Glucose oxidase mutant, preparation method therefor, and use thereof

Abstract

The present invention belongs to the technical field of genetic engineering. Provided are a glucose oxidase mutant, a preparation method therefor, and use thereof. The glucose oxidase mutant comprises disulfide bond mutation sites K37C + A572C relative to a parent glucose oxidase with the amino acid sequence set forth in SEQ ID NO: 1. Compared with the parent glucose oxidase, the thermal stability of the glucose oxidase mutant is significantly improved, exhibiting better heat resistance and facilitating the production and application of gluconate.

Description

A glucose oxidase mutant, its preparation method and application Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a glucose oxidase mutant, its preparation method, and its application. Background Technology

[0002] Glucose oxidase (EC1.1.3.4, GOD) specifically catalyzes the conversion of β-D-glucose to 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 distinct regions: one region, primarily composed of β-sheets, binds tightly to the FAD coenzyme molecule via non-covalent bonds; the other region, consisting of four α-helices supporting one antiparallel β-sheet, binds to the substrate β-D-glucose. This enzyme catalyzes the oxidation of β-D-glucose to gluconolactone and hydrogen peroxide, with gluconolactone being converted to gluconic acid without enzymatic reaction.

[0003] Gluconic acid is a mild, non-corrosive, non-toxic, and readily biodegradable organic acid produced by the microbial oxidation of glucose. Its chemical and physiological properties make gluconic acid and its salts, particularly sodium gluconate, play important roles in the chemical, textile, beverage, pharmaceutical (e.g., iron and calcium deficiencies), construction, and food industries. According to market research reports, the global gluconic acid market was valued at approximately US$1 billion in 2020 and is projected to reach US$1.9 billion by 2028, growing at a CAGR of 5% from 2021 to 2028. Chemical and fermentation processes are the main technologies for producing gluconic acid. Although the one-step synthesis of gluconic acid via chemical and electrolytic oxidation is efficient, high electrolysis costs, environmental toxicity, and biohazards limit the industrial application of chemical methods. Current industrial processes are based on the fermentation process of Aspergillus niger using glucose as a substrate, and some also employ the co-catalytic production of gluconate using glucose oxidase / catalase. The dual-enzyme method for producing sodium gluconate uses glucose as the main raw material; glucose oxidase, in synergy with catalase, directly converts glucose into gluconic acid, which is then neutralized with alkali to obtain sodium gluconate.

[0004] However, existing glucose oxidases still have poor heat resistance, making it difficult to meet the sterilization requirements before production. This heat resistance issue restricts the application of the two-enzyme production method. Therefore, this paper aims to propose a glucose oxidase with good heat resistance for industrial production. Summary of the Invention

[0005] This invention aims to at least solve the technical problems existing in the prior art. To this end, this invention proposes a glucose oxidase mutant, its preparation method, and its application. Compared with the parental glucose oxidase, this glucose oxidase mutant exhibits significantly improved thermal stability and better heat resistance, which is beneficial for the production and application of gluconate.

[0006] The present invention provides a glucose oxidase mutant that has at least 98% and less than 100% sequence identity relative to the parental glucose oxidase derived from Aspergillus niger (whose amino acid sequence is shown in SEQ ID NO: 1), and includes a disulfide bond mutation site K37C+A572C.

[0007] The meaning of K37C+A572C is that the amino acid residues at positions 37 and 572 of the parent glucose oxidase are replaced with cysteine ​​and a disulfide bond is formed.

[0008] Preferably, in some embodiments of the present invention, it further includes a disulfide bond mutation site S53C+T246C or S191C+A479C.

[0009] In this context, S53C+T246C means that the amino acid residues at positions 53 and 246 of the parental glucose oxidase are replaced with cysteine, and a disulfide bond is also formed between positions 53 and 246; S191C+A479C means that the amino acid residues at positions 191 and 479 of the parental glucose oxidase are replaced with cysteine, and a disulfide bond is also formed between positions 191 and 479.

