Glucose oxidase mutant and application thereof

Abstract

The invention provides a thermal-stability glucose oxidase mutant, polynucleotide for encoding the glucose oxidase mutant, a recombinant expression vector containing the polynucleotide, a host cell for expressing the glucose oxidase mutant, and applications of the thermal-stability glucose oxidase mutant, the polynucleotide, the recombinant expression vector and the host cell in the fields of food, chemical industry, medicine, agriculture or feed.

Description

[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 invention relates to the technical field of protein engineering, and 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 can use molecular oxygen as an electron acceptor to specifically catalyze β-D-glucose to produce gluconic acid and hydrogen peroxide. Among glucose oxidases, GOD from Aspergillus niger is the most widely studied. It is a glycosylated dimer protein with a molecular weight of approximately 160 kDa and uses FAD as a cofactor.

[0005] 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. Microorganisms are the main source of GOD because they grow and reproduce quickly and have a wide range of sources. At present, the industrial level of GOD production mainly uses Aspergillus niger and Penicillium as production strains. Compared with Penicillium GOD, the GOD expressed by Aspergillus niger has better thermal stability, but both have problems such as low enzyme activity and complex separation and purification.

[0006] GOD has a wide range of industrial applications in different fields such as food, pharmaceuticals, energy and textiles. As a food additive, GOD has significant antiseptic, antioxidant and stabilizer properties. The hydrogen peroxide produced by the action of GOD has antibacterial effects, and the subsequent decomposition of hydrogen peroxide leads to a net oxygen consumption with antioxidant and stabilizing effects. In the chemical industry, GOD is not only often used in biological bleaching, but also a key enzyme in the production of gluconic acid and its derivatives. In the medical field, GOD is a key raw material for glucose detection 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. With the continuous expansion of the application field of GOD, the market demand at home and abroad has increased sharply. However, GOD often needs to undergo a high temperature process during the production and processing process, which leads to a significant decrease in enzyme activity or even inactivation. For example, a short high temperature process during feed processing will cause GOD to be inactivated, thereby affecting the application effect of GOD. Thermal stability is very critical for the application of GOD. In the preparation process of the enzyme and under extreme high temperature reaction conditions, GOD with strong heat resistance has a relatively large advantage. Therefore, improving the thermal stability of GOD has important practical significance. Summary of the invention

[0007] The 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 beneficial to the application of the enzyme in industrial production.

[0008] 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, and the amino acid sequence of the wild-type Aspergillus niger glucose oxidase is the same as SEQ ID NO: The sequence shown in ID NO: 1 has a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

[0009] 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 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.

[0010] 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 SEQ ID NO: The glucose oxidase mutant has at least 90%, at least 91%, at least 92% or at least 93% sequence identity with the wild-type Aspergillus niger glucose oxidase shown in SEQ ID NO:1.

[0011] 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 shown 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 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 mutations of S53C and T246C relative to the parent glucose oxidase shown in SEQ ID NO: 1.

[0012] In some embodiments, the amino acid sequence of the glucose oxidase mutant is shown in SEQ ID NO:2.

[0013] In some embodiments, the present invention also provides a polynucleotide encoding the glucose oxidase mutant.

[0014] In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO:3.

[0015] In some embodiments, the present invention also provides a recombinant expression vector comprising the polynucleotide.

[0016] 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 a Pichia pastoris or an Aspergillus niger.

[0017] In some embodiments, the present invention also provides a method for preparing the above-mentioned glucose oxidase mutant, comprising the following steps:

[0018] (a) culturing the host cell of the present invention under conditions suitable for the expression of the glucose oxidase mutant;

[0019] (b) Recovery of the expressed glucose oxidase mutant.

[0020] In some embodiments, the present invention also relates to a glucose oxidase mutant obtained by the above preparation method.

[0021] In some embodiments, the present invention provides 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.

[0022] In some embodiments, the method for improving the thermal stability and specific activity of glucose oxidase further comprises introducing at least one pair of amino acid mutations that can form disulfide bonds into the wild-type Aspergillus niger glucose oxidase. In some embodiments, the introduction of at least one pair of mutations that can form 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 introduction of at least one pair of mutations that can form disulfide bonds comprises mutations of S53C and T246C.

[0023] In some embodiments, the present invention also provides the use of the above-mentioned glucose oxidase mutant in the fields of food, chemical industry, medicine, agriculture or feed.

