Glucose oxidase mutant, preparation method and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and discloses a glucose oxidase mutant, a preparation method and application thereof. The glucose oxidase mutant includes a disulfide bond mutant site K37C+A572C relative to the parent glucose oxidase of the amino acid sequence shown in SEQ ID NO:1. Compared with the parent glucose oxidase, the thermal stability of the glucose oxidase mutant is obviously improved, and the glucose oxidase mutant has better heat resistance, which is beneficial to the production and application of gluconate.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a 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, which are 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 manner, which 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 reaction.

[0003] Gluconic acid is a mild, non-corrosive, non-toxic, and easily biodegradable organic acid produced by microbial oxidation of glucose. Its chemical and physiological properties make gluconic acid and its salts, especially sodium gluconate, play an important role in the chemical, textile, beverage, pharmaceutical (such as iron and calcium deficiency), construction, and food industries. According to a market research report, the global gluconic acid market value was about 1 billion US dollars in 2020, and is expected to reach 1.9 billion US dollars by 2028, with a compound annual growth rate of 5% during 2021-2028. Chemical and fermentation processes are the main technologies for producing gluconic acid. Although the one-step synthesis of gluconic acid by chemical and electrolytic oxidation is effective, the high electrolytic cost, environmental toxicity, and biological hazards limit the industrial application of the chemical method. The current industrial process is based on the fermentation process of Aspergillus niger using glucose as the substrate, and there is also a method of using glucose oxidase / hydrogen peroxide enzyme to co-catalyze the production of gluconate. The double-enzyme method for producing sodium gluconate uses glucose as the main raw material, and glucose oxidase converts glucose directly into gluconic acid under the synergistic action of hydrogen peroxide enzyme, and then sodium gluconate is prepared by neutralization with alkali.

[0004] However, the existing glucose oxidase still has poor heat resistance, which is difficult to meet the sterilization requirement before production, and the problem of heat resistance restricts the application of the double-enzyme method for production. Therefore, it is hoped to provide a glucose oxidase with good heat resistance for industrial production. SUMMARY

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

[0006] The present application provides a glucose oxidase mutant, which has at least 98% and less than 100% sequence identity relative to a parent glucose oxidase (the amino acid sequence of which is shown as SEQ ID NO: 1) derived from Aspergillus niger. Aspergillus niger

[0007] Wherein, the meaning of K37C+A572C is that the 37th and 572th amino acid residues of the parent glucose oxidase are substituted with cysteine, and a disulfide bond is formed.

[0008] SEQ ID NO: 1: SNGIEASLLKDPKLVAGRTYDYIIAGGGLAGLTVAEKLTENPNITVLVIESGSYESDRGPIIEDLNAYGEIFGTSVDHAYETVELATNNRTALIRSGNGLGGSTLINGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGLNGTVHVGPRDTGDDYSPLMRALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRADAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKSNTHNVYAKHEVLLSAGSTVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTGGVASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLSAWVEYIPEHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQLSSHVMTVFYAMALKIADAVLADYASMQ.

[0009] ​Preferably, in some embodiments of the present application, the disulfide bond mutation site S53C+T246C or S191C+A479C is further comprised.

[0010] wherein S53C+T246C means that the 53th and 246th amino acid residues of the parent glucose oxidase are substituted with cysteine, and the 53th and 246th amino acid residues also form a disulfide bond; S191C+A479C means that the 191th and 479th amino acid residues of the parent glucose oxidase are substituted with cysteine, and the 191th and 479th amino acid residues also form a disulfide bond.

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

[0012] The present application 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 reverse complementary to (a).

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

[0016] The present application also provides a recombinant cell comprising the above-mentioned nucleic acid molecule or the above-mentioned 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 application also provides a preparation method of the above-mentioned glucose oxidase mutant, comprising the following steps:

[0020] (1) culturing the recombinant cell;

[0021] (2) inducing the recombinant cell to express the above-mentioned glucose oxidase mutant.

[0022] The present application also provides the application of the above-mentioned glucose oxidase mutant in the production of gluconate.

