Heat-resistant glucose oxidase mutant with high catalytic activity as well as construction method and application thereof

Abstract

The invention relates to a heat-resistant glucose oxidase mutant with high catalytic activity as well as a construction method and application thereof, and the construction method comprises the following steps: by taking glucose oxidase AeGoxA as a female parent, mutating Ala12 into Ser12 to obtain a mutant named as AeGoxAA12S; the amino acid sequence of the AeGoxAA12S is as shown in SEQ ID NO. 2. In the aspect of thermal stability, the T50 value of the mutant AeGoxAA12S is 72 DEG C and is increased by 12 DEG C compared with that of a wild type (60 DEG C); the half-life period (t1 / 2) of the mutant AeGoxAA12S at 75 DEG C is 27 min, and is increased by 3.5 times compared with that of a wild enzyme AeGoxA (6 min); in the aspect of catalytic performance, the specific activity and catalytic efficiency of the mutant A12S are 84.28 U / mg and 32.9 mM <-1 >. S <-1 > respectively, and are 1.5 times and 1.0 times higher than those of a wild type respectively.

Description

Technical Field

[0001] The invention relates to the fields of genetic engineering and protein engineering, and in particular to a heat-resistant high-catalytic activity glucose oxidase mutant and a construction method and application thereof. Background Art

[0002] Glucose oxidase (Gox; EC1.1.3.4) is a well-characterized aerobic dehydrogenase that uses oxygen molecules as electron acceptors to catalyze β-D-glucose to produce D-gluconolactone and hydrogen peroxide, which is then hydrolyzed into gluconic acid and water. Glucose oxidase is widely distributed in animals, plants and microorganisms. Microorganisms are its main source, and the main production strains are Aspergillus niger and Penicillium. Glucose oxidase, as a new feed enzyme preparation to replace antibiotics, has multiple functions such as protecting animal intestines, promoting digestion and absorption, and improving the body's immunity.

[0003] Most Gox exhibit maximum enzyme activity at 30-50°C and pH 6.0, but their stability is poor. Currently, most Gox can maintain stability under buffer conditions of pH 4.0-7.0, and have little enzyme activity when the pH value is less than 3.0 or higher than 10.0. Gox from Penicillium is generally stable below 50°C. The catalytic efficiency (k cat / K m =93μM -1 s -1 ) is higher than Gox(k cat / K m =27μM -1 s -1 ), under the condition of 5mM blood glucose concentration, the catalytic efficiency of Penicillium niger Gox is twice that of Aspergillus niger Gox. Therefore, Penicillium niger Gox is a candidate material for industrial development that is more suitable for efficient bioelectrochemical devices under physiological conditions. However, Gox from Aspergillus is more stable than Gox from Penicillium niger, so they are more suitable for use in the food and feed fields that require higher enzyme protein stability, which requires the enzyme to have different characteristics to adapt to different application requirements, such as good stability, high catalytic efficiency and low oxygen sensitivity.

[0004] Protein engineering has been widely used to improve enzyme properties to meet various commercial needs. The main methods involved are directed evolution, rational design and semi-rational design. Directed evolution is a commonly used technique in protein engineering. The key challenge is to find an efficient screening method. Frodanovic et al. developed an ultra-high throughput screening system to obtain t 1 / 2 The mutant F91 increased 2.2-fold. Summary of the invention

[0005] Technical problem to be solved: The present invention provides a heat-resistant and high-catalytic activity glucose oxidase mutant, and a construction method and application thereof. The mutant is obtained by screening the mutant after mutating the key amino acid site A12 of AeGoxA from Aspergillus eucalypticola, specifically a glucose oxidase mutant with improved thermal stability is obtained, which has broad prospects for application in feed mildew prevention and antibiotic replacement industries.

[0006] Technical solution: A high-temperature resistant glucose oxidase mutant, comprising glucose oxidase AeGoxA as the parent, Ala12 is mutated to Ser12 to obtain a mutant named AeGoxA_A12S; the amino acid sequence of the AeGoxA is shown in SEQ ID NO.1; the amino acid sequence of the AeGoxA_A12S is shown in SEQ ID NO.2.

