Glucose oxidase mutant as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and discloses a glucose oxidase mutant as well as a preparation method and application thereof. Relative to parent glucose oxidase with an amino acid sequence as shown in SEQ ID NO: 1, the glucose oxidase mutant comprises a disulfide bond mutation site K37C + A572C. Compared with parent glucose oxidase, the thermal stability of the glucose oxidase mutant is obviously improved, good heat resistance is embodied, and production and application of gluconate are facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a glucose oxidase mutant and a preparation method and application thereof. Background Art

[0002] Glucose oxidase (EC1.1.3.4, GOD) can specifically catalyze β-D-glucose to produce 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 is mainly composed of β-folds, which are tightly bound to the flavin adenine dinucleotide (FAD) coenzyme molecule by non-covalent bonds; the other region is composed of four α-helices supporting an antiparallel β-fold, which can bind to the substrate β-D-glucose. The enzyme can catalyze the oxidation of β-D-glucose into gluconolactone and hydrogen peroxide, among which gluconolactone can be converted into gluconic acid under non-enzymatic reactions.

[0003] Gluconic acid is a mild, non-corrosive, non-toxic, and readily biodegradable organic acid produced by microbial oxidation of glucose. Its chemical and physiological properties make gluconic acid and its salts, especially sodium gluconate, important in the chemical, textile, beverage, pharmaceutical (such as iron and calcium deficiency), construction, and food industries. According to market research reports, the global gluconic acid market was valued at approximately $1 billion in 2020 and is expected to reach $1.9 billion by 2028, growing at a compound annual growth rate of 5% between 2021 and 2028. Chemical and fermentation processes are the main technologies for producing gluconic acid. Although the synthesis of gluconic acid by chemical and electrolytic oxidation in one step is effective, the high electrolysis cost, environmental toxicity, and biological hazards limit the industrial application of chemical methods. The current industrial process is based on the fermentation process of Aspergillus niger with glucose as the substrate, and there is also the use of glucose oxidase / catalase co-catalysis to produce gluconate. The dual enzyme method for producing sodium gluconate uses glucose as the main raw material. Glucose oxidase directly converts glucose into gluconic acid under the synergistic action of catalase, and then neutralizes it with alkali to produce sodium gluconate.

[0004] However, the heat resistance of existing glucose oxidase is still poor, which makes it difficult to meet the sterilization requirements before production. The problem of heat resistance restricts the application of dual enzyme production. Based on this, it is hoped to propose a glucose oxidase with good heat resistance for industrial production. Summary of the invention

[0005] The present invention aims to at least solve the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a glucose oxidase mutant and a preparation method and application thereof. Compared with the parent glucose oxidase, the thermal stability of the glucose oxidase mutant is significantly improved, reflecting good heat resistance, which is conducive to the production and application of gluconate.

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

[0007] K37C+A572C means that the 37th and 572th amino acid residues of the parent glucose oxidase are substituted with cysteine ​​to form a disulfide bond.

[0008] SEQ ID NO: 1: SNGIEASLLKDPKLVAGRTYDYIIAGGGLAGLTVAEKLT ENPNITVLVIESGSYESDRGPIIIEDLNAYGEIFGTSVDHAYETVELATNNRTALIRSGNGLGGSTLINGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGLNGTVHVGPRDTGDDYSPLMRALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRADAAREWLLPNYQRPNLQV LTGQYVGKVLLSQNATTPRAVGVEFGTHKSNTHNVYAKHEVLLSAGSTVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELF LDTGGVASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLSAWVEYIPEHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQLSSHVMTVFYAMALKIADAVLADYASMQ.

[0009] Preferably, in some embodiments of the present invention, it also comprises a disulfide bond mutation site S53C+T246C or S191C+A479C.

[0010] Among them, S53C+T246C means that the 53rd and 246th amino acid residues of the parent glucose oxidase are replaced by cysteine, and the 53rd and 246th positions also form a disulfide bond; S191C+A479C means that the 191st and 479th amino acid residues of the parent glucose oxidase are replaced by cysteine, and the 191st and 479th positions also form a disulfide bond.

[0011] Experiments show that compared with the parent glucose oxidase, the glucose oxidase mutant containing a disulfide bond mutation site of the present invention has significantly improved high temperature stability and heat resistance, and can be applied to the production of gluconate with excellent results.

