Glucose oxidase mutant and use thereof

Abstract

Provided are a thermostable glucose oxidase mutant, a polynucleotide encoding the glucose oxidase mutant, a recombinant expression vector comprising the polynucleotide, a host cell expressing the glucose oxidase mutant, and uses thereof in the field of food, chemical industry, medicine, agriculture or feed.

Description

Glucose oxidase mutant and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number CN202311114925.4 and filing date August 30, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to the technical field of protein engineering, in particular to a glucose oxidase mutant and an application thereof. Background Art

[0004] Glucose oxidase (GOD, EC 1.1.3.4) is an aerobic dehydrogenase that uses molecular oxygen as an electron acceptor to specifically catalyze the conversion of β-D-glucose to gluconic acid and hydrogen peroxide. Among glucose oxidases, GOD from Aspergillus niger is the most extensively studied. It is a glycosylated dimer with a molecular weight of approximately 160 kDa and uses FAD as a cofactor.

[0005] GOD is widely distributed in plants, animals, and microorganisms. Extracting GOD from plant and animal tissues has certain limitations, and the enzyme is not abundant. Microorganisms are the primary source of GOD due to their rapid growth and availability. Currently, industrial-level GOD production primarily utilizes Aspergillus niger and Penicillium as production strains. While Aspergillus niger-expressed GOD exhibits better thermal stability than Penicillium GOD, both strains suffer from low enzyme activity and complex separation and purification.

[0006] GOD has a wide range of industrial applications in diverse sectors, including food, pharmaceuticals, energy, and textiles. As a food additive, GOD exhibits significant antiseptic, antioxidant, and stabilizer properties. The hydrogen peroxide produced by GOD has antimicrobial properties, and the subsequent decomposition of hydrogen peroxide results in a net oxygen consumption with antioxidant and stabilizing effects. In the chemical industry, GOD is not only commonly used in biological bleaching but also serves as a key enzyme in the production of gluconic acid and its derivatives. In the medical field, GOD is a key raw material in glucose test kits and is also added to toothpaste to reduce the incidence of oral diseases. As a new feed additive, glucose oxidase can improve the intestinal environment of animals, increase feed utilization, and promote animal growth. As the application areas of GOD continue to expand, domestic and international market demand for GOD is rapidly increasing. However, during the production and processing of GOD, it often undergoes high temperatures, which can significantly reduce or even inactivate the enzyme activity. For example, brief high-temperature exposure during feed processing can inactivate GOD, thus affecting its effectiveness. Thermal stability is crucial for GOD's application. Heat-resistant GOD offers significant advantages during enzyme preparation and under extremely high-temperature reaction conditions. Therefore, improving the thermal stability of GOD has important practical significance.

[0007] Summary of the Invention

[0008] The present invention provides a glucose oxidase mutant, which has significantly improved enzyme activity and thermal stability compared with wild-type glucose oxidase, and also has improved stability at low pH, which is conducive to the application of the enzyme in industrial production.

[0009] In some embodiments, the glucose oxidase mutant of the present invention is a mutant of wild-type Aspergillus niger glucose oxidase, which comprises the following amino acids: 10K, 14L, 16A, 20Y, 30A, 34V, 36E, 37K, 45T, 70E, 74T, 90R, 106I, 135S, 167L, 173V, 185L, 186M, 187R, 204L, 226A, 259A, 274S, 288S, 292T, 332S, 362T, 405K, 406D, 418G, 446R, 504V, 509E, 556L, 572A, 575V, and 577A, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1, and the amino acid sequence of the wild-type Aspergillus niger glucose oxidase is the same as that of SEQ ID NO: 2. The sequence shown in NO: 1 has a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

[0010] In some embodiments, the glucose oxidase mutant further comprises at least one pair of introduced disulfide bonds. In some embodiments, the disulfide bonds are formed by one or more of the following pairs of amino acids: amino acids at positions 53 and 246, amino acids at positions 25 and 250, amino acids at positions 39 and 242, and amino acids at positions 87 and 508. In a preferred embodiment, the disulfide bonds are formed by amino acids at positions 53 and 246.

[0011] In some embodiments, the glucose oxidase mutant of the present invention comprises the following mutations compared to the wild-type Aspergillus niger glucose oxidase shown in SEQ ID NO: 1: T10K, D14L, S16A, V20Y, T30A, T34V, A36E, R37K, S45T, D70E, S74T, Q90R, V106I, N135S, V167L, A173V, I185L, V186M, K187R, F204L, S226A, G259A, G274S, A288S, A292T, A332S, S362T, N405K, H406D, A418G, H446R, T504V, Y509E, M556L, S572A, I575V, and E577A, wherein the amino acid positions correspond to those in SEQ ID NO: 1. The glucose oxidase mutant has a sequence identity of at least 90%, at least 91%, at least 92% or at least 93% to the wild-type Aspergillus niger glucose oxidase shown in SEQ ID NO: 1.

[0012] In some embodiments, the glucose oxidase mutant further comprises at least one pair of introduced disulfide bonds relative to the wild-type Aspergillus niger glucose oxidase set forth in SEQ ID NO: 1. In some embodiments, the glucose oxidase mutant further comprises one or more of the following groups of mutations relative to the parent glucose oxidase set forth in SEQ ID NO: 1: S53C and T246C, A25C and V250C, T39C and L242C, and T87C and P508C. In a preferred embodiment, the glucose oxidase mutant further comprises mutations of S53C and T246C relative to the parent glucose oxidase set forth in SEQ ID NO: 1.

[0013] In some embodiments, the amino acid sequence of the glucose oxidase mutant is shown in SEQ ID NO:2.

[0014] In some embodiments, the present invention also provides a polynucleotide encoding the glucose oxidase mutant.

[0015] In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO:3.

[0016] In some embodiments, the present invention also provides a recombinant expression vector comprising the polynucleotide.

[0017] In some embodiments, the present invention also provides a host cell comprising the polynucleotide or the recombinant expression vector. In some embodiments, the host cell is a bacterium or a fungus. In some embodiments, the host cell is a filamentous fungus or a yeast, preferably Pichia pastoris or Aspergillus niger.

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

[0019] (a) culturing the host cell of the present invention under conditions suitable for expression of the glucose oxidase mutant;

[0020] (b) Recovery of the expressed glucose oxidase mutant.

[0021] In some embodiments, the present invention also relates to a glucose oxidase mutant obtained by the above preparation method.

[0022] In some embodiments, the present invention provides a method for improving the thermal stability and enzymatic activity of glucose oxidase, comprising: The wild-type Aspergillus niger glucose oxidase shown in NO:1 was subjected to the following mutations: T10K, D14L, S16A, V20Y, T30A, T34V, A36E, R37K, S45T, D70E, S74T, Q90R, V106I, N135S, V167L, A173V, I185L, V186M, K187R, F204L, S226A, G259A, G274S, A288S, A292T, A332S, S362T, N405K, H406D, A418G, H446R, T504V, Y509E, M556L, S572A, I575V and E577A, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO:1.

[0023] In some embodiments, the method for improving the thermostability and specific activity of glucose oxidase further comprises introducing at least one pair of amino acid mutations capable of forming disulfide bonds into the wild-type Aspergillus niger glucose oxidase. In some embodiments, the at least one pair of mutations capable of forming disulfide bonds comprises one or more of the following groups of mutations: S53C and T246C, A25C and V250C, T39C and L242C, and T87C and P508C. In a preferred embodiment, the at least one pair of mutations capable of forming disulfide bonds comprises mutations of S53C and T246C.

[0024] In some embodiments, the present invention also provides applications of the glucose oxidase mutants in the fields of food, chemical industry, medicine, agriculture or feed.