[0010] Experiments show that, compared with the parental glucose oxidase, the glucose oxidase mutant containing the disulfide bond mutation site of this invention has significantly improved high-temperature stability and heat resistance, and can be applied to the production of gluconate with excellent results.

[0011] The present invention also provides a nucleic acid molecule comprising the nucleotide fragments shown in (a) and / or (b):

[0012] (a) The nucleotide fragment encoding the above glucose oxidase mutant;

[0013] (b) A nucleotide fragment that is the reverse complementary to (a).

[0014] The present invention also provides a recombinant expression vector comprising the above-mentioned nucleic acid molecules.

[0015] The present invention also provides a recombinant cell comprising the above-mentioned nucleic acid molecule or the above-mentioned recombinant expression vector.

[0016] Preferably, the recombinant cells comprise bacterial or fungal cells.

[0017] More preferably, the fungal cells are Pichia pastoris cells.

[0018] This invention also provides a method for preparing the above-mentioned glucose oxidase mutant, comprising the following steps:

[0019] (1) Culture recombinant cells;

[0020] (2) Induce the recombinant cells to express the above glucose oxidase mutant.

[0021] The present invention also provides the application of the above-mentioned glucose oxidase mutant in the production of gluconate.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] The glucose oxidase mutant provided by this invention exhibits significantly improved high-temperature stability and heat resistance compared to the parental glucose oxidase with the amino acid sequence shown in SEQ ID NO: 1. Furthermore, the glucose oxidase mutant provided by this invention demonstrates superior performance in the production of gluconate, exhibiting a faster reaction rate and a more complete reaction. Detailed Implementation

[0024] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0025] Unless otherwise specified, the biological materials, reagents, or devices used in the following examples are all available from conventional commercial sources or can be obtained by existing known methods. Molecular biology experimental methods not specifically described in the following examples were performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions.

[0026] Variant, Mutant: The terms "variant" and "mutant" refer to a polypeptide with glucose oxidase activity that contains mutations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the parental glucose oxidase position shown in SEQ ID NO:1. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. The mutants of the present invention have at least 20%, for example, 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.

[0027] This invention provides a glucose oxidase mutant, which, relative to the parental glucose oxidase of the amino acid sequence shown in SEQ ID NO: 1, includes at least the following mutation: K37C+A572C. In a preferred embodiment, the mutant has at least 98%, for example, at least 98% or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 1.

[0028] In one embodiment, the glucose oxidase mutant of the present invention has at least 98% and less than 100% sequence identity compared to the parental glucose oxidase, and includes substitutions of amino acid residues at positions 37 and 572 with cysteine ​​residues, forming disulfide bonds. Furthermore, the glucose oxidase mutant includes substitutions of amino acid residues at positions 53 and 246 with cysteine ​​residues, and disulfide bonds are also formed at positions 53 and 246.

[0029] In another embodiment, the glucose oxidase mutant of the present invention has at least 98% and less than 100% sequence identity compared to the parental glucose oxidase, and includes substitutions of amino acid residues at positions 37 and 572 with cysteine ​​residues, forming disulfide bonds. Furthermore, the glucose oxidase mutant includes substitutions of amino acid residues at positions 191 and 246 with cysteine ​​residues, and disulfide bonds are also formed at positions 191 and 246.

[0030] The term "disulfide bond" refers to a chemical bond that connects different peptide chains or two cysteine ​​residues within the same peptide chain. It is a relatively stable covalent bond formed by the oxidation of the thiol groups of two cysteine ​​residues in an amino acid sequence. Because disulfide bonds can bridge different regions of a peptide chain, they are sometimes also called disulfide bridges. In this invention, the disulfide bonds are not spontaneously generated and are introduced through point mutation.

[0031] 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—for example, to detect increased expression—using techniques known in the art, such as measuring the level of mRNA and / or translated polypeptide.

