A thermostable glucose oxidase

Abstract

Provided is a thermostable glucose oxidase obtained by introducing at least one pair of disulfide bonds into the amino acid sequence of wild-type Aspergillus niger glucose oxidase or mutant Aspergillus niger glucose oxidase. The glucose oxidase is suitable for use in the fields of food, chemical industry, medicine, agriculture and feed.

Description

Technical Field

[0001] The invention belongs to the field of protein engineering, and specifically relates to glucose oxidase derived from filamentous fungi, especially Aspergillus niger glucose oxidase, which has improved thermal stability after being modified by introducing disulfide bonds. Background Art

[0002] Glucose oxidase (GOD, EC 1.1.3.4) is an aerobic dehydrogenase. The enzyme molecule is a dimer containing two subunits with a molecular weight of about 160kDa, and each subunit binds to a FAD molecule. It can use molecular oxygen as an electron acceptor to specifically catalyze β-D-glucose to produce gluconic acid and hydrogen peroxide. GOD is widely distributed in animals, plants and microorganisms. There are certain limitations in extracting GOD from animal and plant tissues, and the amount of enzyme is not abundant. The amount of bacterial GOD enzyme production is small. Microbial fermentation is the main source of GOD production. At present, most commercial products on the market are produced by fermentation of Pichia pastoris and filamentous fungi, such as Aspergillus niger and Aspergillus oryzae.

[0003] Glucose oxidase has the advantages of catalytic specificity and high efficiency, and is widely used in the fields of food, chemical industry, medicine, agriculture, feed, etc. It has attracted much attention in recent years, and the market demand is also increasing. Due to the deoxygenation and antioxidant effects of glucose oxidase, it is widely used in food, medicine, feed, etc. In the food industry, glucose oxidase, as a food preservative, has a significant effect in preventing beer aging, maintaining the original flavor of the product, and extending the shelf life. It can also be used as a flour improver and bread quality improver to improve food quality. In the field of medicine, glucose oxidase electrode method, glucose oxidase-peroxidase coupling method, etc. are widely used to detect glucose content in blood and serum. As a new feed additive, glucose oxidase can improve the intestinal environment of animals, improve feed utilization, and promote animal growth. As glucose oxidase is increasingly used in various fields, the industry, especially the feed industry, has higher and higher requirements for its existing performance, such as maintaining enzyme activity for a long time at room temperature, tolerance to heat and extreme pH conditions, and tolerance to digestive enzymes. The thermal stability of the enzyme is very important for the application of glucose oxidase. In the preparation process of the enzyme and under extreme reaction conditions (high temperature), enzymes with strong heat resistance have a relatively large advantage. Therefore, improving the thermal stability of glucose oxidase has important practical significance for the widespread promotion and application of glucose oxidase. Summary of the invention

[0004] The present inventors have discovered that by introducing one or more pairs of disulfide bonds at specific positions in the amino acid sequence of a wild-type Aspergillus niger glucose oxidase (e.g., one having a sequence identity of greater than 80% with the wild-type Aspergillus niger glucose oxidase shown in SEQ ID NO: 1) or a mutant Aspergillus niger glucose oxidase (e.g., one having a sequence identity of greater than 75% with the mutant Aspergillus niger glucose oxidase shown in SEQ ID NO: 8 or SEQ ID NO: 10), its stability can be improved.

[0005] In some embodiments, one or more pairs of disulfide bonds as shown in Table 1 are introduced at specific positions in the amino acid sequence of a mutant Aspergillus niger glucose oxidase having a sequence identity of greater than 80% with the Aspergillus niger wild-type glucose oxidase as shown in SEQ ID NO: 1. In some preferred embodiments, one or more pairs of disulfide bonds as shown in Table 1 are introduced at specific positions in the amino acid sequence of an Aspergillus niger glucose oxidase having a sequence identity of greater than 85%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the Aspergillus niger wild-type glucose oxidase as shown in SEQ ID NO: 1.