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

[0024] The glucose oxidase mutant provided by the present application has significantly improved high-temperature stability and heat resistance compared with the parent glucose oxidase with the amino acid sequence shown in SEQ ID NO: 1. Meanwhile, the glucose oxidase mutant provided by the present application has more excellent effect in the production of gluconate, showing faster reaction rate and more complete reaction. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the following examples are provided for illustration. It should be noted that the following examples do not constitute a limitation on the scope of protection required by the present application.

[0026] The biological materials, reagents or devices used in the following examples, if not specifically stated, can be obtained from conventional commercial channels or can be obtained by known methods. The molecular biology experimental methods not specifically described in the following examples are carried out according to the specific methods listed in the book "Molecular Cloning Experiment Guide" (third edition) by J. Sambrook, or according to the instructions of the reagent kit and product.

[0027] Variant, mutant: The term "variant" "mutant" means 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 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 amino acids after the amino acid occupying a certain position. The mutant of the present application has at least 20% of the glucose oxidase activity of the mature polypeptide of SEQ ID NO: 1, 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%.

[0028] The present application provides a glucose oxidase mutant, which comprises at least the following mutations: K37C+A572C, relative to the parent glucose oxidase with the amino acid sequence shown in SEQ ID NO: 1. 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 to the polypeptide of SEQ ID NO: 1.

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

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

[0031] The term "disulfide bond" means a chemical bond that links two cysteines in different peptide chains or in the same peptide chain and 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 referred to as disulfide bridges. In the present application, the disulfide bonds are not naturally occurring and are introduced by way of point mutations.

[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 For example, to detect increased expression By techniques known in the art, such as measuring the level of mRNA and / or translated polypeptide.

[0033] Expression vector: The term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide and operably linked to control sequences providing for its expression. The polynucleotide and control sequences can be ligated together to produce a recombinant expression vector, which can include one or more convenient restriction sites allowing for 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 comprising the polynucleotide into an appropriate vector for expression. In creating an expression vector, the coding sequence is located in the vector such that the coding sequence is operably linked to the appropriate control sequences for expression.

[0034] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide(s). 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 linear or closed circular. The term "vector" refers to a piece of DNA that is used to introduce new pieces of DNA into an organism.

[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 a host cell. 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).

[0036] 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 Aspergillus sojae and Aspergillus oryzae Aspergillus oryyzae Suitable hosts can be selected from yeast strains, such as, for example, Pichia pastoris, Saccharomyces cerevisiae and Hansenula polymorpha Hansenula polymorpha

[0037] The cells expressing the glucose oxidase mutants used in the present application can be expressed in a bacterial host, or the protein is 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.

[0038] The glucose oxidases of the present application can be recovered using methods known in the art. For example, the polypeptides can be recovered from the fermentation medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, the whole fermentation broth comprising the glucose oxidases of the present application is recovered.

[0039] ​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 which can or can not be further purified.

[0040] In some embodiments of the application, the glucose oxidase mutants are 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 to obtain high purity of the target protein. In other preferred embodiments, high purity of the target protein is obtained by HPLC purification.

[0041] In other embodiments of the application, the supernatant, or the supernatant partially purified by ultrafiltration, or the concentrated and / or diafiltrated supernatant, 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.

[0042] In further embodiments of the application, the fermentation cell suspension including the 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.

[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 per amount of enzyme under defined reaction conditions.

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

[0045] Using the published three-dimensional structure of the parent glucose oxidase (the amino acid sequence of which is shown as SEQ ID NO: 1), with the three-dimensional structure file PDB ID: 1CF3 as a reference, after creative labor, the following glucose oxidase mutants with a single disulfide bond mutation were finally designed and screened:

[0046] Table 1: Glucose oxidase mutants

[0047]

[0048] Example 2: Construction and expression of glucose oxidase mutants

[0049] Experimental materials and reagents:

[0050] 1. Strains and vectors

[0051] The E. coli strain Top10, Pichia pastoris GS115, the vector pPIC9K, and the antibiotic G418 were purchased from Invitrogen Company.

[0052] 2. Enzymes and kits

[0053] PCR enzymes, plasmid extraction kits, and gel purification kits were purchased from Shanghai Biotechnology Company, and restriction endonucleases were purchased from NEB Company.