[0032] Expression vector: The term "expression vector" refers to a straight or circular DNA molecule containing a polynucleotide encoding a polypeptide and operatively linked to a control sequence provided for its expression. Multiple nucleotides and the control sequence can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is positioned in the vector such that the coding sequence is operatively linked to the suitable control sequence for expression.

[0033] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed circular plasmids.

[0034] Fermentable medium: The term "fermentable medium" or "fermentation medium" refers to a medium containing one or more sugars (e.g., two or more), such as glucose, fructose, sucrose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and / or soluble oligosaccharides, wherein the medium can be partially converted (fermented) by host cells. In some cases, the fermentation medium is derived from natural sources, such as sugarcane, starch, or cellulose; and may be derived from enzymatic hydrolysis (saccharification) pretreatment of such sources. The term fermentation medium is understood herein to refer to the medium prior to the addition of fermenting organisms, for example, media produced by saccharification processes, and media used in simultaneous saccharification and fermentation processes (SSF).

[0035] The recombinant production of the glucose oxidase variant according to the present invention can be carried out in hosts known in the art. Suitable hosts may be selected from filamentous fungal strains, such as Aspergillus niger, Aspergillus sojae, and Aspergillus oryyzae. Suitable hosts may be selected from yeast strains, such as Pichia pastoris, Saccharomyces cerevisiae, and Hansenula polymorpha.

[0036] The cells expressing the glucose oxidase mutant used in this invention are produced by any method known to those skilled in the art. In some embodiments of this invention, the glucose oxidase mutant can be expressed in a bacterial host, or the protein can be secreted into the periplasm or extracellular space. When the glucose oxidase mutant is secreted into the nutrient medium, it can be directly recovered from the medium. If the glucose oxidase mutant is not secreted, it can be recovered from cell lysates.

[0037] The glucose oxidase of the present invention can be recovered using methods known in the art. For example, the polypeptide can be recovered from the fermentation medium by conventional methods, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the whole fermentation broth containing the glucose oxidase of the present invention can be recovered.

[0038] The expression organism is cultured according to standard fermentation methods in appropriate volumes. In a preferred embodiment, cells are grown in a fermenter, with growth conditions such as pH, temperature, oxygen, and / or nutrient supply optionally controlled. The first step of purification involves separating the cells from the supernatant using one or more of several techniques such as sedimentation, microfiltration, centrifugation, or flocculation. In a preferred embodiment, microfiltration is a suitable method. If expressed intracellularly, the cells are treated to release the protein from the intracellular space. These treatments may include, for example, pressurization, enzymatic stimulation, osmotic shock, freezing, sonication, or other treatments, to produce a cell extract, which may be further purified or may not be performed.

[0039] In some embodiments of the invention, after induction culture, the glucose oxidase mutant is secreted into a supernatant. Further protein purification from the supernatant or concentrated supernatant can be performed using one or more methods including: extraction or fractionation methods such as ammonium sulfate, ethanol, or acid precipitation; or chromatography methods, including but not limited to ion exchange, hydrophobic interactions, hydroxyapatite, particle size fractionation by gel filtration, cellulose phosphate or lectin chromatography, and affinity chromatography, or any combination thereof. In some preferred methods, the affinity-labeled protein is purified by metal chelate affinity chromatography to obtain a high-purity target protein. In other preferred embodiments, a high-purity target protein is obtained by HPLC purification.

[0040] In other embodiments of the invention, the supernatant, or the supernatant partially purified by ultrafiltration, or the supernatant concentrated and / or diafiltrated, is further dried by any of the following techniques: such as, but not limited to, spray drying, freeze drying, down-draught evaporation, thin-layer evaporation, centrifugal evaporation, conveyor drying, or any combination thereof.

[0041] In a further embodiment of the invention, the fermentation cell suspension containing expressed glucose oxidase is dried as a whole using methods such as, but not limited to, fluidized bed drying, conveyor drying, spray drying, or drum drying or any combination thereof.