[0006] In other embodiments, one or more pairs of disulfide bonds as shown in Table 1 are introduced at specific positions in the amino acid sequence of a mutant Aspergillus niger glucose oxidase having a sequence identity of greater than 75% with the mutant Aspergillus niger glucose oxidase shown in SEQ ID NO: 8 or SEQ ID NO: 10. In some preferred embodiments, one or more pairs of disulfide bonds as shown in Table 1 are introduced at specific positions in the amino acid sequence of a mutant Aspergillus niger glucose oxidase having a sequence identity of greater than 78%, 79%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the mutant Aspergillus niger glucose oxidase shown in SEQ ID NO:.

[0007] Specifically, the inventors have found that introducing one or more pairs of disulfide bond combinations as shown in Table 1 into the Aspergillus niger wild-type glucose oxidase sequence as shown in SEQ ID NO: 1 or the Aspergillus niger glucose oxidase mutant as shown in SEQ ID NO: 8 or SEQ ID NO: 10, respectively, can improve its thermal stability.

[0008] Table 1: Names and positions of introduced disulfide bonds (amino acid position numbering is based on SEQ ID NO: 1)

[0009] Disulfide bond name Disulfide bond position A S53 / T246 B A25 / V250 C V20 / S45 D T39 / L242 E T87 / P508

[0010] For the purpose of the present invention, "introduction" does not limit the disulfide bond to be generated in any particular manner. For example, "introduction" of a disulfide bond may include replacing the amino acid residue at the corresponding position of the glucose oxidase sequence to be introduced with an amino acid residue capable of forming a disulfide bond (including, for example, cysteine ​​residue Cys, homocysteine ​​residue Hcy, etc.); and / or inserting an amino acid residue capable of forming a disulfide bond at the corresponding position. Such replacement and / or insertion can be achieved, for example, by site-directed mutagenesis methods known in the art. "Introduction" also includes the situation where any one or two amino acid residues forming the disulfide bond are due to natural mutation. In order to produce mutants transformed in this way, microorganisms such as Escherichia coli, fungi such as yeast (Pichia pastoris, Schizosaccharomyces pombe, etc.) and filamentous fungi (such as Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, etc.), and plants (such as corn, soybean, wheat, etc.) can be used as hosts for expression.

[0011] In order to construct the above mutation, conventional site-directed mutagenesis methods can be used based on the existing wild-type nucleic acid sequence, or de novo synthesis can be used using gene synthesis methods. After connecting the promoter and terminator, it is introduced into the host cell and expressed under appropriate culture conditions. The above method is a conventional method in the art.

[0012] Based on this discovery, the present application provides the following technical solutions.

[0013] An object of the present invention is to provide a thermostable glucose oxidase, which comprises at least one pair of introduced disulfide bonds in the amino acid sequence of a wild-type Aspergillus niger glucose oxidase or a mutant Aspergillus niger glucose oxidase, wherein the amino acid sequence of the wild-type glucose oxidase is as shown in SEQ ID NO: 1, the mutant Aspergillus niger glucose oxidase has a mutation at at least one position compared to the wild-type Aspergillus niger glucose oxidase as shown in SEQ ID NO: 1, and the introduced disulfide bonds are selected from:

[0014] (A) a disulfide bond formed between an amino acid residue at a position corresponding to position 53 of SEQ ID NO: 1 and an amino acid residue at a position corresponding to position 246 of SEQ ID NO: 1;

[0015] (B) a disulfide bond formed between an amino acid residue at a position corresponding to position 25 of SEQ ID NO: 1 and an amino acid residue at a position corresponding to position 250 of SEQ ID NO: 1;

[0016] (C) a disulfide bond formed between the amino acid residue at the position corresponding to position 20 of SEQ ID NO: 1 and the amino acid residue at the position corresponding to position 45 of SEQ ID NO: 1;

[0017] (D) a disulfide bond formed between the amino acid residue at the position corresponding to position 39 of SEQ ID NO: 1 and the amino acid residue at the position corresponding to position 242 of SEQ ID NO: 1;

[0018] (E) A disulfide bond formed between the amino acid residue at the position corresponding to position 87 of SEQ ID NO: 1 and the amino acid residue at the position corresponding to position 508 of SEQ ID NO: 1.

