Glucose oxidase mutant with synchronously improved thermal stability and acid tolerance and antibacterial application thereof

Abstract

The invention discloses a glucose oxidase mutant with synchronously improved thermal stability and acid tolerance and antibacterial application of the glucose oxidase mutant, and belongs to the field of gene engineering and protein engineering. According to the present invention, glucose oxidase AiGODL is adopted as a female parent, and Glu148 and Phe283 are respectively mutated into Lys148 and Tyr283 so as to obtain the mutant AiGODDE148K / F283Y; experiments prove that compared with wild type glucose oxidase AiGODL, the mutant AiGODDE148K / F283Y has the advantages that the heat resistance and the acid resistance are improved, the specific activity and the catalytic efficiency of the mutant AiGODL are the same as those of the wild type glucose oxidase AiGODL, and the antibacterial property of the mutant AiGODL is equivalent to that of ampicillin. The mutant AiGODDE148K / F283Y provided by the invention has a huge application prospect in the aspects of antibiosis and antibiotic substitution.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and protein engineering, and in particular to a glucose oxidase mutant with synchronously improved thermal stability and acid tolerance and its antibacterial application. Background Art

[0002] Glucose oxidase (GOD, EC 1.1.3.4) is a flavoprotein whose active center contains a non-covalently bound flavin adenine dinucleotide (FAD) coenzyme. During the catalytic process, FAD acts as an electron carrier, with molecular oxygen as the final electron acceptor, oxidizing β-d-glucose to d-glucose-δ-lactone and generating hydrogen peroxide. Due to this unique catalytic mechanism, GOD has a wide range of applications in the chemical, food, pharmaceutical, and clinical fields, including glucose sensors, food additives, and gluconic acid production. Industrial enzymes must meet three key requirements: high catalytic activity, heat resistance, and acid resistance. Therefore, optimizing enzyme performance through protein engineering has become a research hotspot.

[0003] Protein engineering primarily involves two strategies: directed evolution and rational design. Directed evolution does not rely on protein structural information, but requires the establishment of an efficient screening system, which is labor-intensive and costly. With the advancement of structural biology, rational design based on protein three-dimensional structure is becoming increasingly mature, particularly in improving stability, activity, and environmental tolerance. Currently, commonly used modification strategies, including the introduction of disulfide bonds, optimization of hydrogen bond networks, and hydrophobic cores, have achieved some success but are still unable to fully meet industrial needs.

[0004] It is worth noting that most modifications that improve thermal stability are often accompanied by a decrease in enzyme activity. Therefore, it is crucial to maintain catalytic activity while improving stability. In recent years, computer-aided design has shown great potential in enzyme engineering. Protein stability is closely related to its free energy change (ΔG). By calculating the free energy change (ΔΔG) before and after mutation, changes in thermal stability can be predicted. Molecular dynamics simulation can effectively identify key residues. For example, after modification, the melting point temperature (T m ) increased by 11.7°C and 3.6°C, respectively, verifying the feasibility of this strategy. Therefore, the development of new GODs with high catalytic activity, heat resistance, and acid resistance based on computer-aided design is of great significance for meeting the demand for industrial enzymes. Summary of the Invention

[0005] The purpose of the present invention is to provide a glucose oxidase mutant with simultaneously improved thermal stability and acid tolerance and its antibacterial application, so as to solve the problems existing in the above-mentioned prior art. By performing site-directed mutagenesis on the key amino acid sites Glu148 and Phe283 of AiGODL from Aspergillus, a glucose oxidase mutant with improved thermal stability and acid tolerance was obtained after screening. The mutant has antibacterial activity equivalent to that of antibiotics and has important application and promotion value in antibacterial and antibiotic substitution.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a glucose oxidase mutant with simultaneously improved thermal stability and acid tolerance. The glucose oxidase mutant is obtained by mutating the Glu148 and Phe283 sites of the wild-type glucose oxidase to Lys148 and Tyr283, respectively. The amino acid sequence of the glucose oxidase mutant is shown in SEQ ID NO.2.

[0008] The present invention also provides a gene encoding the glucose oxidase mutant, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0009] The present invention also provides a recombinant vector comprising the gene.

[0010] Preferably, the expression vector pPIC9γ is connected to the target gene to construct a recombinant vector.

[0011] The present invention also provides a recombinant bacterium comprising the recombinant vector.

[0012] Preferably, the starting strain of the recombinant bacteria is Pichia pastoris, more preferably Pichia pastoris GS115.

[0013] The present invention also provides the use of the glucose oxidase mutant, the gene, the recombinant vector or the recombinant bacteria in the preparation of antibacterial agents.