[0024] The beneficial effects of the present invention are as follows: the glucose oxidase mutant GOD-3-1-2C provided by the present invention has significantly improved thermal stability and specific activity compared with 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 with the parent glucose oxidase. In terms of heat resistance, the residual enzyme activity of the parent glucose oxidase in a water bath at 80°C for 5 minutes is 0, while the residual enzyme activity of the glucose oxidase mutant of the present invention in a water bath at 80°C for 5 minutes is 100%. In particular, the residual enzyme activity of the glucose oxidase mutant of the present invention in a water bath at 85°C for 5 minutes is greater than 80%, and the residual enzyme activity in a water bath at 90°C for 5 minutes is greater than 70%, and its heat resistance is significantly better than the existing parent glucose oxidase. In addition, GOD-3-1-2C has higher acid resistance, and GOD-3-1-2C is more able to adapt to the acidic environment of the gastrointestinal tract of animals to function. 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

[0025] Figure 1 The optimal reaction pH of mutant GOD-3-1-2C and parent glucose oxidase GGOX is shown.

[0026] Figure 2 The thermal stability of mutant GOD-3-1-2C and parent glucose oxidase GGOX is shown. DETAILED DESCRIPTION

[0027] 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.

[0028] 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.

[0029] The terms "about" and "approximately" when used in conjunction 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., 95% confidence interval for the mean) or within a wider range of the specified value (e.g., ±5% or ±10%).

[0030] The term "comprise" or its variations such as "contains", "has", "includes" means including the stated steps or elements but not excluding any other steps or elements. "Consisting of" means not excluding steps or elements not listed. "Consisting essentially of" means not excluding steps or elements that do not substantially affect the basic and novel features of the invention being protected. The term "comprising" specific steps or elements and its variations also include "consisting of" and "consisting essentially of" specific steps or elements.

[0031] When a numerical range is mentioned, it should be deemed to specifically disclose the specific values ​​of its upper and lower limits, as well as all intermediate ranges included therein, such as the intermediate range between its upper or lower limit and any intermediate value, or the intermediate range between any two intermediate values ​​thereof. In addition, any intermediate range, subrange and all individual values ​​described in the numerical range can be excluded from the numerical range described.

[0032] The term "and / or" should be understood to refer to any one element or any combination of several elements connected by the term.

[0033] 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 an amino acid occupying a certain position with a different amino acid; deletion refers to removing an 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 activity level. For example, the mutant may remain unchanged or 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.

[0034] The term "thermostability" refers to the fact that glucose oxidase or its mutants still maintain a certain amount of enzymatic activity after a period of time at a specific temperature. When it comes to properties such as thermostability, improved thermostability means that the residual enzymatic activity of glucose oxidase or its mutants after a period of time at a specific temperature is higher than that of other glucose oxidase mutants and / or parent glucose oxidase. The term "residual enzymatic activity" refers to the percentage of the enzymatic activity of glucose oxidase or its mutants after a period of time at a specific temperature relative to the enzymatic activity of the enzyme without corresponding treatment.

[0035] The term "low pH stability" refers to the glucose oxidase or its mutants still maintaining a certain amount of enzymatic activity at a relatively low pH. When it comes to properties such as low pH stability, improved low pH stability means that the relative enzymatic activity of the glucose oxidase or its mutants at a relatively low pH is higher than that of other glucose oxidase mutants and / or parent glucose oxidase. The relative enzymatic activity at a relatively low pH refers to the percentage of the enzymatic activity of the enzyme under relatively low pH conditions relative to the enzymatic activity at a commonly used pH or at an optimal pH.

[0036] 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.

[0037] The term "wild-type" enzyme refers to an enzyme expressed by a naturally occurring organism or cell (eg, bacteria or fungi). The "naturally occurring" means that it has not been artificially induced by mutagenesis or genetic manipulation.

[0038] 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 expresses native genes, but these native genes are otherwise abnormally expressed, under-expressed or not expressed at all.

[0039] 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.

[0040] 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.

[0041] 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 the polynucleotide is operably linked to additional nucleotides (eg, control sequences) that provide for its expression.

[0042] 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.

[0043] The term "control sequence" refers to a nucleic acid sequence necessary for the expression of a polynucleotide encoding a glucose oxidase mutant of the present invention. Each control sequence may 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. The control sequence includes at least a promoter and transcription and translation termination signals.