[0012] The present invention also provides a nucleic acid molecule comprising the nucleotide fragments shown in (a) and / or (b):

[0013] (a) a nucleotide fragment encoding the above-mentioned glucose oxidase mutant;

[0014] (b) A nucleotide fragment that is the reverse complement of (a).

[0015] The present invention also provides a recombinant expression vector, which comprises the above nucleic acid molecule.

[0016] The present invention also provides a recombinant cell, which comprises the above nucleic acid molecule or the above recombinant expression vector.

[0017] Preferably, the recombinant cell comprises a bacterial or fungal cell.

[0018] More preferably, the fungal cell is a Pichia pastoris cell.

[0019] The present invention also provides a method for preparing the above-mentioned glucose oxidase mutant, comprising the following steps:

[0020] (1) Cultivating recombinant cells;

[0021] (2) Inducing the recombinant cell to express the glucose oxidase mutant.

[0022] The present invention also provides the use of the glucose oxidase mutant in producing gluconate.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The glucose oxidase mutant provided by the present invention has significantly improved high temperature stability and heat resistance compared with the parent glucose oxidase of the amino acid sequence shown in SEQ ID NO: 1. At the same time, the glucose oxidase mutant provided by the present invention has a better effect in the production of gluconate, which is manifested in a faster reaction rate and a more thorough reaction. DETAILED DESCRIPTION

[0025] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0026] Unless otherwise specified, the biological materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or by existing known methods. The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the book Molecular Cloning Laboratory Manual (3rd Edition) by J. Sambrook, or according to the kits and product instructions.

[0027] Variant, mutant: The terms "variant" and "mutant" mean a polypeptide having glucose oxidase activity comprising a mutation (i.e., substitution, insertion and / or deletion) at one or more (e.g., several) positions relative to the position of the parent glucose oxidase shown in SEQ ID NO: 1. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a certain position. The mutant of the present invention has at least 20%, such as 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.

[0028] The present invention provides a glucose oxidase mutant, which comprises at least the following mutations relative to the parent glucose oxidase of the amino acid sequence shown in SEQ ID NO: 1: K37C+A572C. In a preferred embodiment, the mutant has at least 98%, such as at least 98% or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 1.

[0029] In one embodiment, the glucose oxidase mutant of the present invention has at least 98% and less than 100% sequence identity compared to the parent glucose oxidase, and includes substitution of amino acid residues at positions 37 and 572 with cysteine, and forms a disulfide bond. In addition, the glucose oxidase mutant includes substitution of amino acid residues at positions 53 and 246 with cysteine, and positions 53 and 246 also form a disulfide bond.

[0030] In another embodiment, the glucose oxidase mutant of the present invention has at least 98% and less than 100% sequence identity compared to the parent glucose oxidase, and includes substitution of amino acid residues at positions 37 and 572 with cysteine, and forms a disulfide bond. In addition, the glucose oxidase mutant includes substitution of amino acid residues at positions 191 and 246 with cysteine, and positions 191 and 246 also form a disulfide bond.

[0031] The term "disulfide bond" means a chemical bond connecting two cysteines in different peptide chains or the same peptide chain, and is a relatively stable covalent bond formed by oxidation of the sulfhydryl groups of two cysteine ​​residues in an amino acid sequence. Since disulfide bonds can bridge different regions of a peptide chain, they are sometimes referred to as disulfide bridges. In the present invention, disulfide bonds are non-naturally occurring and introduced by point mutation.

[0032] 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 levels of mRNA and / or translated polypeptide.

[0033] Expression vector: The term "expression vector" means a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operably linked to a control sequence that provides for its expression. Multiple nucleotides and control sequences 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 an appropriate vector for expression. When generating an expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked to an appropriate control sequence for expression.

[0034] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be easily subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0035] 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 the host cell. In some cases, the fermentation medium is derived from a natural source, such as sugar cane, starch or cellulose; and may be derived from a pretreatment of such sources by enzymatic hydrolysis (saccharification). The term fermentation medium is understood herein to refer to the medium before the addition of the fermenting organism, for example, the medium produced by the saccharification process, and the medium used in a simultaneous saccharification and fermentation process (SSF).