[0054] 3. Culture medium

[0055] 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, and the rest of the components were the same as BMGY). The recombinant yeast fermentation basic salt culture medium: diammonium hydrogen 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 per liter was added. 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%.

[0056] 4. Chemical reagents:

[0057] Glucose oxidase standard, o-anisidine hydrochloride and horseradish peroxidase were purchased from Sigma, glucose was purchased from OXIOD, and other reagents were purchased from Guangzhou Chemical Reagent Factory.

[0058] 5. Glucose oxidase determination method

[0059] The glucose oxidase activity was determined by o-anisidine spectrophotometry (determination conditions: 37℃, pH 5.5). Under the action of glucose oxidase, glucose and oxygen react to generate gluconic acid and hydrogen peroxide. Hydrogen peroxide and colorless reduced o-anisidine generate water and red oxidized o-anisidine 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.

[0060] Table 2: Disulfide bond mutant primers

[0061]

[0062] The amino acid sequence of the parent glucose oxidase (GOX) of the glucose oxidase mutant in the application is shown in SEQ ID NO: 1. The primers were designed to construct the related glucose oxidase mutants in Table 1 using the parent recombinant vector pPIC9K-GOX as a template, and PCR amplification was performed. The related amplification primers are shown in Table 2.

[0063] PCR amplification was detected by agarose electrophoresis, and the target product of PCR amplification was purified and recovered. The template was digested with restriction endonuclease DpnI, and the digested product was transformed into E. coli Top10 competent cells by chemical transformation heat shock method. The recombinant transformants were verified by bacterial liquid PCR, and the plasmid of the verified transformants 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 the electric transformation method was used to transform into Pichia pastoris GS115 competent cells, and YPD+G418 medium was used for screening. The obtained yeast recombinant transformants were picked one by one with a toothpick and added to a 24-well plate, 1 mL of BMGY medium was added to each well, and the culture was incubated at 30℃, 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 above supernatant was taken out and added to a 24-well plate for glucose oxidase heat-resistant property analysis.

[0064] Example 3: Determination of the thermal stability of glucose oxidase disulfide bond mutants

[0065] The 24-well plates were used to measure the residual enzyme activity of the fermentation supernatant of Example 2 after diluting the glucose oxidase enzyme activity to about 10 U / mL with distilled water and treating at 80°C for 3 minutes. The relative enzyme activity was calculated based on the corresponding enzyme activity of the untreated sample as 100%, and the results are shown in Table 3.

[0066] The results show that the introduction of disulfide bonds has a significant impact on the mutants. The thermal stability of the GOX-MUT-S1 and GOX-MUT-S3 mutants is significantly improved, and the residual enzyme activity of the glucose oxidase mutant is more than 50% higher than that of the parent glucose oxidase (GOX), especially GOX-MUT-S1, which has significantly improved residual enzyme activity after heat treatment. The above results show that the introduction of disulfide bond mutations can obtain glucose oxidase mutants with higher thermal stability.

[0067] Table 3: Results of glucose oxidase heat treatment

[0068]

[0069] Example 4: High temperature resistance and thermal storage stability of glucose oxidase disulfide bond combination mutants

[0070] To further improve the heat resistance of glucose oxidase, combination mutations with two disulfide bond mutation sites were performed. pPIC9K-GOX-MUT-S1-S2 and pPIC9K-GOX-MUT-S1-S3 vectors were constructed using pPIC9K-GOX-MUT-S1 as a template. The specific construction method is referred to the construction method in Example 1 for constructing glucose oxidase mutants.

[0071] The high temperature resistance of the glucose oxidase disulfide bond combination mutants was determined according to the scheme of Example 3.

[0072] Table 4: High temperature resistance determination of glucose oxidase disulfide bond combination mutants

[0073]

[0074] As can be seen from Table 4, after treatment at 80°C for 3 minutes, the residual enzyme activity of the glucose oxidase disulfide bond combination mutants is significantly improved compared to the parent glucose oxidase GOX, and the residual enzyme activity of GOX-MUT-S1-S3 after heat treatment is maintained at more than 84%. At the same time, by comparing Table 3, it can be found that the glucose oxidase disulfide bond combination mutants have more excellent high temperature resistance than the glucose oxidase mutants with single disulfide bond mutation.