[0025] The present invention has the following beneficial effects: the glucose oxidase mutant GOD-3-1-2C provided by the present invention has significantly improved thermostability and specific activity compared to the parent glucose oxidase shown in SEQ ID NO: 1. Specifically, the specific activity of the glucose oxidase mutant is increased by at least 190% compared to the parent glucose oxidase. In terms of heat resistance, the parent glucose oxidase has a residual enzyme activity of 0 after a 5-minute water bath at 80°C, while the glucose oxidase mutant of the present invention has a residual enzyme activity of 100% after a 5-minute water bath at 80°C. Particularly, the glucose oxidase mutant of the present invention has a residual enzyme activity of greater than 80% after a 5-minute water bath at 85°C and greater than 70% after a 5-minute water bath at 90°C, significantly outperforming the existing parent glucose oxidase in heat resistance. Furthermore, GOD-3-1-2C has higher acid resistance, making it more adaptable to the acidic environment of the animal gastrointestinal tract and capable of functioning. The glucose oxidase mutant provided by the present invention exhibits excellent performance and is particularly suitable for application in industrial production, for example, in the fields of food, chemical industry, medicine, agriculture and feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 shows the optimal reaction pH of mutant GOD-3-1-2C and parent glucose oxidase GGOX.

[0027] FIG2 shows the thermal stability of mutant GOD-3-1-2C and parent glucose oxidase GGOX. DETAILED DESCRIPTION

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0029] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0030] The terms "about" and "approximately," when used in connection with a numerical variable, generally mean that the value of the variable and all values ​​of the variable are within measurement or experimental error (e.g., a 95% confidence interval for the mean) or within the broader range of the specified value (e.g., ±5% or ±10%).

[0031] The term "comprise" or its variations such as "contains," "has," and "includes" means including the stated steps or elements but not excluding any other steps or elements. "Consisting of" means excluding unrecited steps or elements. "Consisting essentially of" means not excluding steps or elements that do not materially affect the basic and novel characteristics of the claimed invention. The term "comprising" specific steps or elements and its variations also include "consisting of" and "consisting essentially of" specific steps or elements.

[0032] When a numerical range is mentioned, it should be considered that the specific values ​​of its upper and lower limits are specifically disclosed, as well as all intermediate ranges included therein, such as intermediate ranges between its upper or lower limit and any intermediate value, or intermediate ranges between any two intermediate values. In addition, any intermediate ranges, subranges and all individual values ​​described in the numerical range can be excluded from the numerical range.

[0033] The term "and / or" should be understood to refer to any one element or any combination of several elements connected by this term.

[0034] The term "mutant" refers to a polypeptide having one or more amino acid insertions, deletions and / or substitutions relative to a parent polypeptide. 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; insertion refers to adding several (e.g., 1-5) amino acids adjacent to the amino acid occupying a certain position. The mutant retains at least one activity of the parent polypeptide, but may vary in the level of activity. For example, the mutant may remain unchanged or be improved in at least one activity or property relative to the parent polypeptide. In the present invention, the parent polypeptide is glucose oxidase, and its activity or property may include its basic enzymatic activity, i.e., the activity of catalyzing β-D-glucose to produce gluconic acid and hydrogen peroxide, as well as thermal stability, pH stability, etc.

[0035] The term "thermostability" refers to the ability of a glucose oxidase or a mutant thereof to maintain a certain level of enzymatic activity after a period of time at a specific temperature. With respect to properties such as thermostability, improved thermostability refers to a glucose oxidase or a mutant thereof having a higher residual enzymatic activity after a period of time at a specific temperature compared to other glucose oxidase mutants and / or the parent glucose oxidase. The term "residual enzymatic activity" refers to the percentage of enzymatic activity of a glucose oxidase or a mutant thereof after a period of time at a specific temperature relative to the enzymatic activity of the enzyme without the corresponding treatment.

[0036] The term "low pH stability" refers to the ability of a glucose oxidase or a mutant thereof to maintain a certain amount of enzymatic activity at a relatively low pH. When referring to properties such as low pH stability, improved low pH stability refers to the relative enzymatic activity of a glucose oxidase or a mutant thereof at a relatively low pH being higher than that of other glucose oxidase mutants and / or the parent glucose oxidase. Relative enzymatic activity at a relatively low pH refers to the percentage of the enzyme's enzymatic activity at a relatively low pH relative to the enzyme's activity at a commonly used pH or at its optimal pH.

[0037] The term "parent" refers to the glucose oxidase that is altered to produce the enzyme mutant of the present invention. The parent may be a naturally occurring (wild-type) polypeptide or a mutant thereof.

[0038] The term "wild-type" enzyme refers to an enzyme expressed by a naturally occurring organism or cell (such as bacteria or fungi). The "naturally occurring" means that it has not been artificially induced by mutagenesis or genetic manipulation.

[0039] The term "recombinant" when applied to a cell, nucleic acid, protein, or vector indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or by altering a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, a recombinant cell expresses genes that are not present in the native (non-recombinant) form of the cell; or it expresses native genes, but these native genes are otherwise abnormally expressed, underexpressed, or not expressed at all.

[0040] The term "heterologous" means that the nucleic acid or polypeptide is artificially introduced into the cell and / or does not naturally occur in the cell in which it is present.

[0041] The term "expression" in the context of the present invention includes any step involved in the production of the glucose oxidase mutants of the present invention including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion.

[0042] The term "expression vector" is defined herein as a linear or circular DNA molecule comprising a polynucleotide encoding a protein such as the glucose oxidase mutant of the present invention, and operably linked to additional nucleotides (eg, control sequences) that provide for its expression.

[0043] The term "host cell" includes cells transformed, transfected, transduced, etc. with a nucleic acid construct or expression vector comprising a polynucleotide encoding a glucose oxidase mutant, and progeny cells thereof, and the glucose oxidase mutant of the present invention is expressed by the cells.

[0044] The term "control sequence" refers to nucleic acid sequences necessary for expression of a polynucleotide encoding a glucose oxidase mutant of the present invention. Each control sequence can be homologous (i.e., from the same gene) or heterologous (i.e., from a different gene) to the polynucleotide encoding the glucose oxidase mutant, or homologous or foreign to each other. These control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. Control sequences include, at a minimum, a promoter and transcriptional and translational stop signals.

[0045] The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. A mature polypeptide may, for example, be N-terminally processed and not include a signal peptide.

[0046] The term "signal peptide" refers to a peptide linked in-frame to the amino terminus of a mature polypeptide and directs the encoded polypeptide into the cell's secretory pathway.

[0047] The term "sequence identity" is used to describe the correlation between two amino acid sequences or two nucleotide sequences." Sequence identity" refers to the sequence identity percentage value determined when two sequences are optimally aligned (i.e., the sequence identity is maximized) over a comparison window. The two aligned sequences can achieve optimal alignment by addition or deletion (i.e., gaps). In some algorithms, the sequence identity percentage can be calculated in the following manner: determine the number of positions at which the same nucleic acid base or amino acid residue appears in the two sequences under optimal alignment mode to produce the number of matching positions, divide the number of matching positions by the total number of positions compared, and then multiply the result by 100 to produce the sequence identity percentage. In other algorithms, the number of gaps and / or gap lengths are also taken into account. Commonly used sequence comparison algorithms or software include EMBOSS, DANMAN, CLUSTALW, MAFFT, BLAST, MUSCLE, etc. The sequence identity between two amino acid sequences can be determined by using available local alignment tools or global alignment tools, for example, using the CLUSTALW algorithm with preset parameters. For purposes of the present invention, preferably use the Needleman-Wengshi algorithm implemented in the Needle program of EMBOSS bag (EMBOSS:the European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277) to determine the sequence identity between two amino acid sequences.The parameter used is gap opening penalty 10, gap extension penalty 0.5, and EBLOSUM62 (EMBOSS version of EBLOSUM62) substitution matrix.The Needle output (using-nobrief option to obtain) labeled as " longest consistency " is used as percentage consistency and is calculated as follows: (consistent residue × 100) / (the total number of spaces in comparison length-comparison).