[0036] The recombinant production of glucose oxidase variants according to the present invention can be carried out in a host 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.

[0037] The cells expressing the glucose oxidase mutants used in the present invention are produced by any method known to those skilled in the art. In some embodiments of the present invention, the glucose oxidase mutants can be expressed in a bacterial host, or the protein can be secreted into the periplasm or the extracellular space. When the glucose oxidase mutants are secreted into the nutrient medium, they can be directly recovered from the culture medium. If the glucose oxidase mutants are not secreted, they can be recovered from the cell lysate.

[0038] 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 is recovered.

[0039] The cultivation of expression organisms is prepared with appropriate volumes according to standard fermentation methods. In a preferred embodiment, cells are grown in fermentor tanks, and optionally controlled growth conditions such as pH, temperature, oxygen and / or nutrient supply. The first step of purification comprises using several technologies such as sedimentation, microfiltration, centrifugation or flocculation to separate cells from the supernatant. In a preferred embodiment, the method applicable is microfiltration. If expressed in cells, cells are processed to release protein from the intracellular gap. These processes may include, for example, pressurization, enzymatic, osmotic shock, freezing, ultrasound or other processing, thereby producing cell extracts, which may be further purified, or may not be carried out.

[0040] In some embodiments of the present invention, the glucose oxidase mutant is secreted into the supernatant after induction culture, and further protein purification from the supernatant or concentrated supernatant can be performed using one or more of the following methods: extraction or classification methods such as ammonium sulfate or ethanol or acid precipitation, or chromatography, including but not limited to ion exchange, hydrophobic interaction, hydroxyapatite, particle size classification 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 to obtain a high-purity target protein. In other preferred embodiments, the high-purity target protein is obtained by HPLC purification.

[0041] In other embodiments of the present invention, the supernatant, or the supernatant partially purified by ultrafiltration, or the concentrated and / or diafiltrated supernatant, is further dried by any of the following technical methods: the technical methods are, for example, but not limited to, spray drying, freeze drying, down-draught evaporation, thin layer evaporation, centrifugal evaporation, conveyor drying or any combination thereof.

[0042] In a further embodiment of the invention, the fermentation cell suspension comprising the expressed glucose oxidase is dried as a whole using methods such as, but not limited to, fluid bed drying, conveyor drying, spray drying or drum drying or any combination thereof.

[0043] 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 amount of enzyme under defined reaction conditions.

[0044] Example 1: Design and screening of disulfide bond mutations in glucose oxidase

[0045] Using the published three-dimensional structure of wild-type Aspergillus niger glucose oxidase (i.e., parent glucose oxidase, whose amino acid sequence is shown in SEQ ID NO: 1), and taking the three-dimensional structure file PDB ID: 1CF3 as a reference, after creative work, the following glucose oxidase mutants with a single disulfide bond mutation were finally designed and screened:

[0046] Table 1: Glucose oxidase mutants

[0047]

[0048]

[0049] Example 2: Construction and expression of glucose oxidase mutants

[0050] Experimental materials and reagents:

[0051] 1. Strains and vectors

[0052] Escherichia coli strain Top10, Pichia pastoris GS115, vector pPIC9K, and antibiotic G418 were purchased from Invitrogen.

[0053] 2. Enzymes and kits

[0054] PCR enzyme, plasmid extraction kit, and gel purification kit were purchased from Shanghai Bioengineering Company, and restriction endonucleases were purchased from New England Biolabs.

[0055] 3. Culture medium

[0056] The E. coli culture medium is LB (1% peptone, 0.5% yeast extract, 1% NaCL, pH 7.0). LB-Amp is LB medium plus 100 μg / mL ampicillin. LB-Zeocin is LB medium plus 25 μg / mL Zeocin. The yeast culture medium is YPD (1% yeast extract, 2% peptone, 2% glucose). The yeast screening medium is YPDZ (YPD+100 μg / mL Zeocin). The yeast induction medium is 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 ingredients are the same as BMGY). The 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% defoamer. After high pressure, add 4.35 mL PTM1 per liter. PTM1 (trace salt solution): 0.6% copper sulfate, 0.018% potassium iodide, 0.3% manganese sulfate monohydrate, 0.02% sodium molybdate dihydrate, 0.002% boric acid, 0.05% running water cobalt chloride, 2% zinc chloride, 6.5% ferrous sulfate heptahydrate, 0.5% concentrated sulfuric acid, 0.02% biotin.