[0019] In some embodiments of the invention, the mutant Aspergillus niger glucose oxidase has a mutation at at least one of the following positions compared to the wild-type Aspergillus niger as shown in SEQ ID NO: 1: 14, 16, 25, 30, 34, 37, 43, 45, 53, 67, 84, 90, 92, 94, 96, 106, 121, 135, 141, 142, 162, 167, 204, 246, 259, 315, 332, 362, 405, 406, 420, 446, 449, 453, 477, 501, 504, 506, 509, 510, 521, 526, 528, 536, 554, 560, 572, 575, 577.

[0020] In some embodiments of the present invention, the mutant Aspergillus niger glucose oxidase has at least one of the following mutations compared to the wild-type Aspergillus niger as shown in SEQ ID NO: 1: D14E, S16A, A25V, T30V, T34V, R37K, N43D, S45T, S53C, A67Y, E84C, Q90R, A92Q, I94V, S96F, V106I, S121A, N135S, L141K, Q142K, A162T, V167I, F204L, T24 6C, G259A, D315K, A332S, S362T, N405K, H406D, V420E, H446R, A449M, Q453N, S477Y, S501R, T 504V, Y506W, Y509E, H510N, C521A, K526R, M528L, A536L, T554M, V560L, S572A, I575V, E577A.

[0021] In some embodiments of the invention, the mutant Aspergillus niger glucose oxidase has a mutation at at least one of the following positions compared to the wild-type Aspergillus niger as shown in SEQ ID NO: 1: 14, 16, 30, 34, 37, 43, 45, 53, 67, 84, 90, 94, 106, 135, 162, 167, 204, 246, 259, 315, 332, 362, 405, 406, 420, 446, 501, 504, 509, 510, 554, 572, 575, 577.

[0022] In some embodiments of the invention, the mutant Aspergillus niger glucose oxidase has at least one of the following mutations compared to the wild-type Aspergillus niger as shown in SEQ ID NO: 1: D14E, S16A, T30V, T34V, R37K, N43D, S45T, S53C, A67Y, E84C, Q90R, I94V, V106I, N135S, A162T, V167I, F204L, T246C, G259A, D315K, A332S, S362T, N405K, H406D, V420E, H446R, S501R, T504V, Y509E, H510N, T554M, S572A, I575V, E577A

[0023] In some embodiments of the present invention, the mutant Aspergillus niger glucose oxidase has a mutation at at least one of the following positions compared to the wild-type Aspergillus niger as shown in SEQ ID NO: 1: 14, 30, 37, 43, 45, 53, 67, 84, 90, 94, 106, 135, 162, 167, 204, 246, 259, 315, 406, 420, 446, 501, 509, 572, 577.

[0024] In some embodiments of the present invention, the mutant Aspergillus niger glucose oxidase has at least one of the following mutations compared to the wild-type Aspergillus niger as shown in SEQ ID NO: 1: D14E, T30V, R37K, N43D, S45T, S53C, A67Y, E84C, Q90R, I94V, V106I, N135S, A162T, V167I, F204L, T246C, G259A, D315K, H406D, V420E, H446R, S501R, Y509E, S572A, E577A.

[0025] In some embodiments of the present invention, the mutant Aspergillus niger glucose oxidase has a mutation at at least one of the following positions compared to the wild-type Aspergillus niger as shown in SEQ ID NO: 1: 14, 30, 37, 43, 53, 67, 84, 90, 94, 106, 135, 162, 204, 246, 315, 446, 501, 509, 554.

[0026] In some embodiments of the present invention, the mutant Aspergillus niger glucose oxidase has at least one of the following mutations compared to the wild-type Aspergillus niger as shown in SEQ ID NO: 1: D14E, T30V, R37K, N43D, S53C, A67Y, E84C, Q90R, I94V, V106I, N135S, A162T, F204L, T246C, D315K, H446R, S501R, Y509E, T554M.

[0027] In some embodiments of the present invention, the amino acid sequence of the mutant Aspergillus niger glucose oxidase is SEQ ID NO.8 or SEQ ID NO.10.

[0028] In some embodiments of the present invention, the amino acid sequence of the thermostable glucose oxidase satisfies at least one of the above items (A), (B), (C) or (E).