[0048] A "position corresponding to..." is a position that corresponds to a particular position in a reference amino acid sequence when the query amino acid sequence is optimally aligned with the reference amino acid sequence. In the present invention, the positions of amino acids in the glucose oxidase mutants described herein are determined based on the amino acid sequence set forth in SEQ ID NO:1. The glucose oxidase mutants are optimally aligned with SEQ ID NO:1, for example, using the CLUSTALW algorithm with preset parameters, to determine the amino acid position corresponding to any amino acid in SEQ ID NO:1.

[0049] Method for representing mutants of the present invention: Amino acids are represented by conventional single-letter or three-letter nomenclature. As is well known to those skilled in the art, the single-letter nomenclature for amino acids is as follows: A is alanine; C is cysteine; D is aspartic acid; E is glutamic acid; F is phenylalanine; G is glycine; H is histidine; I is isoleucine; K is lysine; L is leucine; M is methionine; N is asparagine; P is proline; Q is glutamine; R is arginine; S is serine; T is threonine; V is valine; W is tryptophan; and Y is tyrosine. Herein, for example, an amino acid substitution can be represented by the form "T10K", where 10 means that the substitution occurs at the amino acid at position 10, the T before 10 is the amino acid that occupied the position before the substitution occurs, and the K after 10 is the amino acid that occupied the position after the substitution occurs. Herein, the presence of a specific amino acid at a specific amino acid position can be represented by the form "10K", which means that the amino acid at position 10 is K.

[0050] Unless otherwise indicated, nucleic acids are written herein in 5' to 3' orientation left to right and amino acid sequences are written left to right in amino to carboxyl orientation.

[0051] The present invention relates to a thermostable mutant of glucose oxidase (EC1.1.3.4), in particular a mutant of wild-type Aspergillus niger glucose oxidase, a polynucleotide encoding the glucose oxidase mutant, an expression vector comprising the polynucleotide, a host cell expressing the glucose oxidase mutant, and uses thereof.

[0052] In particular, the glucose oxidase mutants of the present invention exhibit significantly improved thermal stability and enzymatic activity compared to the parent glucose oxidase. These properties make them particularly useful in industrial production and are widely applicable in the fields of food, feed, chemical production, medicine, pharmaceuticals, energy, textiles, etc. For example, they can be used as food or feed additives, in the production of chemical substances such as hydrogen peroxide, gluconic acid, and its derivatives, in biological bleaching, as toothpaste additives to reduce the incidence of oral diseases, as components of immunoassays and biosensors in the field of medical diagnosis, for example, in the preparation of glucose detection kits, and in the manufacture of micro-biofuel cells that can power biomedical implants such as biosensors and insulin pumps.

[0053] In the present invention, the parent glucose oxidase can be a wild-type Aspergillus niger glucose oxidase, such as the wild-type Aspergillus niger glucose oxidase shown in SEQ ID NO: 1, or a wild-type Aspergillus niger glucose oxidase having a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the sequence shown in SEQ ID NO: 1. In some embodiments, the parent glucose oxidase is a mature polypeptide that does not include a signal peptide. In some embodiments, the parent glucose oxidase includes a signal peptide.

[0054] In some embodiments, the glucose oxidase mutant of the present invention is a mutant of wild-type Aspergillus niger glucose oxidase, which mutant includes the following amino acids: 10K, 14L, 16A, 20Y, 30A, 34V, 36E, 37K, 45T, 70E, 74T, 90R, 106I, 135S, 167L, 173V, 185L, 186M, 187R, 204L, 226A, 259A, 274S, 288S, 292T, 332S, 362T, 405K, 406D, 418G, 446R, 504V, 509E, 556L, 572A, 575V and 577A, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1. In other words, the glucose oxidase mutant of the present invention comprises the following amino acids: K at position 10, L at position 14, A at position 16, Y at position 20, A at position 30, V at position 34, E at position 36, K at position 37, T at position 45, E at position 70, T at position 74, R at position 90, I at position 106, S at position 135, L at position 167, V at position 173, L at position 185, M at position 186, and S at position 187. is R at position 204, L at position 226, A at position 259, S at position 274, S at position 288, T at position 292, S at position 332, T at position 362, K at position 405, D at position 406, G at position 418, R at position 446, V at position 504, E at position 509, L at position 556, A at position 572, V at position 575, and A at position 577, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the wild-type Aspergillus niger glucose oxidase has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1. In some embodiments, except for the amino acids at the aforementioned positions, the amino acids at other positions of the glucose oxidase mutant are the same as those of the parent wild-type Aspergillus niger glucose oxidase.

[0055] In some embodiments, the glucose oxidase mutant further comprises at least one pair of introduced disulfide bonds relative to the parent glucose oxidase. Thus, in some embodiments, the glucose oxidase mutant further comprises two introduced disulfide bond-forming amino acids relative to the parent glucose oxidase. In some embodiments, the disulfide bond-forming amino acids are cysteine ​​or homocysteine. In some embodiments, the glucose oxidase mutant further comprises two amino acid mutations relative to the parent glucose oxidase, wherein the two amino acids are mutated to amino acids capable of forming disulfide bonds.

[0056] For the purposes 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 can include replacing the amino acid at the corresponding position in the glucose oxidase sequence where the disulfide bond is to be introduced with an amino acid capable of forming a disulfide bond (including, for example, cysteine, Cys, homocysteine, Hcy, etc.); and / or inserting an amino acid 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 situations where any one or both of the amino acid residues forming the disulfide bond arise from natural mutation.

[0057] In some embodiments, the disulfide bond is formed by one or more of the following pairs of amino acids: amino acids at positions 53 and 246, amino acids at positions 25 and 250, amino acids at positions 39 and 242, and amino acids at positions 87 and 508. In a preferred embodiment, the disulfide bond is formed by amino acids at positions 53 and 246. In some embodiments, except for the amino acids at the aforementioned 37 positions and the mutated amino acids capable of forming disulfide bonds, the amino acids at other positions of the glucose oxidase mutant are identical to those of the parent wild-type Aspergillus niger glucose oxidase.

[0058] In some embodiments, the glucose oxidase mutant of the present invention is the same as SEQ ID niger glucose oxidase as compared to the wild-type Aspergillus niger glucose oxidase shown in NO:1, comprising the following mutations: T10K, D14L, S16A, V20Y, T30A, T34V, A36E, R37K, S45T, D70E, S74T, Q90R, V106I, N135S, V167L, A173V, I185L, V186M, K187R, F204L, S226A, G259A, G274S, A288S, A292T, A332S, S362T, N405K, H406D, A418G, H446R, T504V, Y509E, M556L, S572A, I575V and E577A, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO:1. In some embodiments, the glucose oxidase mutant has at least 90%, at least 91%, at least 92%, or at least 93% sequence identity to the wild-type Aspergillus niger glucose oxidase set forth in SEQ ID NO: 1. In some embodiments, the glucose oxidase mutant comprises only the aforementioned mutations compared to the wild-type Aspergillus niger glucose oxidase set forth in SEQ ID NO: 1.