[0014] Preferably, the antimicrobial agent is an agent that inhibits Escherichia coli.

[0015] The present invention also provides an antibacterial agent, the main active ingredient of which is the glucose oxidase mutant.

[0016] Preferably, the antimicrobial agent is an agent that inhibits Escherichia coli.

[0017] The present invention discloses the following technical effects:

[0018] The present invention uses glucose oxidase AiGODL from Aspergillus as the parent, and simultaneously mutates amino acid residues Glu148 and Phe283 by computer-aided site-directed mutagenesis to obtain a mutant. Specifically, a recombinant strain containing the mutant is constructed, and after induction culture, a glucose oxidase mutant AiGODL_E148K / F283Y with improved thermal stability and acid tolerance is screened out. In terms of thermal stability, the optimal temperature (40°C) of the mutant AiGODL_E148K / F283Y is 5°C higher than that of the wild-type AiGODL (35°C), and T 50 The saturation temperature and half-life at 70°C were increased by 9°C and 12 min, respectively, compared with the wild type (64°C and 25 min for the mutant vs. 55°C and 13 min for the wild type). In terms of pH performance, compared with the wild type AiGODL, the optimal pH of the mutant AiGODL_E148K / F283Y was pH 5.5, which was 1 pH unit shifted towards the acidic environment compared with the wild type (pH 6.0), and the relative enzyme activity in the pH 2.0-5.0 range was increased by 19%-74% compared with the wild type. In terms of pH tolerance, the residual enzyme activity of the mutant AiGODL_E148K / F283Y in acidic environment (pH 1.0 and pH 2.0) was 18% and 35%, respectively, which was 200% and 84% higher than that of the wild type (6% and 19%). The present invention uses computer-aided design and modified target scoring to obtain a mutant AiGODL_E148K / F283Y with enhanced thermal stability and acid tolerance. The mutant AiGODL_E148K / F283Y with high thermal stability and acid tolerance shows great application prospects in antibacterial and antibiotic substitution. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 The polyacrylamide gel electrophoresis of glucose oxidase AiGODL and its mutant AiGODL_E148K / F283Y; wherein M is a marker, 1 and 2 are AiGODL and AiGODL_E148K / F283Y, respectively;

[0021] Figure 2 The results of the optimal pH determination of glucose oxidase AiGODL and its mutant AiGODL_E148K / F283Y are shown;

[0022] Figure 3 The results of pH stability test of glucose oxidase AiGODL and its mutant AiGODL_E148K / F283Y are shown;

[0023] Figure 4 The results of the optimal temperature determination of glucose oxidase AiGODL and its mutant AiGODL_E148K / F283Y;

[0024] Figure 5 T is the T of glucose oxidase AiGODL and its mutant AiGODL_E148K / F283Y 50 Measurement results;

[0025] Figure 6 is the half-life of glucose oxidase AiGODL and its mutant AiGODL_E148K / F283Y at 70℃ (t 1 / 2 ) measurement results;

[0026] Figure 7 These are the results of antibacterial activity determination of glucose oxidase AiGODL and its mutant AiGODL_E148K / F283Y. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The test materials used in the following examples are:

[0033] 1. Strains and vectors: The expression host is Pichia pastoris GS115, and the expression plasmid vector pPIC9γ is prepared in the laboratory.

[0034] 2. Enzymes and other biochemical reagents: Taq enzyme and endonuclease were purchased from Quanshijin Company, o-dianisidine and peroxidase were purchased from Sigma Company, and the others were domestic analytical grade reagents (all purchased from Sinopharm Group).

[0035] 3. Culture medium:

[0036] (1) YPD medium: 1% yeast extract, 2% peptone, and 2% glucose.

[0037] (2) MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00005% Biotin.

[0038] (3) BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (V / V), 1.34% YNB, 0.00005% Biotin.

[0039] (4) LB medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0.

[0040] (5) MM solid medium: 1.5% agarose, 1.34% YNB, 0.00005% Biotin, 0.5% methanol.

[0041] (6) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00005% Biotin, 0.5% methanol (V / V).