[0042] The term "activity" or "catalytic activity" quantitatively describes the conversion of a given substrate under specified reaction conditions. The term "specific activity" quantitatively describes the catalytic activity relative to the amount of enzyme under specified reaction conditions.

[0043] Example 1: Design and screening of disulfide bond mutations in glucose oxidase

[0044] Using the publicly available three-dimensional structure of wild-type Aspergillus niger glucose oxidase (i.e., the parental glucose oxidase, whose amino acid sequence is shown in SEQ ID NO:1), and with reference to the three-dimensional structure file PDB ID: 1CF3, through creative effort, the following glucose oxidase mutants with a single disulfide bond mutation were finally designed and screened:

[0045] Table 1: Glucose oxidase mutants

[0046] Example 2: Construction and expression of glucose oxidase mutant

[0047] Experimental materials and reagents:

[0048] 1. Strains and vectors

[0049] Escherichia coli strain Top10, Pichia pastoris GS115, vector pPIC9K, and antibiotic G418 were purchased from Invitrogen.

[0050] 2. Enzymes and kits

[0051] PCR enzymes, plasmid extraction kits, and gel purification kits were purchased from Shanghai Sangon Biotech Co., Ltd., and restriction endonucleases were purchased from NEB Corporation.

[0052] 3. Culture medium

[0053] E. coli culture medium was LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). LB-Amp was LB medium with 100 μg / mL ampicillin. LB-Zeocin was LB medium with 25 μg / mL Zeocin. Yeast culture medium was YPD (1% yeast extract, 2% peptone, 2% glucose). Yeast selection medium was YPDZ (YPD + 100 μg / mL Zeocin). 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, other components the same as BMGY). Basic salt medium for recombinant yeast fermentation: 5% diammonium hydrogen phosphate, 0.5% potassium dihydrogen phosphate, 1.5% magnesium sulfate heptahydrate, 1.95% potassium sulfate, 0.1% calcium sulfate, and 0.03% antifoaming agent. After pressurization, add 4.35 mL of PTM1 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%, cobalt chloride in running water 0.05%, zinc chloride 2%, ferric sulfate heptahydrate 6.5%, concentrated sulfuric acid 0.5%, biotin 0.02%.

[0054] 4. Chemical reagents:

[0055] Glucose oxidase standard, o-anisidine hydrochloride and horseradish peroxide were purchased from Sigma-Aldrich, glucose was purchased from OXIOD, and other reagents were purchased from Guangzhou Chemical Reagent Factory.

[0056] 5. Glucose oxidase assay method

[0057] Glucose oxidase activity was determined using the o-anisidine spectrophotometric method (measurement conditions: 37℃, pH 5.5). Under the action of glucose oxidase, glucose reacts with oxygen to produce gluconic acid and hydrogen peroxide. Hydrogen peroxide then reacts with colorless reduced o-anisidine under the action of peroxidase to produce water and red oxidized o-anisidine. The absorbance of the reaction solution was measured at 540 nm, and the enzyme activity of glucose oxidase was calculated based on a standard curve.

[0058] Table 2: Disulfide bond mutation primers

[0059] The amino acid sequence of the parent glucose oxidase (GOX) of the glucose oxidase mutant in this invention is shown in SEQ ID NO:1. Using the parental recombinant vector pPIC9K-GOX as a template, primers were designed to construct the relevant glucose oxidase mutants listed in Table 1, and PCR amplification was performed. The relevant amplification primers are shown in Table 2.