[0059] In some embodiments, the glucose oxidase mutant further comprises at least one pair of introduced disulfide bonds relative to the parent glucose oxidase shown in SEQ ID NO: 1. Therefore, in some embodiments, the glucose oxidase mutant further comprises two introduced amino acids capable of forming disulfide bonds relative to the parent glucose oxidase shown in SEQ ID NO: 1. In some embodiments, the amino acids capable of forming disulfide bonds are cysteine ​​or homocysteine. In some embodiments, the glucose oxidase mutant further comprises two amino acid mutations relative to the parent glucose oxidase, wherein the two amino acids are mutated to amino acids capable of forming disulfide bonds.

[0060] In some embodiments, the glucose oxidase mutant further comprises one or more of the following groups of mutations relative to the parent glucose oxidase shown in SEQ ID NO: 1: S53C and T246C, A25C and V250C, T39C and L242C, and T87C and P508C. In a preferred embodiment, the glucose oxidase mutant further comprises S53C and T246C mutations relative to the parent glucose oxidase shown in SEQ ID NO: 1.

[0061] In some embodiments, the glucose oxidase mutant is the same as SEQ ID The wild-type Aspergillus niger glucose oxidase shown in NO:1 only includes the following mutations: T10K, D14L, S16A, V20Y, T30A, T34V, A36E, R37K, S45T, S53C, D70E, S74T, Q90R, V106I, N135S, V167L, A173V, I185L, V186M, K187R, F204L, S226A, T246C, G259A, G274S, A288S, A292T, A332S, S362T, N405K, H406D, A418G, H446R, T504V, Y509E, M556L, S572A, I575V and E577A. In a preferred embodiment, the sequence of the glucose oxidase mutant is shown as SEQ ID NO: 2.

[0062] In some embodiments, the glucose oxidase mutant of the present invention is a mature polypeptide that does not comprise a signal peptide and / or a leader sequence. In some embodiments, the glucose oxidase mutant of the present invention comprises a signal peptide and / or a leader sequence.

[0063] In some embodiments, the glucose oxidase mutants of the present invention have improved enzyme activity, improved thermal stability, and / or improved low pH stability (particularly stability under pH 3-5.5 conditions) compared to the parent glucose oxidase or other glucose oxidase mutants. The thermal stability refers to stability under high temperature conditions, where "high temperature" refers to 70°C-100°C, preferably 80°C-95°C. The "low pH" refers to pH 3-5.5.

[0064] In some embodiments, the enzymatic activity of the glucose oxidase mutants of the present invention is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher than that of the parent glucose oxidase or other glucose oxidase mutants. The enzymatic activity can be detected, for example, by o-dianisidine-horseradish peroxidase coupled spectrophotometry based on the change in absorbance at 540 nm and a standard curve. In some embodiments, the enzyme activity is measured at 37° C. and pH 5.5. The “o-dianisidine-horseradish peroxidase coupled spectrophotometry” refers to the process of catalyzing the dehydrogenation of glucose by glucose oxidase to produce H2O2 under aerobic conditions, and oxidizing the oxygen donor o-dianisidine (DH2) under the action of horseradish peroxidase (POD) to produce a brown product. The glucose oxidase activity is calculated based on the change in absorbance at 540 nm and a standard curve.

[0065] In some embodiments, the glucose oxidase mutants of the present invention have a higher residual enzyme activity at high temperature conditions, particularly at 80°C-95°C, than that of the parent glucose oxidase or other glucose oxidase mutants. In some embodiments, the glucose oxidase mutants of the present invention have a higher residual enzyme activity at 80°C than that of the parent glucose oxidase or other glucose oxidase mutants. In some embodiments, the glucose oxidase mutants of the present invention have a higher residual enzyme activity after being kept at 80°C for 5 minutes than that of the parent glucose oxidase or other glucose oxidase mutants. In some embodiments, the glucose oxidase mutants of the present invention have a higher residual enzyme activity at 85°C than that of the parent glucose oxidase or other glucose oxidase mutants. In some embodiments, the glucose oxidase mutants of the present invention have a higher residual enzyme activity after being kept at 85°C for 5 minutes than that of the parent glucose oxidase or other glucose oxidase mutants. In some embodiments, the glucose oxidase mutants of the present invention have a higher residual enzyme activity at 90°C than that of the parent glucose oxidase or other glucose oxidase mutants. In some embodiments, the glucose oxidase mutants of the present invention have a higher residual enzyme activity after being kept at 90°C for 5 minutes than that of the parent glucose oxidase or other glucose oxidase mutants.

[0066] In some embodiments, the glucose oxidase mutant of the present invention has a residual enzyme activity of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95% or 100% after being kept at 80°C for 5 minutes. In some embodiments, the glucose oxidase mutant of the present invention has a residual enzyme activity of at least 60%, at least 65%, at least 69%, at least 70%, at least 75%, at least 80% or at least 85% after being kept at 85°C for 5 minutes. In some embodiments, the glucose oxidase mutant of the present invention has a residual enzyme activity of at least 50%, at least 54%, at least 55%, at least 60%, at least 65%, at least 70% or at least 74% after being kept at 90°C for 5 minutes. The residual enzyme activity refers to the relative enzyme activity of the glucose oxidase mutant after being kept at the corresponding temperature for the corresponding time, that is, the percentage of the enzyme activity of the glucose oxidase mutant after being kept at the corresponding temperature for the corresponding time relative to the enzyme activity of the mutant that has not been treated with the corresponding method. The enzyme activity before and after treatment is determined by the same method. In some embodiments, the determination of the residual enzyme activity after the heat treatment is performed at pH 5.5, and the determination of the enzyme activity of the mutant that has not undergone the corresponding treatment is performed at 37° C. and pH 5.5.

[0067] In some embodiments, the optimal pH of the glucose oxidase mutant of the present invention is about 5.5. The "optimum pH" refers to the pH value at which the enzyme exhibits maximum activity when other assay conditions are the same.

[0068] In some embodiments, the relative enzyme activity of the glucose oxidase mutants of the present invention at pH 3-5.5 is greater than that of the parent glucose oxidase or other glucose oxidase mutants. Relative enzyme activity under specific pH conditions refers to the percentage of the enzyme activity under those pH conditions relative to the enzyme activity under the optimal pH conditions for the same enzyme. When determining the relative enzyme activity under specific pH conditions, other measurement conditions are the same except for the pH. In some embodiments, the relative enzyme activity under specific pH conditions is determined at 37°C.

[0069] The present invention also relates to a method for improving the thermal stability and enzymatic activity of glucose oxidase, comprising performing the above-mentioned mutation on wild-type Aspergillus niger glucose oxidase. The above-mentioned mutation can be performed using any method known in the art, such as site-directed mutagenesis, homologous recombination, etc.

[0070] The present invention also relates to a polynucleotide encoding the glucose oxidase mutant of the present invention, and a recombinant expression vector comprising the polynucleotide for expressing the glucose oxidase mutant. In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 3. The polynucleotide may further comprise a coding sequence for a signal peptide to allow the mutant to be secreted extracellularly after expression.

[0071] The expression vector comprises a polynucleotide encoding the glucose oxidase mutant of the present invention, and the polynucleotide is operably linked to one or more control sequences, which can direct the expression of the coding sequence of the glucose oxidase mutant in a suitable host cell.

[0072] Control sequences include, but are not limited to, promoters, terminators, leader sequences, polyadenylation sequences, and the like. Constitutive or inducible promoters may be used. Control sequences suitable for use with different host cells are well known to those skilled in the art.