[0029] In some embodiments of the present invention, the amino acid sequence of the thermostable glucose oxidase satisfies any two, three or all of the above items (A), (B), (C) or (E).

[0030] In some embodiments of the present invention, the amino acid sequence of the thermostable glucose oxidase satisfies the above-mentioned item (A), item (B) or item (C).

[0031] In some embodiments of the present invention, the amino acid sequence of the thermostable glucose oxidase satisfies the above item (A).

[0032] In some embodiments of the present invention, the amino acid sequence of the thermostable glucose oxidase satisfies the above item (B).

[0033] In some embodiments of the present invention, the amino acid sequence of the thermostable glucose oxidase satisfies the above item (C).

[0034] In some embodiments of the present invention, the thermostable glucose oxidase comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 11-15, 17, 19, 21 or 23.

[0035] In some embodiments of the present invention, the thermostable glucose oxidase is heterologously expressed in a Pichia pastoris host.

[0036] In some embodiments of the present invention, the amino acid residue capable of forming a disulfide bond is a cysteine ​​residue or a homocysteine ​​residue.

[0037] Another object of the present invention is to provide a polynucleotide encoding the thermostable glucose oxidase.

[0038] In some embodiments of the present invention, the polynucleotide sequence encoding the thermostable glucose oxidase is codon-optimized for expression in Pichia pastoris.

[0039] In some embodiments of the present invention, the polynucleotide comprises the nucleotide sequence shown in any one of SEQ ID NOs: 3-7, 16, 18, 20 or 22.

[0040] Another object of the present invention is to provide a host cell comprising the polynucleotide encoding the thermostable glucose oxidase.

[0041] In some embodiments of the present invention, the host cell is a fungal cell, a bacterial cell or a plant cell.

[0042] In some embodiments of the present invention, the host cell is a yeast cell or a filamentous fungal cell.

[0043] In some embodiments of the present invention, the host cell is a Pichia pastoris cell or an Aspergillus niger cell.

[0044] Another object of the present invention is to provide the application of the thermostable glucose oxidase in the fields of food, chemical industry, medicine, agriculture or feed.

[0045] As used herein, A, R, C, Q, N, L, K, M, F, P, S, T, W, Y, V, G, and E are abbreviations for alanine Ala, arginine Arg, cysteine ​​Cys, glutamine Gln, asparagine Asn, leucine Leu, lysine Lys, methionine Met, phenylalanine Phe, proline Pro, serine Ser, threonine Thr, tryptophan Trp, tyrosine Tyr, valine Val, glycine Gly, and glutamic acid Glu, respectively.

[0046] As used herein, the term "glucose oxidase mutant", "mutant" or "mutant glucose oxidase" refers to a polypeptide having glucose oxidase activity that contains changes, i.e., substitutions, insertions and / or deletions of one or more (several) amino acid residues at one or more (several) positions. Substitution refers to replacing the amino acid occupying a certain position with a different amino acid; deletion refers to removing the amino acid occupying a certain position; and insertion refers to adding 1-5 amino acids adjacent to and after the amino acid occupying a certain position. Mutating the wild-type glucose oxidase also refers to amino acid substitution, insertion and / or deletion at at least one position compared to the wild-type glucose oxidase, preferably, refers to amino acid substitution at at least one position, such as "T30V", which means that the threonine at position 30 of the wild-type glucose oxidase is substituted with valine.

[0047] As used herein, the term "thermostability" refers to the glucose oxidase mutants of the present invention, which maintain a certain amount of enzyme activity after a given period of time at a specific temperature. When referring to properties such as thermostability, the term "increased thermostability" refers to higher enzyme activity after a period of time compared to other glucose oxidase variants and / or wild-type glucose oxidase.

[0048] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Such terms include, but are not limited to, single-stranded, double-stranded or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrids or polymers containing purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derived nucleotide bases.

[0049] As used herein, the term "vector" refers to a polynucleotide construct designed to introduce a nucleic acid into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, cassettes, and the like.

[0050] As used herein, the term "gene" refers to a polynucleotide encoding a polypeptide, including regions before and after the coding region, and intervening sequences (introns) located between individual coding segments (exons).