[0057] 4. Chemical reagents:

[0058] Glucose oxidase standard, o-dianisidine hydrochloride and horseradish peroxide were purchased from Sigma, glucose was purchased from OXIOD, and other reagents were purchased from Guangzhou Chemical Reagent Factory.

[0059] 5. Glucose oxidase assay method

[0060] Glucose oxidase activity was determined using o-dianisidine spectrophotometry (determination conditions: 37°C, pH 5.5). Under the action of glucose oxidase, glucose reacts with oxygen to generate gluconic acid and hydrogen peroxide. Hydrogen peroxide and colorless reduced o-dianisidine generate water and red oxidized o-dianisidine under the action of peroxidase. The absorbance of the reaction solution was measured at 540nm, and the glucose oxidase activity was calculated based on the standard curve.

[0061] Table 2: Disulfide bond mutation primers

[0062]

[0063]

[0064] The amino acid sequence of the parent glucose oxidase (GOX) of the glucose oxidase mutant of the present invention is shown in SEQ ID NO: 1. Using the parent recombinant vector pPIC9K-GOX as a template, primers were designed to construct the relevant glucose oxidase mutants in Table 1, and PCR amplification was performed. The relevant amplification primers are shown in Table 2.

[0065] Agarose electrophoresis was used to detect PCR amplification, and the target product of PCR amplification was purified and recovered. The template was digested with restriction endonuclease DpnI, and the decomposed product was transferred into Escherichia coli Top10 competent cells by chemical conversion heat shock method. The recombinant transformant was verified by bacterial liquid PCR, and the plasmid of the correct transformant was extracted for sequencing to determine the corresponding mutation. The mutant plasmid with correct sequencing was linearized with PmeI, the linear plasmid fragment was purified, and it was transferred into Pichia pastoris GS115 competent cells by electrotransformation method, and screened by YPD+G418 medium. The obtained yeast recombinant transformants were picked one by one with a toothpick to a 24-well plate, 1 mL of BMGY medium was added to each well, and cultured at 30°C and 220 rpm for about 24 hours, and the supernatant was centrifuged to remove the supernatant. Then 1.6 mL of BMMY medium was added for induction culture. After 24 hours of culture, the supernatant was centrifuged and 200 μL of the above supernatant was taken out to a 24-well plate for analysis of the heat resistance of glucose oxidase.

[0066] Example 3: Determination of thermal stability of disulfide bond mutants of glucose oxidase

[0067] The fermentation supernatant in Example 2 was diluted to about 10 U / mL with distilled water in a 24-well plate, and the residual enzyme activity was measured after treatment at 80°C for 3 minutes. The corresponding enzyme activity of the untreated sample was taken as 100%, and the relative enzyme activity was calculated to obtain the results shown in Table 3.

[0068] The results showed that the introduction of disulfide bonds had a significant effect on the mutants. The thermal stability of GOX-MUT-S1 and GOX-MUT-S3 mutants was significantly improved, and the residual enzyme activity of the glucose oxidase mutants was increased by more than 50% compared with the parent glucose oxidase (GOX), especially GOX-MUT-S1, the residual enzyme activity after heat treatment was significantly improved. The above results show that the introduction of disulfide bond mutations can obtain glucose oxidase mutants with higher thermal stability.

[0069] Table 3: Glucose oxidase heat treatment results

[0070] serial number Residual enzyme activity after heat treatment (%) GOX 41.5 GOX-MUT-S1 73.4 GOX-MUT-S2 50.1 GOX-MUT-S3 62.5

[0071] Example 4: High temperature resistance and thermal storage stability of disulfide bond combination mutants of glucose oxidase

[0072] In order to further improve the heat resistance 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 respectively. The specific construction method refers to the construction method in Implementation Case 1 to construct the glucose oxidase mutant.

[0073] According to the protocol of Example 3, the high temperature resistance of the disulfide bond combination mutant of glucose oxidase was determined.