[0044] 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.

[0045] 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.

[0046] The term "sequence identity" is used to describe the correlation between two amino acid sequences or between two nucleotide sequences."Sequence identity" refers to the sequence identity percentage value determined by performing optimal alignment (i.e., making the sequence identity the maximum) on the comparison window for two sequences, and the two sequences compared can achieve optimal alignment by addition or deletion (i.e., gap). In some algorithms, the sequence identity percentage can be calculated in the following manner: determine the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences under the 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 the length of the gap 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, such as 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 EuropeanMolecular 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) marked as "longest consistency" is used as percentage consistency and calculated as follows: (consistent residue × 100) / (total number of gaps in comparison length-comparison).

[0047] "The position corresponding to..." is the position corresponding to a specific position in the reference amino acid sequence when the queried amino acid sequence is optimally aligned with the reference amino acid sequence. In the present invention, the positions of the amino acids in the described glucose oxidase mutants are determined based on the amino acid sequence shown in the reference amino acid sequence 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, thereby determining the amino acid position corresponding to any amino acid in SEQ ID NO: 1.

[0048] Method for representing mutants of the present invention: Amino acids are represented by conventional single-letter or three-letter nomenclature. As known to those skilled in the art, the single-letter nomenclature of 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 in the form of "T10K", wherein 10 means that the substitution occurs at the amino acid at position 10, the T before 10 is the amino acid occupying the position before the substitution occurs, and the K after 10 is the amino acid occupying the position after the substitution occurs. Herein, a specific amino acid can be represented in the form of "10K", which means that the amino acid at position 10 is K.

[0049] Unless otherwise indicated, nucleic acids are written herein from left to right in 5' to 3' orientation and amino acid sequences are written from left to right in amino to carboxyl orientation.

[0050] 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.

[0051] In particular, the glucose oxidase mutant of the present invention shows significantly improved thermal stability and enzyme activity compared with the parent glucose oxidase, and these characteristics make it particularly useful in industrial production and can be widely used in the fields of food, feed, chemical production, medical treatment, pharmaceuticals, energy, textiles, etc. For example, it can be used as a food or feed additive, and can also be used to produce chemical and chemical substances such as hydrogen peroxide, gluconic acid and its derivatives, and can also be used for biological bleaching, and can also be used as a toothpaste additive to reduce the incidence of oral diseases, and can also be used as a component of immunoassays and biosensors in the field of medical diagnosis, for example, for the preparation of glucose detection kits, and can also be used to manufacture micro biofuel cells that can power biomedical implants such as biosensors and insulin pumps.

[0052] 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.

[0053] 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. 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 with the sequence shown in SEQ ID NO: 1. In some embodiments, except for the amino acids at the above 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.

[0054] In some embodiments, the glucose oxidase mutant further comprises at least one pair of introduced disulfide bonds relative to the parent glucose oxidase. 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. In some embodiments, the amino acid capable of forming disulfide bonds is cysteine ​​or homocysteine. In some embodiments, the glucose oxidase mutant further comprises two amino acid mutations relative to the parent glucose oxidase, and the two amino acids are mutated to amino acids capable of forming disulfide bonds.

[0055] For the purpose of the present invention, "introduction" does not limit the disulfide bond to be generated in any specific manner. For example, "introduction" of a disulfide bond can include replacing the amino acid at the corresponding position of the glucose oxidase sequence 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 the situation where any one or two amino acid residues forming the disulfide bond are due to natural mutations.

[0056] In some embodiments, the disulfide bond is 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. 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 that can form disulfide bonds, 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.

[0057] In some embodiments, the glucose oxidase mutants of the present invention are identical to those of SEQ ID Compared with the wild-type Aspergillus niger glucose oxidase shown in NO:1, it includes 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 with the wild-type Aspergillus niger glucose oxidase shown in SEQ ID NO: 1. In some embodiments, the glucose oxidase mutant only includes the above mutations compared to the wild-type Aspergillus niger glucose oxidase shown in SEQ ID NO: 1.

[0058] 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 acid capable of forming disulfide bonds is cysteine ​​or homocysteine. In some embodiments, the glucose oxidase mutant further comprises mutations of two amino acids relative to the parent glucose oxidase, and the two amino acids are mutated to amino acids capable of forming disulfide bonds.