[0051] As used herein, the term "percent (%) sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Sequence alignment can be performed in a variety of ways within the skill of the art to determine percent amino acid sequence identity, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0052] As used herein, the term "host cell" refers to a host suitable for an expression vector containing the DNA according to the present invention.

[0053] Beneficial effects of the present invention:

[0054] The present invention introduces one or more pairs of disulfide bonds to the wild-type glucose oxidase, and after being treated at 70°C for 3 minutes, the thermal stability thereof is significantly improved compared with the wild-type glucose oxidase, preferably the enzyme activity of the glucose oxidase mutant is increased by more than 70%, more preferably the enzyme activity of the glucose oxidase mutant is increased by more than 120%, and particularly preferably the enzyme activity of the glucose oxidase mutant is increased by 180%. The present invention introduces one or more pairs of disulfide bonds to the glucose oxidase mutant, and after being treated at 70°C for 3 minutes, the enzyme activity of the mutant is increased by more than 34%; after being treated at 80°C for 3 minutes, the enzyme activity is greatly improved from zero to one; the present invention introduces one or more pairs of disulfide bonds to the glucose oxidase mutant from other Aspergillus niger sources, and after being treated at 80°C for 3 minutes, the enzyme activity of the mutant is increased by more than 350%, which is significantly better than the existing wild-type glucose oxidase. Therefore, the technical scheme of the present invention can improve the enzymatic activity of glucose oxidase, especially in terms of heat stability, and is suitable for industrial production, for example, in the fields of food, chemical industry, medicine, agriculture and feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is the pPIC9K-GOD-wt plasmid map.

[0056] Figure 2 It is a graph showing the results of thermal stability measurement of the wild type and disulfide bond mutants. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be pointed out that the embodiments are only used to explain the present invention, rather than to limit the scope of the present invention.

[0058] Example 1 Construction of disulfide bond mutants

[0059] The 3D structure of wild-type glucose oxidase from Aspergillus niger has been published (see Wohlfahrt, G et al, (1999) Acta Crystallogr., Sect. D 55:969-977). Based on the 3D structure file PDB ID 1CF3 as a reference, the disulfide bonds described in the following table were designed and introduced.

[0060] Table 2: Names and positions of introduced disulfide bonds (amino acid position numbering is based on SEQ ID NO: 1)

[0061]

[0062]

[0063] The amino acid sequence of wild-type glucose oxidase is shown in SEQ ID NO: 1, the expression vector is pPIC9K, and the Alpha factor of Saccharomyces cerevisiae is used as a signal peptide. The synthesized wild-type sequence of glucose oxidase gene GOD-wt (containing EcoR I and Not I restriction sites at both ends of the sequence) and pPIC9K are digested with EcoR I and Not I respectively, and then connected and transformed into Escherichia coli DH5α competent cells. The transformants are selected for sequencing verification to obtain the wild-type glucose oxidase expression plasmid pPIC9K-GOD-wt as shown in FIG. Figure 1 As shown. The mutant sequences were named GOD-A to GOD-M according to the disulfide bond names in the above table. The glucose oxidase gene GOD-wt (such as SEQ ID NO: 2) and mutant sequences GOD-A to GOD-M were synthesized by the whole gene synthesis method, wherein the sequences of mutants GOD-A to GOD-E are shown in SEQ ID NO: 3-SEQ ID NO: 7, and the gene synthesis was completed by Nanjing GenScript Biotechnology Co., Ltd. The synthesized gene has EcoR I and Not I restriction sites at both ends.