[0073] The expression vector can be an autonomously replicating vector, i.e., one that replicates independently of the chromosome, such as a plasmid or an artificial chromosome, or a vector that is integrated into the genome and replicates together with the chromosome when introduced into the host cell. The expression vector can contain one or more selectable markers that allow for easy selection of transformed cells, transfected cells, or transduced cells, such as markers that can provide resistance to antibiotics, heavy metals, etc. The expression vector can also contain an origin of replication to allow the expression vector to replicate autonomously in the host cell. The selection of these elements is a common technique in the art, and methods for constructing the recombinant expression vector of the present invention using these elements are well known in the art, for example, see J. Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition. In some embodiments, the polynucleotide encoding the glucose oxidase mutant of the present invention is inserted into the plasmid vector pPICZαA to prepare an expression vector for expressing the glucose oxidase mutant.

[0074] The present invention also relates to recombinant host cells for expressing the glucose oxidase mutants of the present invention. The host cell can be a prokaryotic cell or a eukaryotic cell, for example, a bacterial cell, a fungal cell, a plant cell, an insect cell, or an animal cell (e.g., a mammalian cell). In some embodiments, the host cell can be a filamentous fungus or a yeast cell. The filamentous fungus can be, for example, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, etc. The yeast can be, for example, a methanol-trophic yeast, for example, Pichia pastoris or Komagataella pastoris, such as Pichia pastoris or Komagataella pastoris. In some embodiments, Pichia pastoris X-33 is used as a host cell. Methods for introducing polynucleotides into host cells are well known to those skilled in the art, such as electroporation, gene guns, etc.

[0075] The present invention also relates to a method for preparing the above-mentioned glucose oxidase mutant, comprising: (a) culturing the host cell of the present invention under conditions suitable for the expression of the glucose oxidase mutant; and (b) recovering the glucose oxidase mutant.

[0076] In some embodiments, step (a) comprises inducing expression of the glucose oxidase mutant. The inducer used depends on the control sequence (e.g., promoter) used to direct expression of the glucose oxidase mutant. In some embodiments, the inducer is methanol, and the coding sequence of the glucose oxidase mutant is operably linked to a methanol-inducible promoter (e.g., AOX1).

[0077] In some embodiments, the method for preparing the glucose oxidase mutant comprises: (a) inserting a polynucleotide encoding the glucose oxidase mutant of the present invention into the plasmid vector pPICZαA to prepare a recombinant expression vector for expressing the glucose oxidase mutant; (b) transferring the recombinant expression vector prepared in step (a) into Pichia pastoris to obtain a host cell expressing the glucose oxidase mutant; (c) culturing and inducing the host cell to express the glucose oxidase mutant; and (d) recovering the glucose oxidase mutant.

[0078] Those skilled in the art can determine conditions suitable for expressing the glucose oxidase mutant. For example, a nutrient medium containing a carbon source and inorganic salts can be used to culture the host cell, and growth conditions such as pH, temperature, oxygen and / or nutrient supply can be controlled. The host cell can be cultured in a shake flask or fermented in a laboratory or industrial fermentor, such as continuous fermentation, batch fermentation, fed-batch fermentation or solid fermentation culture. In some embodiments, the host cell is cultured in a fermentor. When an inducible promoter (e.g., alcohol oxidase promoter AOX1) is used, methanol can be added during the culture process to induce expression of the mutant.

[0079] The glucose oxidase mutant can be secreted into the culture medium and then recovered from the culture medium. The glucose oxidase mutant can also be non-secreted, that is, expressed and retained intracellularly, in which case the glucose oxidase mutant can be recovered after cell lysis.

[0080] The glucose oxidase mutant can be recovered using methods known in the art. For example, the glucose oxidase mutant can be recovered from the nutrient medium by a variety of conventional procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation and / or precipitation. In some embodiments, the recombinantly expressed glucose oxidase mutant is secreted into the culture medium, and a supernatant containing the glucose oxidase mutant can be obtained by one or more techniques such as sedimentation, flocculation or centrifugation. In some embodiments, the recombinantly expressed glucose oxidase mutant is located intracellularly, and the cells can be subjected to a crushing process to release the target product from the cells. The crushing process can include, for example, pressurization, osmotic shock, freezing, ultrasound or other treatments to produce a cell lysate, and the cell lysate can be centrifuged to obtain a supernatant containing the glucose oxidase mutant. The supernatant containing the glucose oxidase mutant can be further purified to obtain a higher purity glucose oxidase mutant. The mutant can be purified by various procedures known in the art to obtain a substantially pure mutant, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, size exclusion chromatography, or any combination thereof), electrophoresis (e.g., preparative isoelectric focusing or SDS-PAGE), fractional precipitation (e.g., using ammonium sulfate, ethanol, or acid), or extraction, etc. (see Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966).

[0081] In some embodiments, the glucose oxidase mutant may not be recovered from the host cells, but the host cells may be used directly as a source of the glucose oxidase mutant. In some embodiments, the cultured cell suspension containing the glucose oxidase mutant may be dried, for example, by fluidized bed drying, spray drying, drum drying, or any combination thereof, for further use.

[0082] The generated glucose oxidase mutants can be tested using various methods known in the art. For example, the activity of the glucose oxidase mutants can be tested by enzyme activity assays, such as the activity of catalyzing glucose dehydrogenation to produce H2O2. This can be measured, for example, by o-dianisidine-horseradish peroxidase-coupled spectrophotometry based on changes in absorbance at 540 nm and a standard curve.

[0083] The glucose oxidase mutants of the present invention have high enzymatic activity, good thermal stability, and stability under low pH conditions, and can be widely used in various fields, such as the food, chemical, pharmaceutical, agricultural, or feed sectors. The use of glucose oxidase in these fields is well known, and the glucose oxidase of the present invention, due to its excellent performance, will achieve superior results in these fields compared to other glucose oxidase mutants or the parent glucose oxidase.

[0084] For example, in the food industry, the glucose oxidase mutant of the present invention can be used as a food additive with preservative, antioxidant and stabilizer properties. For example, it can be used as a food preservative to prevent beer aging, maintain the original flavor of the product, and extend the shelf life. It can also be used as a flour improver and bread quality improver.

[0085] For example, in the chemical industry, the glucose oxidase mutant of the present invention can be used for biological bleaching and for producing gluconic acid and its derivatives, such as sodium gluconate or calcium gluconate.

[0086] For example, in the medical field, the glucose oxidase mutant of the present invention can be used to detect the glucose content in blood and serum, for example, by the glucose oxidase electrode method, the glucose oxidase-peroxidase coupling method, etc., and can also be added to toothpaste to reduce the incidence of oral diseases.

[0087] For example, in the field of feed, the glucose oxidase mutant of the present invention can be used as a feed additive to improve the intestinal environment of animals and increase feed utilization.

[0088] In some embodiments, the use of the glucose oxidase mutants of the present invention is for non-therapeutic and / or non-diagnostic purposes.

[0089] The present invention is further described by the following examples, which should not be construed as limiting the present invention.

[0090] Unless otherwise specified, all reagents used in the following examples are commercially available. Molecular biology experimental methods not specifically described in the examples were performed according to the specific methods listed in J. Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition, or according to the kits and product instructions.

[0091] The experimental materials and reagents used in the following examples are as follows:

[0092] 1. Strains and vectors: The expression host Pichia pastoris X-33 (Invitrogen), the expression plasmid vector pPICZαA (Invitrogen), the Escherichia coli strain DH5α or Top10, and the strain containing the glucose oxidase gene and expression plasmid are maintained in our laboratory.

[0093] 2. Enzymes and other biochemical reagents: Endonucleases were purchased from Fermentas, ligases from Promaga, and DNA polymerases from Beijing Quanshijin Biotechnology. Plasmid extraction and purification kits were purchased from Shanghai Shenggong Company. The antibiotic Zeocin was purchased from Invitrogen. All other reagents were domestically produced, analytical grade reagents available from common biochemical reagent companies.