[0059] 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 mutations of S53C and T246C relative to the parent glucose oxidase shown in SEQ ID NO: 1.

[0060] In some embodiments, the glucose oxidase mutant is identical to 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.

[0061] 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.

[0062] In some embodiments, the glucose oxidase mutant of the present invention has improved enzyme activity, improved thermal stability and / or improved low pH stability (especially stability under pH 3-5.5 conditions) compared with the parent glucose oxidase or other glucose oxidase mutants. The thermal stability refers to stability under high temperature conditions, and the "high temperature" refers to 70°C-100°C, preferably 80°C-95°C. The "low pH" refers to pH 3-5.5.

[0063] In some embodiments, the enzyme activity of the glucose oxidase mutant 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 enzyme activity can be detected, for example, by o-dianisidine-horseradish peroxidase coupled spectrophotometry, based on the change in absorbance at 540nm and a standard curve. In some embodiments, the enzyme activity is determined at 37°C and pH 5.5. The "o-dianisidine-horseradish peroxidase coupled spectrophotometry" refers to the process of catalyzing glucose dehydrogenation to produce H2O2 by glucose oxidase under aerobic conditions, and oxidizing the oxygen donor o-dianisidine (DH2) under the action of horseradish peroxidase (POD) to obtain a brown product, and converting the activity of glucose oxidase based on the change in absorbance at 540nm and the standard curve.

[0064] In some embodiments, the glucose oxidase mutants of the present invention have a higher residual enzyme activity under high temperature conditions, particularly at 80°C-95°C, than 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 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 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 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 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 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 the parent glucose oxidase or other glucose oxidase mutants.

[0065] 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 accordingly, and the enzyme activity before and after the treatment is determined by the same method. In some embodiments, the determination of the residual enzyme activity after the above heat treatment is carried out at pH 5.5, and the determination of the enzyme activity of the mutant that has not undergone the corresponding treatment is carried out at 37° C. and pH 5.5.

[0066] In some embodiments, the optimal pH of the glucose oxidase mutant of the present invention is about 5.5. The "optimal pH" refers to the pH value at which the enzyme exhibits maximum activity when other assay conditions are the same.

[0067] In some embodiments, the relative enzyme activity of the glucose oxidase mutant of the present invention under the conditions of pH 3-5.5 is higher than that of the parent glucose oxidase or other glucose oxidase mutants. The relative enzyme activity under specific pH conditions refers to the percentage of the enzyme activity of the same enzyme under the pH conditions relative to the enzyme activity under the optimal pH conditions. When the relative enzyme activity under specific pH conditions is determined, except for the pH, the other determination conditions are the same. In some embodiments, the relative enzyme activity under specific pH conditions is determined at 37°C.

[0068] The present invention also relates to a method for improving the thermal stability and enzyme activity of glucose oxidase, comprising performing the above mutation on wild-type Aspergillus niger glucose oxidase. The above mutation can be performed using any method known in the art, such as site-directed mutagenesis, homologous recombination, etc.

[0069] 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 a sequence as shown in SEQ ID NO: 3. The polynucleotide may further comprise a coding sequence of a signal peptide so that the mutant is secreted into the extracellular space after expression.

[0070] 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, and the control sequence can direct the expression of the coding sequence of the glucose oxidase mutant in a suitable host cell.

[0071] Control sequences include, but are not limited to, promoters, terminators, leader sequences, polyadenylation sequences, etc. Constitutive promoters or inducible promoters can be used. Control sequences suitable for different host cells are well known to those skilled in the art.

[0072] The expression vector can be an autonomously replicating vector, i.e., self-replicating independently of the chromosome, such as a plasmid or an artificial chromosome, or can be a vector that is integrated into the genome and replicated together with the chromosome when introduced into the host cell. The expression vector can contain one or more selectable markers that allow convenient selection of transformed cells, transfected cells, and transduced cells, and the marker can, for example, provide resistance to antibiotics, heavy metals, etc. The expression vector can also contain a replication origin 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 the method for constructing the recombinant expression vector of the present invention with these elements is 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.

[0073] The present invention also relates to a recombinant host cell for expressing the glucose oxidase mutant 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 (for example, 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. Yeast can be, for example, a methanol-nutritional yeast, for example, Pichia or Komagataella, such as Pichia pastoris or Komagataella pastoris. In some embodiments, Pichia pastoris X-33 is used as a host cell. The method of introducing a polynucleotide into a host cell is well known to those skilled in the art, such as electroporation, gene gun, etc.