[0042] Example 1: Synthesis of Glucose Oxidase Mutant Gene and Acquisition of Recombinant Plasmid

[0043] To improve the enzymatic performance of Aspergillus spp. glucose oxidase AiGODL, molecular dynamics simulations and computer-aided design were used to analyze key differential amino acids, score modification targets, and screen mutants. Using the recombinant expression vector pPic9γ-AiGODL containing the glucose oxidase gene AiGODL (nucleotide sequence shown in SEQ ID NO. 3, amino acid sequence shown in SEQ ID NO. 1) as a template, site-directed mutagenesis was used to mutate Glu148 and Phe283 to Lys and Tyr, respectively. The resulting mutant, named AiGODL_E148K / F283Y, has the nucleotide sequence shown in SEQ ID NO. 4. Primer design is shown in Table 1. Mutation and cloning methods are based on the literature (Characterization, stability improvement, and bread baking applications of a novel cold-adapted glucose oxidase from Cladosporium neopsychrotolerans SL16. Ge et al., 2020).

[0044] Table 1 Glucose oxidase AiGODL site-directed mutagenesis primers

[0045]

[0046] Example 2: Preparation of recombinant heat-resistant and acid-resistant glucose oxidase mutants

[0047] The linear recombinant expression vector obtained by PCR in Example 1 was directly transformed into DMT competent cells, and colony PCR was performed to verify the nucleic acid sequence of the target site mutant. The recombinant plasmid was linearized and transformed into Pichia pastoris GS115 to obtain the recombinant yeast strain GS115 / AiGODL_E148K / F283Y.

[0048] The GS115 strain containing the recombinant plasmid was inoculated into 2 mL of BMGY medium in a 10 mL test tube and incubated at 30°C, 220 rpm, and shaken for 48 hours. The culture was then centrifuged at 3000 g for 5 minutes, the supernatant discarded, and the pellet resuspended in 2 mL of BMMY medium containing 0.5% methanol. The culture was then incubated again at 30°C, 220 rpm, for 48 hours. The supernatant was used for enzyme activity assays, and a mutant, AiGODL_E148K / F283Y, was identified (amino acid sequence shown in SEQ ID NO. 2, nucleotide sequence shown in SEQ ID NO. 4), exhibiting enhanced specific activity compared to the wild-type enzyme.

[0049] The wild-type GS115 / AiGODL and mutant GS115 / AiGODL_E148K / F283Y were scaled up to form a fermentation system. First, the seed culture was inoculated into YPD medium, and then inoculated into a 1L Erlenmeyer flask containing 300mL of BMGY medium at a 1% inoculum. The culture was cultured at 30°C and 220rpm for 48h. The culture was then centrifuged at 3000g for 5min, the supernatant was discarded, and the precipitate was resuspended in 200mL of BMMY medium containing 0.5% methanol and again induced at 30°C and 220rpm. 1mL of methanol was added every 12h to maintain the methanol concentration in the bacterial solution at 0.5%. The supernatant was taken for enzyme activity detection. Finally, the supernatant was concentrated to 20mL for enzyme property determination and comparison. The protein was purified using anion exchange method. The molecular weight of the expressed wild-type and mutant glucose oxidase was determined by polyacrylamide gel electrophoresis ( Figure 1 ) as shown.

[0050] Example 3: Comparative analysis of enzymatic properties of recombinant thermostable and acid-resistant glucose oxidase mutants and wild type

[0051] 1. Determination by o-dianisidine method

[0052] The specific method is as follows: Under standard conditions (pH 6.0, 40°C), a 3 mL reaction system consisting of: 2.5 mL o-dianisidine buffer, 300 μL substrate, 100 μL peroxidase (90 U / mL), and 100 μL diluted enzyme solution is incubated for 3 minutes. The reaction is terminated by the addition of 2 mL of 2M H₂SO₄. OD values are measured at 540 nm. One unit (U) of enzyme activity is defined as the amount of enzyme required to decompose 1 μmol of substrate per minute to produce hydrogen peroxide under standard conditions.

[0053] 2. Determination of the properties of recombinant thermostable and acid-stable glucose oxidase mutants and wild type

[0054] 1. Kinetic parameter determination method for recombinant glucose oxidase mutants and wild type

[0055] The detection method refers to the literature (Characterization, stability improvement, and breadbaking applications of a novel cold-adapted glucose oxidase from Cladosporium neopsychrotolerans SL16. Ge, et al., 2020).

[0056] Glucose solutions of varying concentrations (3.125 mM-1000 mM) were prepared as substrates using 0.1 mol / L citric acid-sodium hydrogen phosphate buffer at pH 6.0. Enzyme activity was determined under standard conditions (40°C, pH 6.0). The measured enzyme activity data were analyzed using GraphPad Prism 5.01 software to obtain the K values of the wild-type recombinant high-specific-activity glucose oxidase and its mutants. m Value and V max .