[0094] 3. Culture medium:

[0095] LB solid medium: 0.5% yeast extract, 1% peptone, 1% NaCl, 1% agar powder, pH 7.0.

[0096] LB-A resistance medium is LB medium with ampicillin added to a final concentration of 100 μg / mL.

[0097] LB-Z resistance medium is LB medium with Zeocin added to a final concentration of 25 μg / mL.

[0098] YPD medium: 1% yeast extract, 2% peptone, 2% glucose.

[0099] YPD-Z resistance medium is YPD medium with Zeocin added to a final concentration of 100 μg / mL.

[0100] Induction medium BMGY: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 1% glycerol (v / v).

[0101] Induction medium BMMY: 0.5% methanol replaces glycerol, and the rest of the ingredients are the same as BMGY.

[0102] Fermentation basic salt medium: 5% diammonium hydrogen phosphate, 0.5% potassium dihydrogen phosphate, 1.5% magnesium sulfate heptahydrate, 1.95% potassium sulfate, 0.1% calcium sulfate, 0.03% defoamer, add 4.35mL PTM1 per liter after sterilization.

[0103] PTM1 (trace salt solution): copper sulfate 0.6%, potassium iodide 0.018%, manganese sulfate monohydrate 0.3%, sodium molybdate dihydrate 0.02%, boric acid 0.002%, cobalt chloride hexahydrate 0.05%, zinc chloride 2%, ferric sulfate heptahydrate 6.5%, concentrated sulfuric acid 0.5%, biotin 0.02%.

[0104] 4. Determination of glucose oxidase activity

[0105] Under aerobic conditions, GOD catalyzes the dehydrogenation of glucose to produce H2O2. Under the action of horseradish peroxidase (POD), the oxygen donor o-dianisidine (DH2) is oxidized to a brown product. The activity of GOD can be calculated based on the change in absorbance at 540nm and the standard curve. The enzyme activity assay system contains 2.5mL o-dianisidine solution, 0.3mL of 18% glucose, and 0.1mL of 90U / mL horseradish peroxidase. After incubation at 37°C for 2 minutes, 0.1mL of the diluted enzyme solution sample is added to a test tube. After a 3-minute reaction, 2mol / L sulfuric acid is added to terminate the reaction. The test tube is removed and the OD540 absorbance is measured. A heat-inactivated enzyme solution is used as a blank control. Based on the results of the standard curve, the glucose oxidase activity units are calculated.

[0106] Reagents and solutions:

[0107] 0.1 mol / L pH 5.5 disodium hydrogen phosphate-sodium citrate buffer: Accurately weigh 14.32 g disodium hydrogen phosphate and 8.4056 g citric acid monohydrate, dissolve them in 400 mL distilled water, and adjust the pH to 5.5 with disodium hydrogen phosphate.

[0108] o-Dianisidine solution: Accurately weigh 0.1 g of o-dianisidine and dissolve it in 10 mL of methanol. This is the stock solution, which is effective for 3 days at 4°C. Before the experiment, take 0.1 mL of the stock solution and dissolve it in 12 mL of the above 0.1 mol / L, pH 5.5 phosphate buffer.

[0109] 18% glucose: Accurately weigh 9.00 g of glucose (AR) dried to constant weight, dissolve in a small amount of distilled water, and dilute to 50 mL with distilled water. Store at 4°C.

[0110] 2 mol / L H2SO4: Accurately weigh 40.00 g H2SO4 and slowly add it to 160 mL of distilled water to make up to 200 mL.

[0111] 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 until use.

[0112] 90 U / mL horseradish peroxidase: Purchase 100 mg of horseradish peroxidase standard (enzyme activity > 250 units / mg), accurately add 1 mL of distilled water to fully dissolve the horseradish peroxidase, and store at -20°C until ready to use. Dilute an appropriate amount of the standard to an activity of 90 U / mL before use. Always dilute immediately before use.

[0113] Determination of enzyme activity:

[0114] (1) Preparation of standard curve

[0115] Dilute the GOD standard to 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 U / mL, respectively. 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 37°C for 2 minutes, then add 0.1 mL of the diluted GOD standard at 15-second intervals. After an accurate reaction for 3 minutes, immediately add 2 mL of 2 mol / L H2SO4 to terminate the reaction. Take out and mix thoroughly, 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.

[0116] (2) Sample determination

[0117] Add 2.5 mL of o-dianisidine solution and 0.3 mL of 18% glucose solution to a test tube, then add 0.1 mL of 90 U / mL horseradish peroxidase solution. Preheat at 37°C for 2 minutes, then add 0.1 mL of diluted sample (the dilution standard is that the absorbance value of the sample is within the linear range) at 15 second intervals. After accurate reaction for 3 minutes, immediately add 2 mL of 2 mol / L H2SO4 to terminate the reaction. Take out and mix thoroughly, measure the absorbance A at 540 nm, and calculate the enzyme activity.

[0118] (3) Calculation of enzyme activity

[0119] X=(K*A+b)*n

[0120] X: enzyme activity of the sample U / mL, A: absorbance of the sample, n: dilution factor of the enzyme solution, K: slope of the standard curve, b: intercept of the standard curve.

[0121] Example 1 Parent Glucose Oxidase GGOX Gene Synthesis and Vector Construction

[0122] The parent glucose oxidase GGOX is the mature form of glucose oxidase (GenBank: EHA27180.1) from Aspergillus niger ATCC 1015, and its amino acid sequence is shown in SEQ ID NO: 1. The glucose oxidase gene was synthesized using a full gene synthesis method, which was completed by Nanjing GenScript Biotechnology Co., Ltd. The synthesized gene has EcoRI and XbaI restriction sites at both ends. The synthesized gene was digested with EcoRI and XbaI, the target gene fragment was recovered by gel extraction, the product was purified and recovered, and ligated to the expression vector pPICZαA. The ligation product was transformed into Escherichia coli Top10 competent cells using a chemical conversion heat shock method, coated on LB-Z resistant plates, and inverted and cultured at 37°C. After transformants appeared on the plates, single colonies were picked and inoculated into LB-Z resistant test tubes and cultured at 37°C. The recombinant transformants were verified by bacterial liquid PCR, and the plasmids of the verified correct transformants were extracted and sequenced to obtain the expression vector pPICZαA-GGOX.

[0123] Example 2 Random mutation of glucose oxidase GGOX by error-prone PCR

[0124] Using the above-mentioned pPICZαA-GGOX as a template, mutations were randomly introduced using the GeneMorph II Random Mutation PCR Kit (Stratagene).

[0125] The primers used were:

[0126] GGOX-F 5'-CCGGAATTCTCCAACGGTATTGAGGCCTCTTTG-3'(SEQ ID NO:4)

[0127] GGOX-R 5'-CTAGTCTAGATTACTGCATGGAAGCGTAGTCAGC-3'(SEQ ID NO:5)

[0128] The underlined areas are the EcoRI and XbaI restriction sites, respectively.

[0129] The reaction conditions were as follows: pre-denaturation at 94°C for 10 min, denaturation at 94°C for 60 s, annealing at 58°C for 60 s, and extension at 72°C for 2 min, for a total of 30 cycles.