[0064] In order to express glucose oxidase and mutants, Pichia pastoris GS115 and plasmids were manipulated according to the instruction manual of Pichia expression kit (Invitrogen). Specifically, the Pichia pastoris GS115 strain was cultured on a YPD (1% yeast extract, 2% protein, 2% glucose and 1.5% agar) plate at 30°C for 48 hours, and then a single clone was picked up and transferred to 4 mL of YPD liquid culture medium (1% yeast extract, 2% protein, 2% glucose), and cultured at 30°C and 200 rpm for 12 hours. It was then transferred to a 30 mL YPD liquid culture medium flask and cultured at 30°C and 220 rpm for 4-5 hours. After the OD600 value was detected to be in the range of 1.1-1.3, the culture solution was centrifuged at 4 degrees and 9,000 rpm for 2 minutes, and 4 mL of bacteria were collected into sterilized EP tubes respectively, the supernatant was gently discarded, the residual supernatant was absorbed with sterilized filter paper, and the bacteria were resuspended with 1 mL of pre-cooled sterile water, and centrifuged at 4°C and 9,000 rpm for 2 minutes, and the supernatant was discarded. Repeat the above steps, resuspend the cells in 1 mL of precooled sorbitol (1 mol / L); centrifuge at 4°C and 9,000 rpm for 2 min, discard the supernatant, and resuspend the cells in 100-150 μl of precooled sorbitol (1 mol / L). The competent state preparation is now complete. The expression plasmid pPIC9K-GOD-wt and the remaining 13 disulfide bond mutants were linearized with PmeI, and the linearized fragments were purified and recovered and transformed into the above-mentioned Pichia GS115 competent state by electroporation. The mixture was evenly spread on the MDH plate, inverted and cultured at 30°C for 2-3 days, and all the colonies on the plate were washed with sterile water and spread on YPD plates (0.5 mg / mL-8 mg / mL) containing different concentrations of geneticin to screen for multi-copy transformants. The recombinant strains of Pichia pastoris obtained by screening on MDH plates were named GOD-A, GOD-B, GOD-C, GOD-D, GOD-E, GOD-F, GOD-G, GOD-H, GOD-I, GOD-J, GOD-K, GOD-L, and GOD-M. The clones obtained by the above screening were respectively transferred to BMGY medium, cultured in a shaking incubator at 30°C and 250rpm for 24h, and then transferred to BMMY medium, maintained at 30°C and 250rpm, and 0.5% methanol was added every day to induce expression for 120h; centrifuged at 9000-12000rpm for 10min to remove the bacteria, and the fermentation supernatant containing glucose oxidase GOD-wt and its 13 mutants was obtained. The SDS-PAGE results showed that the four mutants of GOD-G, GOD-I, GOD-K and GOD-L failed to express, and the remaining 9 mutants were expressed.

[0065] Example 2 Determination of glucose oxidase activity

[0066] Under aerobic conditions, GOD catalyzes glucose dehydrogenation to produce H2O2. Under the action of horseradish peroxidase (POD), the oxygen donor o-dianisidine (DH2) is oxidized into a brown product. The activity of GOD can be converted according to the change in its absorbance at 540nm and the standard curve. The enzyme activity assay system contains 2.5mL o-dianisidine solution, 0.3mL 18% glucose, 0.1mL 90U / mL horseradish peroxidase, and after 2 minutes of incubation at 35℃, 0.1mL of the diluted enzyme solution sample is added to the test tube. After reacting for 3 minutes, 2mol / L sulfuric acid is added to terminate the reaction, the test tube is taken out, and the OD is measured. 540 The absorbance value of the standard curve was calculated using the heat-inactivated enzyme solution as a blank control. The glucose oxidase activity units were calculated based on the results of the standard curve.

[0067] Reagents and solutions

[0068] 0.1mol / L pH5.5 disodium hydrogen phosphate-sodium citrate buffer: Accurately weigh 14.32g disodium hydrogen phosphate and 8.4056g citric acid monohydrate, dissolve in 400ml distilled water, adjust the pH to 5.5 with disodium hydrogen phosphate, and set aside.

[0069] o-Dianisidine solution: Accurately weigh 0.1g o-dianisidine and dissolve it in 10ml methanol. This is the storage solution, which is effective for 3 days at 4℃. Before the experiment, take 0.1ml of the storage solution and dissolve it in 12ml of the above 0.1mol / L, pH 5.5 phosphate buffer.

[0070] 18% glucose: Accurately weigh 9.0000 g of glucose (AR) dried to constant weight, dissolve it in a small amount of distilled water, and make up to 50 ml with distilled water, and store at 4°C.