[0060] PCR amplification was detected by agarose gel electrophoresis, and the target product of PCR amplification was purified and recovered. Template was digested with restriction endonuclease DpnI, and the digested product was transformed into *E. coli* Top10 competent cells using a chemical transformation heat shock method. Recombinant transformants were verified by colony PCR, and plasmids from verified transformants were extracted and sequenced to identify the corresponding mutations. The correctly sequenced mutant plasmids were linearized with PmeI, the linear plasmid fragments were purified, and transformed into *Pichia pastoris* GS115 competent cells using electroporation. Selection was performed using YPD+G418 medium. Each yeast recombinant transformant was picked up with a toothpick and transferred to a 24-well plate. 1 mL of BMGY medium was added to each well, and the plates were incubated at 30°C and 220 rpm for approximately 24 hours. The supernatant was then discarded by centrifugation. 1.6 mL of BMGY medium was added to each well for induction culture. After 24 hours of culture, the supernatant was collected by centrifugation, and 200 μL of the supernatant was transferred to each well for analysis of glucose oxidase thermostability.

[0061] Example 3: Determination of the thermal stability of glucose oxidase disulfide bond mutant

[0062] The fermentation supernatant from Example 2 was diluted with distilled water to approximately 10 U / mL for each glucose oxidase in a 24-well plate. After treatment at 80°C for 3 minutes, the residual enzyme activity was measured. The relative enzyme activity was calculated with the corresponding enzyme activity of the untreated sample as 100%, and the results are shown in Table 3.

[0063] The results showed that the introduction of disulfide bonds had a significant impact on the mutants. The thermostability of the GOX-MUT-S1 and GOX-MUT-S3 mutants was significantly improved, and the residual enzyme activity of the glucose oxidase mutants was more than 50% higher than that of the parental glucose oxidase (GOX), especially GOX-MUT-S1, which showed a significant increase in residual enzyme activity after heat treatment. These results indicate that introducing disulfide bond mutations can yield glucose oxidase mutants with higher thermostability.

[0064] Table 3: Results of heat treatment of glucose oxidase

[0065] Example 4: High-temperature resistance and thermal storage stability of glucose oxidase disulfide bond combinatorial mutants

[0066] To further enhance the thermostability of glucose oxidase, a combined mutation with two disulfide bond mutation sites was performed. Using pPIC9K-GOX-MUT-S1 as a template, pPIC9K-GOX-MUT-S1-S2 and pPIC9K-GOX-MUT-S1-S3 vectors were constructed. The specific construction method was the same as that in Example 1 for constructing glucose oxidase mutants.

[0067] The high-temperature resistance of the glucose oxidase disulfide bond combination mutant was determined according to the scheme in Example 3.

[0068] Table 4: High-temperature stability determination of glucose oxidase disulfide bond combinatorial mutants

[0069] As shown in Table 4, after treatment at 80℃ for 3 minutes, the residual enzyme activity of the glucose oxidase disulfide bond combination mutant was significantly higher than that of the wild-type glucose oxidase GOX, and the residual enzyme activity after heat treatment with GOX-MUT-S1-S3 remained above 84%. Furthermore, comparing with Table 3, it can be found that the glucose oxidase disulfide bond combination mutant exhibits superior heat resistance compared to the glucose oxidase mutant with a single disulfide bond mutation.

[0070] Following the methods described in Example 2, the fermentation supernatant of the 24-well plate was diluted with distilled water, and the activity of each glucose oxidase was diluted to approximately 10 U / mL. The plates were then incubated at 45°C for 48 hours, and the residual enzyme activity was measured. The relative enzyme activity was calculated with the corresponding enzyme activity of the untreated sample as 100%. The results are shown in Table 5.

[0071] Table 5: Thermal stability determination of glucose oxidase mutants

[0072] The results showed that after heat treatment at 45℃ for 48 hours, the residual enzyme activity of wild-type glucose oxidase GOX was 74.9%, and the thermal storage stability of the glucose oxidase mutant GOX-MUT-S1 with a single disulfide bond mutation also reached 87.7%. However, it can be seen that the thermal storage stability of the glucose oxidase disulfide bond combination mutants GOX-MUT-S1-S2 and GOX-MUT-S1-S3 was significantly higher, with almost no loss after heat treatment. These results indicate that introducing two pairs of disulfide bond mutations can obtain mutants with higher thermal stability.