[0130] PCR amplification results were verified by agarose gel electrophoresis, and the amplified target product was purified and recovered. The target fragment was double-digested with EcoRI and XbaI and ligated into the pPICZαA vector. The ligation product was transformed into Escherichia coli Top10 competent cells using the chemical transformation heat shock method. The ligation product was plated on LB-Z resistant plates and incubated upside down at 37°C. Once transformants appeared on the plates, single colonies were selected and inoculated into LB-Z resistant tubes and incubated at 37°C. Recombinant transformants were verified by PCR in the culture medium. Plasmids from confirmed transformants were extracted and sequenced to identify the corresponding mutants. The mutant plasmids that were sequenced correctly were linearized with the restriction endonuclease PmeI. The linearized plasmid fragment was recovered and purified, and transformed into Pichia pastoris X33 competent cells by electroporation. Recombinant transformants were selected using YPD-Z resistant medium to obtain yeast.

[0131] Preparation of Pichia pastoris X-33 competent cells: 1) Inoculate a single colony of Pichia pastoris X-33 into 5 mL of YPD medium and culture at 30°C and 250 rpm / min for 24 h; 2) Take 1 mL of culture solution and inoculate into a 500 mL shake flask containing 100 mL of YPD medium and culture for 12-16 h. When OD600 reaches 0.8-1.2, transfer the culture solution into a 50 mL pre-cooled centrifuge tube and centrifuge at 4°C and 5000 g. Centrifuge for 5 minutes and discard the supernatant; 3) Resuspend the cells in 25 mL of pre-cooled sterile water, centrifuge at 4°C and 5000g for 5 minutes, discard the supernatant, and repeat this step 2-3 times; 4) Resuspend the cells in 5 mL of pre-cooled 1 mol / L sorbitol per tube, centrifuge at 4°C and 5000g for 5 minutes, discard the supernatant, and repeat this step 2-3 times; 5) Finally, resuspend the cells in 200 μL of sorbitol and divide the volume into 1.5 mL centrifuge tubes, 80 μL per tube.

[0132] Transformation screening: 1) Take one tube of competent cells and add 10 μL of linearized and concentrated DNA, mix gently, and transfer to a pre-cooled 0.2 cm electrode cup and bathe on ice for 5 minutes; 2) Perform electric shock at the shock parameters of 1.5 kV, 25 μF, 200 Ω. After the electric shock is completed, quickly add 1 mL of pre-cooled 1 mol / L sorbitol, then transfer the mixture to a 1.5 mL centrifuge tube and resuscitate at 30°C for 1 hour; 3) Pipette 200 μL and spread it on YPD-Z solid medium containing 100 μg / ml Zeocin. Incubate the plate at 30°C for 3 days, observe positive transformants, identify positive single colonies by PCR, and send PCR-positive colonies for sequencing verification.

[0133] Example 3 High-throughput screening of high-specific-activity mutant strains

[0134] Use toothpicks to pick the yeast recombinant transformants obtained in Example 2 to 24-well plates, add 1 mL of BMGY medium to each well in advance, culture at 30°C, 200 rpm for about 24 hours, and centrifuge to remove the supernatant. Then add 1.6 mL of BMMY medium to each well for induction culture. Add 1% of the culture volume of methanol every 24 hours. After induced expression for 4 days, centrifuge the culture solution to obtain the supernatant, take out 100 μL of the above supernatant, and perform glucose oxidase activity assay to calculate the specific activity. Take the strain with higher activity than the parent glucose oxidase to a new 24-well culture plate and repeat the screening. After high-throughput screening, yeast recombinant transformants with improved specific activity are obtained.

[0135] Example 4 High-throughput screening of glucose oxidase mutants with improved heat resistance

[0136] Yeast recombinant transformants with activity close to or higher than that of the parent glucose oxidase GGOX obtained in Example 3 were picked individually with toothpicks into a 24-well plate. 1 mL of BMGY medium was added to each well in advance, and the cells were cultured at 30°C, 200 rpm for approximately 24 hours. The supernatant was then removed by centrifugation. 1.6 mL of BMMY medium was then added to each well for induction culture. Methanol was added at 1% of the culture volume every 24 hours. After 4 days of induced expression, the culture was centrifuged to obtain the supernatant. 100 μL of the supernatant was taken out for glucose oxidase activity assay and heat treatment residual enzyme activity assay. Through multiple rounds of screening and comparison, mutants with significantly improved heat resistance compared to the parent glucose oxidase GGOX were screened out.

[0137] Example 5 Combination Mutation and Screening

[0138] In Example 3 and Example 4, based on the specific activity or heat-resistant forward mutation site, double-site or multi-site combination mutation was performed, and the combination mutant sequence was subjected to full gene synthesis by gene synthesis. The combination mutant vector was constructed in the manner of Example 1. The combination mutant plasmid with correct sequencing was linearized with the restriction endonuclease PmeI, the purified linear plasmid fragment was recovered, and the electroporation method was used to transform it into Pichia pastoris X33 competent cells. Yeast recombinant transformants were screened using YPD-Z resistance culture medium to obtain yeast recombinant transformants. The recombinant transformants were picked and transferred to 50mL BMGY culture medium respectively. After shaking culture at 30°C and 220rpm for 18h, the cells were centrifuged and transferred to 50mL BMMY culture medium to make the cell concentration reach OD600=1. The shaking culture was continued at 30°C and 250rpm, and 1% methanol of the culture volume was added every 24h. After 4 days of induction, the culture medium was centrifuged to obtain the supernatant, and the parent glucose oxidase GGOX and the combined mutant were purified by nickel affinity chromatography. The purified samples were tested for glucose oxidase activity and the specific activity was calculated. The relative specific activity of the mutant was calculated by dividing the specific activity of the combined mutant by the specific activity of the parent glucose oxidase GGOX. After combined mutation and screening, the optimal combined mutant was finally screened out. The mutation sites were: T10K, D14L, S16A, V20Y, T30A, T34V, A36E, R37K, S45T, D70E, S74T, Q90R, V106I, N135S, V167L, A173V, I185L, V186M, K187R, F 204L, S226A, G259A, G274S, A288S, A292T, A332S, S362T, N405K, H406D, A418G, H446R, T504V, Y509E, M556L, S572A, I575V, E577A. The mutant was designated GOD-3-1, and its specific activity assay results are shown in Table 1. The purified samples were diluted to approximately 100 U / mL with pH 5.5 phosphate buffer and treated at different temperatures for 5 minutes. The residual enzyme activity was then measured, and the relative enzyme activity was calculated, with the enzyme activity of the untreated sample assumed to be 100%. The results are shown in Table 2.

[0139] Example 6: Combination mutant GOD-3-1 introduces disulfide bonds to further improve heat resistance

[0140] Based on the amino acid sequence of mutant GOD-3-1, the disulfide bonds S53C and T246C were designed and introduced, ultimately resulting in a new combined mutant GOD-3-1-2C. The mutant vector was constructed and transformed into Pichia pastoris X33 as in Example 5. The mutant GOD-3-1-2C was purified using nickel affinity chromatography. The purified sample was assayed for glucose oxidase activity and the specific activity was calculated, as shown in Table 1. The purified sample was diluted to approximately 100 U / mL with pH 5.5 phosphate buffer and treated at 80°C, 85°C, and 90°C for 5 minutes, respectively. The residual enzyme activity was then measured, and the relative enzyme activity was calculated, with the enzyme activity of the untreated sample taken as 100%. The results are shown in Table 2.