[0071] 2mol / L H2SO4: Accurately weigh 40.00g H2SO4, slowly add it into 160mL distilled water, make up to 200mL, and set aside.

[0072] GOD standard: Purchase sigma glucose oxidase standard with an enzyme activity of 10,000 units, accurately add 5 mL of distilled water, mix well, and store at -20°C for later use.

[0073] 90U / mL horseradish peroxidase: Purchase horseradish peroxidase standard (enzyme activity> 250units / mg, 100mg), accurately add 1mL of distilled water to fully dissolve the horseradish peroxidase, and store at -20℃ for later use. When using, take an appropriate amount of the standard and dilute it to an enzyme activity of 90U / ml before use. It must be diluted and used immediately.

[0074] Determination of enzyme activity

[0075] (1) Preparation of standard curve

[0076] Dilute GOD standard to 0.4, 0.8, 1.2, 1.6, 2.0, 2.4 U / mL, add 2.5 mL of o-dianisidine solution and 0.3 mL of 18% glucose solution into a test tube, then add 0.1 mL of 90 U / mL horseradish peroxidase solution, preheat at 35°C for 2 min, add 0.1 mL of diluted GOD standard at 15 s intervals, react accurately for 3 min, then immediately add 2 ml of 2 mol / L H2SO4 to terminate the reaction, take out and mix, measure the absorbance at 540 nm, and draw a standard curve y=Kx+b with the absorbance as the horizontal axis and the standard enzyme activity as the vertical axis.

[0077] (2) Sample determination

[0078] Add 2.5mL of o-dianisidine solution and 0.3mL of 18% glucose solution into a test tube, add 0.1mL of 90U / mL horseradish peroxidase solution, preheat at 35℃ for 2min, then add 0.1mL of diluted sample to be tested (the dilution standard is that the detection absorbance value of the sample is within the linear range) at intervals of 15s, react accurately for 3min, then immediately add 2ml of 2mol / LH2SO4 to terminate the reaction, take out and mix, measure the absorbance value A at 540nm, and calculate the enzyme activity.

[0079] (3) Calculation of enzyme activity

[0080] X=(K*A+b)*n

[0081] Where:

[0082] X---enzyme activity of the sample U / ml A---absorbance value of the sample

[0083] n---Dilution multiple of enzyme solution K----Slope of standard curve

[0084] b----Standard curve intercept

[0085] Example 3 Determination of thermal stability of glucose oxidase and its mutants

[0086] The fermentation supernatant described in Example 1 was diluted with distilled water to about 100 U / mL, and treated at 70°C for 3 min. The residual enzyme activity was measured, and the relative enzyme activity was calculated with the enzyme activity of the untreated sample as 100%. Figure 2As shown in the table below, the results show that the introduction of disulfide bonds has a significant effect on the mutants. The introduction of some disulfide bonds leads to the mutants not being able to express normally, such as GOD-G, GOD-I, GOD-K and GOD-L; the introduction of some other specific disulfide bonds will cause the mutants to have lower thermal stability than the wild type, such as GOD-F, GOD-H, GOD-J and GOD-M; the introduction of some other disulfide bonds will lead to a significant improvement in the thermal stability of the mutants. For example, the thermal stability of GOD-A, GOD-B, GOD-C, GOD-D and GOD-E has been significantly improved. Preferably, the residual enzyme activity of the glucose oxidase mutant is increased by more than 70% compared with the wild-type glucose oxidase, more preferably, the enzyme activity of the glucose oxidase mutant is increased by more than 120%, and particularly preferably, the enzyme activity of the glucose oxidase mutant is increased by 180%. The above results show that the appropriate introduction of the above disulfide bond combination can obtain mutants with higher thermal stability.