[0074] 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.

[0075] In some embodiments, step (a) comprises inducing the expression of the glucose oxidase mutant. The inducer used depends on the control sequence (e.g., promoter) used to direct the 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).

[0076] 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 a 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.

[0077] 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 an inorganic salt can be used to cultivate the host cell, and growth conditions such as pH, temperature, oxygen and / or nutrient supply can be controlled. Can be cultured in a shake flask or in a laboratory or industrial fermenter, the host cell is subjected to fermentation culture, such as continuous fermentation, batch fermentation, fed-batch fermentation or solid fermentation culture. In some embodiments, the host cell is cultivated in a fermenter. When an inducible promoter (e.g., alcohol oxidase promoter AOX1) is used, methanol can be added during the culture to induce the mutant to express.

[0078] 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 lysing the cells.

[0079] 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 in the cell, and the cell can be crushed to release the target product from the cell. The crushing process can include, for example, pressurization, osmotic shock, freezing, ultrasound or other treatments to produce a cell lysate, and the cell lysate is 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 substantially pure mutants, 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).

[0080] In some embodiments, the glucose oxidase mutant may not be recovered from the host cells, and the host cells may be directly used as the 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 or drum drying or any combination thereof, for further use.

[0081] The generated glucose oxidase mutant can be detected by various methods known in the art. For example, the activity of the glucose oxidase mutant can be detected by enzyme activity assay, such as the activity of catalyzing glucose dehydrogenation to produce H2O2, for example, by o-dianisidine-horseradish peroxidase coupled spectrophotometry, according to the change in absorbance at 540nm and a standard curve.

[0082] The glucose oxidase mutant of the present invention has high enzyme activity and good thermal stability and stability under low pH conditions, and can be widely used in multiple fields, such as food, chemical, pharmaceutical, agricultural or feed fields. The application of glucose oxidase in these fields is known, and the glucose oxidase of the present invention has good performance, so that its application in these fields will achieve better results than other glucose oxidase mutants or parent glucose oxidase.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] In some embodiments, the use of the glucose oxidase mutants of the present invention is for non-therapeutic and / or non-diagnostic purposes.

[0088] The present invention is further described by the following examples, which should not be construed as limiting the present invention.

[0089] Unless otherwise specified, all reagents used in the following examples are commercially available products. Molecular biology experimental methods not specifically described in the examples are performed with reference to the specific methods listed in J. Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition, or in accordance with the kits and product instructions.

[0090] The experimental materials and reagents used in the following examples are as follows:

[0091] 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 the expression plasmid were maintained in our laboratory.

[0092] 2. Enzymes and other biochemical reagents: Endonucleases were purchased from Fermentas, ligases were purchased from Promaga, and DNA polymerases were purchased from Beijing Quanshijin Biotechnology. Plasmid extraction kits and purification kits were purchased from Shanghai Shenggong Company. Antibiotic Zeocin was purchased from Invitrogen, and the others were domestic analytical reagents (all available from ordinary biochemical reagent companies).

[0093] 3. Culture medium:

[0094] LB solid medium: 0.5% yeast extract, 1% peptone, 1% NaCl, 1% agar powder, pH 7.0.

[0095] LB-A resistance medium is LB medium with ampicillin added to a final concentration of 100 μg / mL.

[0096] LB-Z resistance medium is LB medium with Zeocin added to a final concentration of 25 μg / mL.

[0097] YPD medium: 1% yeast extract, 2% peptone, 2% glucose.

[0098] YPD-Z resistance medium is YPD medium with Zeocin added to a final concentration of 100 μg / mL.

[0099] Induction medium BMGY: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 1% glycerol (v / v)).

[0100] Induction medium BMMY: 0.5% methanol replaces glycerol, and the rest of the ingredients are the same as BMGY.

[0101] 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% defoaming agent, add 4.35mL PTM1 per liter after sterilization.

[0102] 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%.

[0103] 4. Determination of glucose oxidase activity

[0104] 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 to a brown product. The activity of GOD can be converted based on 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 2min of incubation at 37℃, 0.1mL of the diluted enzyme solution sample is added to the test tube. After 3min of reaction, 2mol / L sulfuric acid is added to terminate the reaction, the test tube is taken out, and the absorbance value of OD540 is measured. The heat-inactivated enzyme solution is used as a blank control. According to the results of the standard curve, the glucose oxidase activity unit is calculated.