[0057] Under standard conditions, with glucose as substrate, the specific activity of glucose oxidase mutant AiGODL_E148K / F283Y was 160.9 U / mg, which was 10% higher than that of wild-type AiGODL (146.1 U / mg); the catalytic efficiency of mutant AiGODL_E148K / F283Y was 25.9 mM -1 ·s -1 , compared with wild-type AiGODL (21.8 mM -1 ·s -1 ) remained at a comparable level. The specific activities and kinetic parameters of the mutants and the wild type are shown in Table 2.

[0058] Table 2 Comparison of specific activities and kinetic parameters of recombinant glucose oxidase mutants and wild type

[0059]

[0060] 2. Optimal pH determination method for recombinant thermostable and acid-resistant glucose oxidase mutants and wild type

[0061] The glucose oxidase mutant and wild-type glucose oxidase from Example 2 were subjected to enzymatic reactions at various pH values (1.0-8.0) to determine their optimal pH. Glucose oxidase activity was determined at 40°C using 0.1 mol / L citric acid-disodium hydrogen phosphate buffer at various pH values (1.0-8.0) using the substrate β-D-glucose.

[0062] The results are as follows Figure 2 As shown, the optimal reaction pH of wild-type glucose oxidase AiGODL and glucose oxidase mutant AiGODL_E148K / F283Y are pH 6.0 and pH 5.5, respectively. In addition, the relative enzyme activity of the mutant in acidic environment (pH 2.0-5.0) is increased by 19%-74% compared with the wild-type.

[0063] 3. pH Stability Determination of Recombinant Thermo- and Acid-Resistant Glucose Oxidase Mutants and Wild-Type

[0064] The glucose oxidase mutants and wild-type glucose oxidase were diluted with 0.1 mol / L citric acid-disodium hydrogen phosphate buffer at different pH values (1.0-9.0) and placed in a 37°C constant temperature water bath for 1 h. The relative residual enzyme activity was then measured at pH 6.0 and 40°C. The untreated enzyme activity was set as 100% of the control.

[0065] The results are as follows Figure 3 As shown, in terms of acid tolerance, the residual enzyme activity of the mutant AiGODL_E148K / F283Y in acidic environment (pH 1.0 and pH 2.0) was 18% and 35%, respectively, which were increased by 200% and 84% compared with the wild type (6% and 19%).

[0066] 4. Method for determining the optimal temperature of recombinant heat-resistant and acid-resistant glucose oxidase mutants and wild type

[0067] The optimal temperatures of the recombinant glucose oxidase mutant and the wild-type glucose oxidase were determined by performing the enzymatic reaction in a 0.1 mol / L citric acid-disodium hydrogen phosphate buffer (pH 6.0) buffer system at different temperatures (10° C.-70° C.).

[0068] The results are as follows Figure 4 As shown, the optimum temperature of the recombinant wild-type glucose oxidase AiGOD is 35°C, and the optimum temperature of the mutant AiGODL_E148K / F283Y is 40°C. Moreover, the relative enzyme activity of the mutant under high temperature (50-70°C) conditions is significantly higher than that of the wild-type enzyme.

[0069] 5. Thermal stability determination method of recombinant heat-resistant and acid-resistant glucose oxidase mutants and wild type

[0070] T at 40-70℃ 50 Value: After the mutant AiGODL_E148K / F283Y and the wild-type enzyme were treated at different temperatures of 40-70°C for 30 min, the residual enzyme activities of each were detected.

[0071] Half-life at 70℃ (t 1 / 2 ): The mutant AiGODL_E148K / F283Y and the wild-type enzyme were treated at 70°C for different time periods, up to 30 min, and their respective residual enzyme activities were detected.

[0072] T at 40-70℃ 50 The results of the value determination are as follows Figure 5 As shown, the T of glucose oxidase mutant AiGODL_E148K / F283Y 50 The half-life value is 64℃, which is 9℃ higher than that of the wild type (55℃); the half-life determination results at 70℃ are as follows Figure 6As shown, the half-life of mutant AiGODL_E148K / F283Y at 70°C (t 1 / 2 ) is 25 min, which is 12 min longer than that of the wild enzyme AiGODL (13 min), indicating that the thermal stability of the glucose oxidase mutant AiGODL_E148K / F283Y is significantly improved.

[0073] Example 4: Antibacterial activity analysis of recombinant thermostable glucose oxidase mutants and wild type

[0074] The E. coli culture solution cultured at 37°C for 16 h was diluted to a concentration of 2 × 10 7 CFU·mL -1 The enzyme solution (AiGODL and AiGODL_E148K / F283Y) obtained in Example 2 was mixed with an equal amount of bacterial solution, 10% glucose substrate was added, and the mixture was cultured at 37°C for 24 h before measuring the OD value. 600 In addition, ampicillin was mixed with an equal amount of bacterial solution and cultured at 37°C for 24 h before measuring OD 600 The IC values of enzyme solution and ampicillin were calculated using GraphPad Prism 5.01 software. 50 (half-maximal inhibitory concentration) to evaluate the antibacterial activity of the glucose oxidase mutant enzyme.