[0087] Table 3: Residual enzyme activity at 70℃

[0088]

[0089] Example 4

[0090] F91 is a heat-resistant excellent mutant obtained by mutation screening of wild-type glucose oxidase (as described in US2016 / 0068824), which introduces 5 mutations on the basis of the wild type, and the specific sequence is shown in SEQ ID NO: 8. In order to test whether the disulfide bond mutant described in Example 1 can also function on the glucose oxidase mutant, the stability is further improved. According to the method in Example 1, disulfide bonds A and B were introduced on the basis of sequence F91, respectively, and the two mutants were named F19-A and F19-B, respectively. Pichia pastoris was used to express the two mutants, and then the thermal stability was determined according to the method in Example 3, and they were incubated at 70°C and 80°C for 3 minutes. The results are shown in Table 2. Both disulfide bonds A and B further improve the thermal stability of the mutant, which is 34% and 35% higher than mutant F91 at 70°C, respectively, and especially improves the stability of the mutant at 80°C. F91 has no residual enzyme activity at 80°C, while the residual enzyme activity can reach 12% and 7% after adding disulfide bonds, which has achieved a substantial improvement from scratch. It can be seen that the introduction of disulfide bonds proposed by the inventors can more effectively improve the thermal stability of the glucose oxidase mutant.

[0091] Table 4: Residual enzyme activity results before and after the introduction of disulfide bonds into F91

[0092]

[0093] Example 5 Determination of thermal stability after introducing disulfide bonds into other Aspergillus niger derived glucose oxidase mutants

[0094] The wild-type glucose oxidase sequences from other Aspergillus niger sources are shown in SEQ ID NO: 9, and the similarity to SEQ ID NO: 1 is 97%. The wild-type glucose oxidase from different Aspergillus niger sources is obtained by mutation screening. The heat-resistant excellent mutant (as described in CN 108893453) introduces 5 mutations on the basis of the wild type to obtain GOD-M5, and the specific sequence is shown in SEQID NO: 10. In order to test whether the disulfide bond mutant described in Example 1 can also function on glucose oxidase mutants from different Aspergillus niger sources, and further improve the thermal stability. According to the method in Example 1, disulfide bonds A and B are introduced on the basis of the sequence GOD-M5, respectively, and named as GOD-M5-A and GOD-M5-B, respectively. The two mutants are expressed using Pichia pastoris, and then the thermal stability is determined according to the method in Example 3, and the incubation is carried out at 70°C and 80°C for 3 minutes, respectively. The results are shown in Table 3. Both disulfide bonds A and B further improved the thermal stability of the mutant, especially at 80°C, by 350% and 400% respectively compared with the mutant GOD-M5. It can be seen that the introduction of disulfide bonds proposed by the inventors showed an effect of improving the thermal stability of glucose oxidase mutants from different Aspergillus niger sources.

[0095] Table 5: Residual enzyme activity results of GOD-M5 before and after the introduction of disulfide bonds

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Claims

1. A thermostable glucose oxidase, comprising introducing a pair of disulfide bonds into the amino acid sequence of a wild-type Aspergillus niger glucose oxidase or a mutant Aspergillus niger glucose oxidase, wherein the amino acid sequence of the wild-type Aspergillus niger glucose oxidase is as shown in SEQ ID NO: 1, and the amino acid sequence of the mutant Aspergillus niger glucose oxidase is as shown in SEQ ID NO. 8 or SEQ ID NO. 10, and the introduced disulfide bonds are formed by replacing the amino acid residues at the S53 and T246 positions corresponding to SEQ ID NO: 1 with cysteine.

2. The thermostable glucose oxidase according to claim 1, wherein the thermostable glucose oxidase is obtained by heterologous expression in a Pichia pastoris host.

3. A polynucleotide encoding the thermostable glucose oxidase according to claim 1.

4. The polynucleotide of claim 3, which is codon-optimized for expression in Pichia pastoris. 5 . The polynucleotide according to claim 4 , which is the nucleotide sequence shown in any one of SEQ ID NO: 3, 16 or 20. A host cell comprising the polynucleotide according to any one of claims 3 to 5.

7. The host cell of claim 6, wherein the host cell is a fungal cell, a bacterial cell or a plant cell. The host cell according to claim 7 , which is a yeast cell or a filamentous fungal cell. The host cell according to claim 8, which is a Pichia pastoris cell or an Aspergillus niger cell.

10. Use of the thermostable glucose oxidase according to any one of claims 1 to 2 in the fields of food, chemical industry, medicine, agriculture or feed.

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