[0074] Table 4: Determination of the high temperature stability of disulfide bond combination mutants of glucose oxidase

[0075]

[0076] As can be seen from Table 4, after being treated at 80°C for 3 minutes, the residual enzyme activity of the glucose oxidase disulfide bond combination mutant was significantly improved compared with the wild-type glucose oxidase GOX, and the residual enzyme activity of GOX-MUT-S1-S3 after heat treatment was maintained at more than 84%. At the same time, compared with Table 3, it can be found that the glucose oxidase disulfide bond combination mutant has better high temperature resistance than the glucose oxidase mutant with a single disulfide bond mutation.

[0077] Referring to the relevant method in Example 2, the fermentation supernatant of the 24-well plate was diluted with distilled water, and the glucose oxidase activity of each was diluted to about 10U / mL, and the samples were placed at 45°C for 48 hours to measure the residual enzyme activity. The corresponding enzyme activity of the untreated sample was taken as 100%, and the relative enzyme activity was calculated. The results are shown in Table 5.

[0078] Table 5: Thermal storage stability assay of glucose oxidase mutants

[0079]

[0080] The results showed that after heat treatment at 45°C for 48 hours, the wild-type glucose oxidase GOX had a residual enzyme activity of 74.9%, and the thermal storage stability of the glucose oxidase mutant GOX-MUT-S1 with a single disulfide bond mutation could reach 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 is significantly higher, with basically no loss after heat treatment. The above results show that the introduction of two pairs of disulfide bond mutations can obtain mutants with higher thermal stability.

[0081] Example 5: Application of glucose oxidase in the production of sodium gluconate

[0082] The residual sugar and reaction cycle in the double enzyme method for producing sodium gluconate were used as evaluation indicators. The double enzyme method for producing sodium gluconate uses glucose as the main raw material. Glucose oxidase directly converts glucose into gluconic acid under the synergistic action of catalase, and then neutralizes with alkali to produce sodium gluconate. Catalase (CAT) was compared with different glucose oxidase mutants in the sodium gluconate application experiment to study the application effect of different glucose oxidase mutants in sodium gluconate. Under the conditions of 45°C, pH 5.5, speed 500rpm, pressure 0.1MPa, 36% glucose solution, and 40% NaOH solution, glucose oxidase can oxidize glucose into gluconic acid and hydrogen peroxide, and gluconic acid further reacts with sodium hydroxide to generate sodium gluconate, and hydrogen peroxide can be catalyzed by catalase to generate water and oxygen.

[0083] Experimental equipment and materials:

[0084] Experimental equipment: 20L reactor

[0085] Experimental reagents:

[0086] (1) 36% glucose solution: Dissolve 4 kg of glucose monohydrate in water and make up to 10 L;

[0087] (2) 40% NaOH solution: Dissolve 2 kg NaOH in water and make up to 5 L;

[0088] (3) Catalase, enzyme activity: 600,000 U / g.

[0089] Table 6: Catalytic effect of glucose oxidase

[0090]

[0091] As shown in Table 6, under the condition of 45°C and the same amount of glucose oxidase and catalase added, the glucose has not reacted completely after 26 hours of reaction of GOX. Compared with GOX, GOX-MUT-S1, GOX-MUT-S1-S2, and GOX-MUT-S1-S3 can all react completely (residual sugar content is less than 0.1%). Among them, the reaction time of GOX-MUT-S1-S2 and GOX-MUT-S1-S3 is 21 hours and 20 hours respectively, and the residual sugar content of the two is similar, and both react completely, which is shortened by 3 hours to 6 hours compared with glucose oxidase GOX and GOX-S1. Comprehensive comparison shows that GOX-MUT-S1-S2 and GOX-MUT-S1-S3 have very good effects in the production of sodium gluconate. Combined with their high temperature resistance and thermal storage stability, GOX-MUT-S1-S2 and GOX-MUT-S1-S3 have better effects in the production of gluconate.

[0092] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

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

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

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

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

5. A recombinant cell, characterized in that The recombinant cell comprises 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: The following steps are involved: (1) culturing the recombinant cell according to any one of claims 5 to 7; (2) Inducing the recombinant cell to express the glucose oxidase mutant.

9. Use of the glucose oxidase mutant according to claim 1 or 2 in producing gluconate.

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