pH of glucose oxidase mutants

[0082] The enzyme activity of each glucose oxidase was taken as 100% at pH 5.5 and 30°C. The relative enzyme activity of glucose oxidase was then determined at pH 3-pH 7.5 at 30°C, and the results are shown in Figure 2 and Table 7. The optimum reaction pH range of GOX2 and the glucose oxidase mutants was pH 4.5-pH 6.5, and the curve trends were consistent, and the mutants maintained the original pH characteristics.

[0083] Table 7: Optimum reaction pH of glucose oxidase mutants

[0084] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent of the present application. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the scope of protection of the patent of the present application should be subject to the appended claims.

Claims

1. A glucose oxidase mutant, characterized in that, The glucose oxidase mutant comprises the following mutations relative to the parent glucose oxidase of the amino acid sequence set forth in SEQ ID NO: 1: K37C, A572C.

2. The glucose oxidase mutant of claim 1, wherein, The glucose oxidase mutant further comprises at least one of the following mutations relative to the parent glucose oxidase of the amino acid sequence set forth in SEQ ID NO: 1: H277F, M305L, W350Y, Q453K, Y80V, Q248V, K252V, P443C, A292L, T296V.

3. The glucose oxidase mutant of claim 2, wherein, The glucose oxidase mutant has any one of the following mutations (1)-(12) relative to the parent glucose oxidase of the amino acid sequence set forth in SEQ ID NO: 1: (1) K37C+A572C+H277F; (2) K37C+A572C+M305L; (3) K37C+A572C+W350Y; (4) K37C+A572C+Q453K; (5) K37C+A572C+Q453K+W350Y; (6) K37C+A572C+Q453K+M305L; (7) K37C+A572C+Y80V; (8) K37C+A572C+Q248V; (9) K37C+A572C+K252V; (10) K37C+A572C+P443C; (11) K37C+A572C+A292L; (12) K37C+A572C+T296V.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises the nucleotide fragments set forth in (a) and / or (b): (a) a nucleotide fragment encoding the glucose oxidase mutant of any one of claims 1-3; (b) a nucleotide fragment reverse complementary to (a).

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 4.

6. A recombinant cell, characterized in that, The recombinant cell comprises the nucleic acid molecule of claim 4 or the recombinant expression vector of claim 5.

7. The recombinant cell of claim 6, wherein, The recombinant cell comprises a bacterial or fungal cell.

8. The recombinant cell of claim 7, wherein, The fungal cell is a Pichia pastoris cell.

9. A method for producing the glucose oxidase mutant according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: (1) culturing the recombinant cell of any one of claims 6-8; (2) inducing the recombinant cell to express the glucose oxidase mutant.

10. Use of the glucose oxidase mutant of any one of claims 1-3 in the field of food, medical diagnosis or feed.

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