[0105] Reagents and Solutions:

[0106] 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 them in 400mL distilled water, and adjust the pH to 5.5 with disodium hydrogen phosphate.

[0107] o-Dianisidine solution: Accurately weigh 0.1 g of o-Dianisidine and dissolve it in 10 mL of methanol. This is the storage solution, which is effective for 3 days at 4°C. Before the experiment, take 0.1 mL of the storage solution and dissolve it in 12 mL of the above 0.1 mol / L, pH 5.5 phosphate buffer.

[0108] 18% glucose: Accurately weigh 9.00 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.

[0109] 2mol / L H2SO4: Accurately weigh 40.00g H2SO4 and slowly add it into 160mL distilled water to make up to 200mL.

[0110] 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.

[0111] 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.

[0112] Determination of enzyme activity:

[0113] (1) Preparation of standard curve

[0114] Dilute the 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 37°C for 2 min, add 0.1 mL of the 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.

[0115] (2) Sample determination

[0116] 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 37℃ 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. After accurate reaction for 3min, 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.

[0117] (3) Calculation of enzyme activity

[0118] X=(K*A+b)*n

[0119] X: Enzyme activity of the sample U / mL, A: Absorbance of the sample, n: Dilution multiple of the enzyme solution, K: Slope of the standard curve, b: Intercept of the standard curve.

[0120] Example 1 Parent Glucose Oxidase GGOX Gene Synthesis and Vector Construction

[0121] The parent glucose oxidase GGOX is a mature form of glucose oxidase (GenBank: EHA27180.1) derived from Aspergillus nigerATCC 1015, and its amino acid sequence is shown in SEQ ID NO: 1. The glucose oxidase gene was synthesized by a total gene synthesis method, and the gene synthesis 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, the product was purified and recovered, and connected to the expression vector pPICZαA, and the connection product was transferred into Escherichia coli Top10 competent cells by chemical conversion heat shock method, coated with LB-Z resistance plate, and inverted cultured at 37°C. After the transformant appeared on the plate, a single clone was picked and inoculated into an LB-Z resistance test tube and cultured at 37°C. The recombinant transformant was verified by bacterial liquid PCR, and the plasmid of the correct transformant was extracted and sequenced to obtain the expression vector pPICZαA-GGOX.

[0122] Example 2 Random mutation of glucose oxidase GGOX error-prone PCR

[0123] Using the above pPICZαA-GGOX as a template, mutations were randomly introduced using the GeneMorph II Random Mutation PCR Kit (Stratagene).

[0124] The primers used were:

[0125] GGOX-F 5'-CCG GAATTC TCCAACGGTATTGAGGCCTCTTTG-3'(SEQ ID NO:4)

[0126] GGOX-R 5'-CTAG TCTAGA TTACTGCATGGAAGCGTAGTCAGC-3'(SEQ ID NO:5)

[0127] The underlined parts are EcoRI and XbaI restriction sites, respectively.

[0128] The reaction conditions were: 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.

[0129] The PCR amplification results were detected by agarose electrophoresis, and the target product of PCR amplification was purified and recovered. The target fragment was digested with EcoRI and XbaI and then connected to the pPICZαA vector. The ligation product was transferred into Escherichia coli Top10 competent cells by chemical transformation heat shock method, coated with LB-Z resistance plate, and inverted cultured at 37°C. After the transformants appeared on the plate, a single clone was picked and inoculated into LB-Z resistance test tube and cultured at 37°C. The recombinant transformants were verified by bacterial liquid PCR, and the plasmids of the correct transformants were extracted and sequenced to determine the corresponding mutants. The mutant plasmids with correct sequencing were linearized with restriction endonuclease PmeI, and the linearized plasmid fragments were recovered and purified. They were transferred into Pichia pastoris X33 competent cells by electrotransformation, and screened with YPD-Z resistance medium to obtain yeast recombinant transformants.