[0073] Example 5: Application of glucose oxidase in sodium gluconate production

[0074] The residual sugar and reaction cycle in the two-enzyme method for producing sodium gluconate were used as evaluation indicators. The two-enzyme method for producing sodium gluconate uses glucose as the main raw material. Glucose oxidase, in synergistic action with catalase, directly converts glucose into gluconic acid, which is then neutralized with alkali to obtain sodium gluconate. The application experiments of catalase (CAT) and different glucose oxidase mutants in sodium gluconate production were compared to study the effects of different glucose oxidase mutants on sodium gluconate production. Under the conditions of 45℃, pH 5.5, rotation speed 500 rpm, pressure 0.1 MPa, 36% glucose solution, and 40% NaOH solution, glucose oxidase can oxidize glucose to gluconic acid and hydrogen peroxide. Gluconic acid further reacts with sodium hydroxide to neutralize and produce sodium gluconate, while hydrogen peroxide is catalyzed by catalase to produce water and oxygen.

[0075] Experimental equipment and materials:

[0076] Experimental equipment: 20L reactor

[0077] Experimental reagents:

[0078] (1) 36% glucose solution: Dissolve 4 kg of glucose monohydrate in water and bring the volume up to 10 L.

[0079] (2) 40% NaOH solution: Dissolve 2 kg of NaOH in water and bring the volume up to 5 L.

[0080] (3) Catalase, enzyme activity: 600,000 U / g.

[0081] Table 6: Catalytic effect of glucose oxidase

[0082] As shown in Table 6, under conditions of 45℃ and with the same amounts of glucose oxidase and catalase, GOX reaction still showed incomplete glucose reaction after 26 hours. Compared to GOX, GOX-MUT-S1, GOX-MUT-S1-S2, and GOX-MUT-S1-S3 all achieved complete reaction (residual sugar content less than 0.1%). Specifically, GOX-MUT-S1-S2 and GOX-MUT-S1-S3 had reaction times of 21 hours and 20 hours, respectively, with similar residual sugar contents, indicating complete reaction. This represents a reduction of 3 to 6 hours compared to GOX and GOX-S1. In summary, GOX-MUT-S1-S2 and GOX-MUT-S1-S3 demonstrate excellent performance in the production of sodium gluconate. Considering their high-temperature resistance and thermal storage stability, GOX-MUT-S1-S2 and GOX-MUT-S1-S3 exhibit superior performance in the production of gluconate.

[0083] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A glucose oxidase mutant, characterized in that, The glucose oxidase mutant has at least 98% and less than 100% sequence identity with the parental glucose oxidase with the amino acid sequence shown in SEQ ID NO: 1, and includes the disulfide bond mutation site K37C+A572C.

2. The glucose oxidase mutant according to claim 1, characterized in that, It also includes disulfide bond mutation sites S53C+T246C or S191C+A479C.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule contains the nucleotide fragments shown in (a) and / or (b): (a) A nucleotide fragment encoding the glucose oxidase mutant of claim 1 or 2; (b) A nucleotide fragment that is the reverse complementary to (a).

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 3.

5. A recombinant cell, characterized in that, The recombinant cells comprise the nucleic acid molecule of claim 3 or the recombinant expression vector of claim 4.

6. The recombinant cell according to claim 5, characterized in that, The recombinant cells include bacterial or fungal cells.

7. The recombinant cell according to claim 6, characterized in that, The fungal cells are Pichia pastoris cells.

8. The method for preparing the glucose oxidase mutant according to claim 1 or 2, characterized in that, Includes the following steps: (1) Culturing the recombinant cells according to any one of claims 5-7; (2) Inducing the recombinant cells to express glucose oxidase mutant.

9. The use of the glucose oxidase mutant according to claim 1 or 2 in the production of gluconate.

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