[0141] As can be seen from Table 1, there is no significant difference in the specific activity of mutants GOD-3-1-2C and GOD-3-1. Compared with the parent glucose oxidase GGOX, the specific activity of both mutants is significantly improved, reaching a relative specific activity of 190%. As can be seen from Table 2, after the introduction of a disulfide bond into mutant GOD-3-1, the thermal stability of the new mutant GOD-3-1-2C is further improved. The parent glucose oxidase has a residual enzyme activity of 0 after a 5-minute water bath at 80°C or above, while the thermal stability of the glucose oxidase GOD-3-1 of the present invention is significantly improved, reaching 88% after a 5-minute water bath at 80°C. Mutant GOD-3-1-2C exhibits even better heat resistance, with a residual enzyme activity of 100%. After a 5-minute water bath at 85°C, the residual enzyme activity of mutant GOD-3-1-2C reaches 85%, and after a 5-minute water bath at 90°C, the residual enzyme activity of mutant GOD-3-1-2C reaches 74%. Therefore, compared with the parent glucose oxidase, the mutant GOD-3-1-2C exhibited excellent heat resistance.

[0142] Table 1 Comparison of specific activities of parent glucose oxidase GGOX and combined mutants

[0143] Table 2 Comparison of thermal stability of parent glucose oxidase GGOX and combined mutants

[0144] Example 7 Determination of the Optimal Reaction pH of Parent Glucose Oxidase GGOX and Mutant GOD-3-1-2C

[0145] The glucose oxidase activity was measured at 37°C in various buffer solutions (sodium hydrogen phosphate-citrate buffer at pH 3.0, 3.5, 4.0, 4.5, and 5.0, and phosphate buffer at pH 5.5, 6.0, 6.5, 7.0, and 7.5). The results were expressed as relative activity, with the pH at which the maximum activity was observed being 100%. As shown in Figure 1, the optimal reaction pH for both the parent glucose oxidase GGOX and the mutant GOD-3-1-2C was approximately 5.5. The mutant GOD-3-1-2C exhibited higher relative activity than the parent glucose oxidase GGOX at pH 3-5.5, indicating that GOD-3-1-2C has a higher acid resistance and is better able to adapt to the acidic environment of the animal gastrointestinal tract.

[0146] Example 8 Determination of thermal stability of parent glucose oxidase GGOX and mutant GOD-3-1-2C

[0147] To investigate the stability of glucose oxidase GGOX and its mutant GOD-3-1-2C at different temperatures, purified samples were incubated at 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, and 90°C for 5 minutes. The relative enzyme activity of the unheated sample at 37°C and pH 5.5 was considered 100%. As shown in Figure 2, the parent glucose oxidase GGOX retained only 90% of its relative activity after incubation at 50°C for 5 minutes. However, GOD-3-1-2C retained >70% of its residual activity after incubation at 90°C for 5 minutes, >80% after incubation at 85°C for 5 minutes, and 100% after incubation at 80°C or below for 5 minutes, demonstrating significantly superior heat resistance to the parent GGOX.

[0148] The sequences involved in the examples herein are as follows:

[0149] Parent glucose oxidase GGOX, amino acid sequence, SEQ ID NO: 1

[0150] Glucose oxidase mutant GOD-3-1-2C with improved thermostability and specific activity, amino acid sequence, SEQ ID NO: 2:

[0151] Gene encoding a glucose oxidase mutant GOD-3-1-2C with improved thermostability and specific activity, nucleotide sequence, SEQ ID NO: 3

[0152] The embodiments of the present invention are not limited to the above embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and details, and these are all considered to fall within the scope of protection of the present invention.

Claims

1. A glucose oxidase mutant, which is a mutant of wild-type Aspergillus niger glucose oxidase, the mutant comprising the following amino acids: 10K, 14L, 16A, 20Y, 30A, 34V, 36E, 37K, 45T, 70E, 74T, 90R, 106I, 135S, 167L, 173V, 185L, 186M, 187R, 204L, 226A, 259A, 274S, 288S, 292T, 332S, 362T, 405K, 406D, 418G, 446R, 504V, 509E, 556L, 572A, 575V and 577A, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1, and the amino acid sequence of the wild-type Aspergillus niger glucose oxidase has at least 95% sequence identity with the sequence shown in SEQ ID NO:

1.

2. The glucose oxidase mutant of claim 1, further comprising at least one pair of introduced disulfide bonds, wherein the disulfide bonds are formed by one or more pairs of amino acids: amino acids at positions 53 and 246, amino acids at positions 25 and 250, amino acids at positions 39 and 242, and amino acids at positions 87 and 508.

3. The glucose oxidase mutant according to claim 2, wherein the disulfide bond is formed by the amino acids at positions 53 and 246.

4. A glucose oxidase mutant, which comprises the following mutations compared to the wild-type Aspergillus niger glucose oxidase shown in SEQ ID NO: 1: T10K, D14L, S16A, V20Y, T30A, T34V, A36E, R37K, S45T, D70E, S74T, Q90R, V106I, N135S, V167L, A173V, I185L, V186M, K187R, F204L, S226A, G259A, G274S, A288S, A292T, A332S, S362T, N405K, H406D, A418G, H446R, T504V, Y509E, M556L, S572A, I575V and E577A, wherein the amino acid positions correspond to SEQ ID NO: The glucose oxidase mutant has at least 90% sequence identity with the wild-type Aspergillus niger glucose oxidase shown in SEQ ID NO:

1.

5. The glucose oxidase mutant according to claim 4, further comprising one or more of the following mutations relative to the wild-type Aspergillus niger glucose oxidase shown in SEQ ID NO: 1: S53C and T246C, A25C and V250C, T39C and L242C, and T87C and P508C. 6 . The glucose oxidase mutant according to claim 5 , wherein the glucose oxidase mutant further comprises mutations of S53C and T246C relative to the wild-type Aspergillus niger glucose oxidase shown in SEQ ID NO:

1.

7. The glucose oxidase mutant according to claim 4, whose amino acid sequence is shown in SEQ ID NO:

2.

8. A polynucleotide encoding the glucose oxidase mutant according to any one of claims 1 to 7.

9. The polynucleotide of claim 8, comprising the sequence shown in SEQ ID NO:

3.

10. A recombinant expression vector comprising the polynucleotide according to claim 8 or 9.

11. A host cell comprising the polynucleotide of claim 8 or 9 or the recombinant expression vector of claim 10.

12. The host cell of claim 11, which is a bacterium or a fungus.

13. The host cell of claim 11, which is a filamentous fungus or a yeast.

14. The host cell of claim 11, which is Pichia pastoris or Aspergillus niger.

15. The method for preparing the glucose oxidase mutant according to any one of claims 1 to 7, comprising the following steps: (a) culturing the host cell according to any one of claims 11 to 14 under conditions suitable for the expression of the glucose oxidase mutant; (b) Recovery of the expressed glucose oxidase mutant.

16. A glucose oxidase mutant obtained by the preparation method according to claim 15.

17. A method for improving the thermal stability and enzyme activity of glucose oxidase, comprising: The wild-type Aspergillus niger glucose oxidase shown in NO:1 is subjected to the following mutations: T10K, D14L, S16A, V20Y, T30A, T34V, A36E, R37K, S45T, D70E, S74T, Q90R, V106I, N135S, V167L, A173V, I185L, V186M, K187R, F204L, S226A, G259A, G274S, A288S, A292T, A332S, S362T, N405K, H406D, A418G, H446R, T504V, Y509E, M556L, S572A, I575V and E577A, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO:

1.

18. The method of claim 17, further comprising introducing at least one pair of amino acid mutations capable of forming a disulfide bond into the wild-type Aspergillus niger glucose oxidase, wherein the mutations comprise one or more of the following groups of mutations: S53C and T246C, A25C and V250C, T39C and L242C, and T87C and P508C.

19. The method of claim 18, wherein the amino acid mutations capable of forming a disulfide bond are S53C and T246C.

20. Use of the glucose oxidase mutant according to any one of claims 1 to 7 in the fields of food, chemical industry, medicine, agriculture or feed.

Images