[0130] Preparation of Pichia pastoris X-33 competent cells: 1) Inoculate a single colony of Pichia pastoris X-33 into 5 mL YPD medium and culture at 30°C and 250 rpm / min for 24 h; 2) Take 1 mL of culture solution and inoculate it into a 500 mL shake flask containing 100 mL YPD medium and culture for 12-16 h. When OD600 = 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 min and discard the supernatant; 3) Resuspend the cells with 25 mL of pre-cooled sterile water, centrifuge at 4°C and 5000 g for 5 min, discard the supernatant, and repeat this step 2-3 times; 4) Resuspend the cells with 5 mL of pre-cooled 1 mol / L sorbitol in each tube, centrifuge at 4°C and 5000 g for 5 min, discard the supernatant, and repeat this step 2-3 times; 5) Finally, resuspend with 200 μL of sorbitol and divide into 1.5 mL centrifuge tubes, 80 μL per tube.

[0131] Transformation screening: 1) Take a 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 place on ice for 5 minutes; 2) Perform electric shock at the electric shock parameters of 1.5Kv, 25μF, 200Ω. After the electric shock is completed, quickly add 1mL of pre-cooled 1mol / L sorbitol, and then transfer the mixed solution to a 1.5mL centrifuge tube and resuscitate at 30℃ for 1h; 3) Take 200μL and spread it on YPD-Z solid culture medium containing 100μg / ml Zeocin, culture the plate at 30℃ for 3 days, observe positive transformants, use PCR to identify positive single colonies, and send PCR positive colonies for sequencing verification.

[0132] Example 3 High-throughput screening of high specific activity mutant strains

[0133] 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 remove the supernatant by centrifugation. Then add 1.6 mL of BMMY medium to each well for induction culture. Add 1% methanol of the culture volume 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 assay the glucose oxidase activity 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.

[0134] Example 4 High-throughput screening of glucose oxidase mutants with improved heat resistance

[0135] The yeast recombinant transformants with enzyme activity close to or improved to the parent glucose oxidase GGOX obtained in Example 3 were picked one by one with toothpicks to a 24-well plate, and 1 mL of BMGY medium was added to each well in advance, and cultured at 30 ° C, 200 rpm for about 24 hours, and the supernatant was removed by centrifugation. Then 1.6 mL of BMMY medium was added to each well for induction culture. 1% of the culture volume of methanol was added every 24 hours. After induced expression for 4 days, the culture solution was centrifuged to obtain the supernatant, and 100 μL of the above supernatant was taken out for glucose oxidase enzyme activity determination and heat treatment residual enzyme activity determination. Through multiple rounds of screening and comparison, mutants with significantly improved heat resistance relative to the parent glucose oxidase GGOX were screened out.

[0136] Example 5 Combination mutation and screening

[0137] In Example 3 and Example 4, based on the specific activity or heat-resistant forward mutation site, double-site or multi-site combined mutation is performed, and the combined mutant sequence is fully gene-synthesized by gene synthesis. The combined mutant vector construction is completed in the manner of Example 1. The combined mutant plasmid with correct sequencing is linearized with restriction endonuclease PmeI, the purified linear plasmid fragment is recovered, and the electroporation method is used to transfer it into Pichia pastoris X33 competent cells, and YPD-Z resistance medium is used for screening to obtain yeast recombinant transformants. The recombinant transformants are picked and transferred to 50mL BMGY medium respectively, and after shaking and culturing at 30°C and 220rpm for 18h, the cells are obtained by centrifugation, and the appropriate amount of cells are transferred to 50mL BMMY medium to make the cell concentration reach OD600=1, and the shaking culture is continued at 30°C and 250rpm, and 1% methanol of the culture volume is added every 24h. After induced expression for 4 days, 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, and 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, and the mutant was named GOD-3-1. The specific activity determination results are shown in Table 1. The purified samples were diluted to about 100 U / mL with pH 5.5 phosphate buffer, and the residual enzyme activity was determined after being treated for 5 min at different temperatures. The relative enzyme activity was calculated with the enzyme activity of the untreated sample as 100%. The results are shown in Table 2.

[0138] Example 6: Combination mutant GOD-3-1 introduces disulfide bonds to further improve heat resistance

[0139] Based on the amino acid sequence of mutant GOD-3-1, disulfide bonds S53C and T246C were designed and introduced, and finally a new combined mutant GOD-3-1-2C was obtained. The mutant vector was constructed and transformed into Pichia pastoris X33 in the manner of Example 5, and the mutant GOD-3-1-2C was purified by nickel affinity chromatography purification method. The purified sample was subjected to glucose oxidase activity determination and specific activity was calculated. The results are shown in Table 1. The purified sample was diluted to about 100 U / mL with pH 5.5 phosphate buffer, and the residual enzyme activity was determined after being treated at 80°C, 85°C and 90°C for 5 minutes, respectively. The enzyme activity of the untreated sample was taken as 100%, and the relative enzyme activity was calculated. The results are shown in Table 2.