[0075] The results are as follows Figure 7 As shown, the IC of mutant AiGODL_E148K / F283Y against Escherichia coli 50 The value was 23.3 mg / L, and its antibacterial performance was significantly better than that of the wild type (IC 50 value is 29.1mg / L), and compared with ampicillin (IC 50 value was 22.5 mg / L) showed similar antibacterial properties.

[0076] The present invention relates to the sequence:

[0077] SEQ ID NO.1:

[0078] LPHYIRSSGIEASLLTDPKDVAGRTVDYIIAGGGLTGLTTAARLTENPNITVLVIESGFYESDRGPIIEDLNTYGEIFGSNVDHAYQTVELATNNLTELIRSGNGLGGSTLVNGGTWTRPHKVQVDSWETVFGNEGWNWDTVAAYSLEAELARAPNAKQIAAGHYFDESCHGTNGTVHVGPRDTGDDYTPIIEALMTTVEKRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQLVGKVLLDQKATVPRAIGVEFGTHRDTTFNVYAKHEVLLAAGSAVSPTILEHSGIGLKSVLDSVGIDTVVELPVGLNLQDQTTVAVSSRITSAGAGQGQAAFFATFNETFGDYAPQAHQLLNSKLAQWAEETVARGGFHNTTALLIQYENYRDWLVNHNVAYSELFLDTAGAVSFTIWDLIPFTRGYVHITDPDPYLRLFAYDPQYFLNELDLYGQAAASQLARNLSNSDAMSAYFAGETVPGDNLAYDADLSDWADYVRYNFRPNYHGVGTCSMMPKELGGVVDSSARVYGVDSLRVIDGSIPPTQVSSHVMTVFYAMALKISDAILKDYAASQ。

[0079] SEQ ID NO.2:

[0080] LPHYIRSSGIEASLLTDPKDVAGRTVDYIIAGGGLTGLTTAARLTENPNITVLVIESGFYESDRGPIIEDLNTYGEIFGSNVDHAYQTVELATNNLTELIRSGNGLGGSTLVNGGTWTRPHKVQVDSWETVFGNEGWNWDTVAAYSLKAELARAPNAKQIAAGHYFDESCHGTNGTVHVGPRDTGDDYTPIIEALMTTVEKRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQLVGKVLLDQKATVPRAIGVEFGTHRDTTYNVYAKHEVLLAAGSAVSPTILEHSGIGLKSVLDSVGIDTVVELPVGLNLQDQTTVAVSSRITSAGAGQGQAAFFATFNETFGDYAPQAHQLLNSKLAQWAEETVARGGFHNTTALLIQYENYRDWLVNHNVAYSELFLDTAGAVSFTIWDLIPFTRGYVHITDPDPYLRLFAYDPQYFLNELDLYGQAAASQLARNLSNSDAMSAYFAGETVPGDNLAYDADLSDWADYVRYNFRPNYHGVGTCSMMPKELGGVVDSSARVYGVDSLRVIDGSIPPTQVSSHVMTVFYAMALKISDAILKDYAASQ。

[0081] SEQ ID NO.3:

[0082]

[0083] SEQ ID NO.4:

[0084]

[0085] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A glucose oxidase mutant with improved thermostability and acid tolerance, characterized in that: The glucose oxidase mutant is obtained by mutating the Glu148 and Phe283 sites of the wild-type glucose oxidase to Lys148 and Tyr283, respectively. The amino acid sequence of the glucose oxidase mutant is shown in SEQ ID NO.

2.

2. The gene encoding the glucose oxidase mutant according to claim 1, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

3. A recombinant vector, characterized in that It comprises the gene according to claim 2.

4. A recombinant bacterium, characterized in that It comprises the recombinant vector according to claim 3.

5. Use of the glucose oxidase mutant according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, or the recombinant bacterium according to claim 4 in the preparation of an antibacterial agent.

6. The use according to claim 5, characterized in that The antimicrobial agent is a bacterial agent that inhibits Escherichia coli.

7. An antibacterial agent, characterized in that The main active ingredient is the glucose oxidase mutant according to claim 1.

8. The antibacterial agent according to claim 7, wherein The antimicrobial agent is a bacterial agent that inhibits Escherichia coli.

Images