[0075] The 24-well plate fermentation supernatant was diluted with distilled water, and the glucose oxidase enzyme activity was diluted to about 10 U / mL, and then placed at 45°C for 48 hours, and the residual enzyme activity was measured. The relative enzyme activity was calculated based on the corresponding enzyme activity of the untreated sample as 100%. The results are shown in Table 5.

[0076] Table 5: Thermal storage stability determination of glucose oxidase mutants

[0077]

[0078] The results show that the residual enzyme activity of the parent glucose oxidase GOX after 45°C, 48 hours of heat treatment is 74.9%, and the thermal storage stability of the glucose oxidase mutant GOX-MUT-S1 with a single disulfide bond mutation can also 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, and there is basically no loss after heat treatment. The above results show that introducing two pairs of disulfide bond mutations can obtain mutants with higher thermal stability.

[0079] Example 5: Application of glucose oxidase in sodium gluconate production

[0080] The residual sugar and reaction cycle in the experiment of producing sodium gluconate by double enzyme method were used as evaluation indexes. The double enzyme method for producing sodium gluconate uses glucose as the main raw material, and glucose oxidase is used to directly convert glucose into gluconic acid under the synergistic action of catalase, and then sodium gluconate is prepared by neutralization with alkali. The application experiment of catalase (CAT) and different glucose oxidase mutants for sodium gluconate was compared, and the application effects of different glucose oxidase mutants for sodium gluconate were studied. Under the conditions of 45°C, 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, and gluconic acid can further react with sodium hydroxide to generate sodium gluconate, and hydrogen peroxide can be catalyzed by catalase to generate water and oxygen.

[0081] Experimental apparatus and materials:

[0082] Experimental equipment: 20L reactor

[0083] Experimental reagents:

[0084] (1) 36% glucose solution, 4 kg of glucose was dissolved in water, and the volume was made up to 10 L;

[0085] (2) 40% NaOH solution, 2 kg of NaOH was dissolved in water, and the volume was made up to 5 L;

[0086] (3) Catalase, enzyme activity: 600,000 U / g.

[0087] Table 6: Catalytic effect of glucose oxidase

[0088]

[0089] As shown in Table 6, under the condition of 45℃ and the same amount of glucose oxidase and catalase, the glucose was not completely reacted after 26 hours of GOX reaction. Compared with GOX, GOX-MUT-S1, GOX-MUT-S1-S2 and GOX-MUT-S1-S3 can all be completely reacted (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 close, both of which are completely reacted, which is 3 hours to 6 hours shorter than glucose oxidase GOX and GOX-S1. In summary, GOX-MUT-S1-S2 and GOX-MUT-S1-S3 have very good effect in the production of sodium gluconate. Combined with their high temperature resistance and heat storage stability, GOX-MUT-S1-S2 and GOX-MUT-S1-S3 have more excellent effect in the production of gluconate.

[0090] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be understood as the limitation of the patent scope of the present application. It should be noted that for ordinary skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the patent protection scope of the present application should be subject to the appended claims.

Claims

1. A glucose oxidase mutant, characterized in that, The glucose oxidase mutant is based on a parent glucose oxidase having the amino acid sequence set forth in SEQ ID NO: 1, and has introduced disulfide bond mutation sites K37C+A572C and S53C+T246C, or K37C+A572C and S191C+A479C.

2. 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 claim 1; (b) a nucleotide fragment reverse complementary to (a).

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 2.

4. A recombinant cell, characterized in that, The recombinant cell comprises the nucleic acid molecule of claim 2 or the recombinant expression vector of claim 3.

5. The recombinant cell of claim 4, wherein, The recombinant cell comprises a bacterial or fungal cell.

6. The recombinant cell of claim 5, wherein, The fungal cell is a Pichia pastoris cell.

7. A method for producing the glucose oxidase mutant of claim 1, characterized by, The method comprises the following steps: (1) culturing the recombinant cell of any one of claims 4-6; (2) inducing the recombinant cell to express the glucose oxidase mutant.

8. Use of the glucose oxidase mutant of claim 1 in the production of gluconate.

Images