[0140] As can be seen from Table 1, there is no significant difference between the specific activities of mutants GOD-3-1-2C and GOD-3-1. Compared with the parent glucose oxidase GGOX, the specific activities of the two mutants are significantly improved, and the relative specific activity reaches 190%. As can be seen from Table 2, after the introduction of disulfide bonds on the basis of mutant GOD-3-1, the thermal stability of the new mutant GOD-3-1-2C is further improved. The residual enzyme activity of the parent glucose oxidase is 0 after 5 minutes of water bath at 80°C and above, while the thermal stability of the glucose oxidase GOD-3-1 of the present invention is significantly improved. After 5 minutes of water bath at 80°C, the residual enzyme activity reaches 88%, while the mutant GOD-3-1-2C shows better heat resistance, and the residual enzyme activity reaches 100%. After 5 minutes of water bath at 85°C, the residual enzyme activity of the mutant GOD-3-1-2C reaches 85%, and after 5 minutes of water bath at 90°C, the residual enzyme activity of the mutant GOD-3-1-2C reaches 74%. Therefore, compared with the parent glucose oxidase, the mutant GOD-3-1-2C exhibited excellent heat resistance.

[0141] Table 1 Comparison of specific activities of parent glucose oxidase GGOX and combined mutants

[0142] Sample name Relative specific activity (%) GGOX 100 GOD-3-1 193.33±5.73 GOD-3-1-2C 190.27±5.54

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

[0144]

[0145] Example 7 Determination of the optimal reaction pH of the parent glucose oxidase GGOX and the mutant GOD-3-1-2C

[0146] The enzyme activity of glucose oxidase was measured at 37°C in various buffers (sodium hydrogen phosphate-citric acid buffer pH 3.0, 3.5, 4.0, 4.5, 5.0, phosphate buffer pH 5.5, 6.0, 6.5, 7.0, 7.5). The results were expressed as relative activity with the pH value showing the maximum activity value as 100%. Figure 1 As shown, the optimal reaction pH of the parent glucose oxidase GGOX and the mutant GOD-3-1-2C is around 5.5. The relative activity of the mutant GOD-3-1-2C is higher than that of the parent glucose oxidase GGOX under pH 3-5.5 conditions, which indicates that GOD-3-1-2C has higher acid resistance and is more able to adapt to the acidic environment of the animal gastrointestinal tract to function.

[0147] Example 8 Determination of thermal stability of parent glucose oxidase GGOX and mutant GOD-3-1-2C

[0148] In order to study the stability of glucose oxidase GGOX and mutant GOD-3-1-2C at different temperatures, the purified samples were placed 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 min. The samples at 37°C and pH 5.5 were taken as the unheat-treated samples, and the relative enzyme activity of the unheat-treated samples was 100%. The results are shown in Table 1. Figure 2 As shown in the figure, the parent glucose oxidase GGOX only retained 90% of the relative enzyme activity after being placed at 50°C for 5 minutes. However, after being placed at 90°C for 5 minutes, the residual enzyme activity of GOD-3-1-2C can still be maintained at more than 70%; after being placed at 85°C for 5 minutes, the residual enzyme activity can still be maintained at more than 80%; after being placed at 80°C and below for 5 minutes, the residual enzyme activity can be maintained at 100%, and its heat resistance is significantly better than that of the parent GGOX.

[0149] The sequences involved in the embodiments of this article are as follows:

[0150] Parent glucose oxidase GGOX, amino acid sequence, SEQ ID NO: 1

[0151]

[0152] Glucose oxidase mutant GOD-3-1-2C with improved thermal stability and specific activity, amino acid sequence, SEQ ID NO: 2:

[0153]

[0154] Gene encoding a glucose oxidase mutant GOD-3-1-2C with improved thermal stability and specific activity, nucleotide sequence, SEQ ID NO: 3

[0155]

[0156]

[0157] The implementation of the present invention is 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 detail, and these are considered to fall within the protection scope 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.

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