[0007] The nucleotide sequence encoding the above-mentioned glucose oxidase mutant, the nucleotide sequence of AeGoxA is shown in SEQ ID NO.3; the nucleotide sequence of AeGoxA_A12S is shown in SEQ ID NO.4.

[0008] A recombinant vector, characterized in that the recombinant vector comprises the nucleotide sequence according to claim 2.

[0009] A recombinant strain, characterized in that it contains the recombinant vector according to claim 3

[0010] The application of the above recombinant strain in antibacterial treatment.

[0011] The application of the above recombinant strain in the preparation of antibacterial drugs.

[0012] An antibacterial drug, the active ingredient of which is prepared from the above recombinant strain.

[0013] Beneficial effects: The present invention obtains a mutant by performing site-directed mutagenesis on the A12 site of glucose oxidase AeGoxA derived from Aspergillus eucalypticola as the parent, and specifically constructs a recombinant strain containing the mutant, and screens out a glucose oxidase mutant AeGoxA_A12S with improved thermal stability after induction culture. In terms of thermal stability, the T of the mutant AeGoxA_A12S is 50 The values ​​were 72℃, 12℃ higher than the wild type (60℃); the half-life of mutant AeGoxA_A12S at 75℃ (t 1 / 2) was 27 min, which was 3.5 times higher than that of the wild enzyme AeGoxA (6 min). In terms of catalytic performance, the specific activity and catalytic efficiency of the mutant A12S were 84.28 U / mg and 32.9 mM, respectively. -1 ·s -1 , 1.5 times and 1.0 times higher than the wild type, respectively; the optimum pH value and optimum temperature are basically the same as those of the wild type. The present invention constructs a mutant library by site-directed mutagenesis to obtain a high-specific-activity heat-resistant mutant AeGoxA_A12S. Therefore, the application of the heat-resistant glucose oxidase mutant of the present invention in the feed additive industry has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Polyacrylamide gel electrophoresis of glucose oxidase AeGoxA and its mutant AeGoxA_A12S;

[0015] Figure 2 The results of the optimum temperature determination of glucose oxidase AeGoxA and its mutant AeGoxA_A12S;

[0016] Figure 3 The results of the determination of the optimum pH for glucose oxidase AeGoxA and its mutant AeGoxA_A12S;

[0017] Figure 4 The results of pH stability test of glucose oxidase AeGoxA and its mutant AeGoxA_A12S;

[0018] Figure 5 T is the T of glucose oxidase AeGoxA and its mutant AeGoxA_A12S 50 Measurement results;

[0019] Figure 6 is the half-life of glucose oxidase AeGoxA and its mutant AeGoxA_A12S at 75°C 1 / 2 Measurement results;

[0020] Figure 7 These are the results of antibacterial activity assay of glucose oxidase AeGoxA and its mutant AeGoxA_A12S. DETAILED DESCRIPTION

[0021] The test materials used in the following examples are:

[0022] 1. Strains and vectors: The expression host is Pichia pastoris GS115, and the expression plasmid vector pPIC9r is prepared in the laboratory;

[0023] 2. Enzymes and other biochemical reagents: Taq enzyme was purchased from Quanshijin Company, endonuclease was purchased from Quanshijin Company, o-dianisidine was purchased from Sigma Company, and peroxidase was purchased from Yuanye Company; other reagents were domestic analytical grade reagents (all purchased from Sinopharm Group);

[0024] 3. Culture medium:

[0025] (1) LB medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;

[0026] (2) YPD medium: 1% yeast extract, 2% peptone, 2% glucose;

[0027] (3) MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin;

[0028] (4) MM solid medium: 1.5% agarose, 1.34% YNB, 0.00004% Biotin, 0.5% methanol;

[0029] (5) BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (V / V), 1.34% YNB, 0.00004% Biotin;

[0030] (6) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 0.5% methanol (V / V).

[0031] Example 1 Synthesis of Glucose Oxidase Mutant Gene and Acquisition of Recombinant Plasmid

[0032] In order to improve the glucose oxidase from Aspergillus eucalypticola, the 12th amino acid site was mutated, and the 12th amino acid was changed from A to S. The glucose oxidase mutant containing the above mutation site was named AeGoxA_A12S; the amino acid sequence of AeGoxA_A12S is shown in SEQ ID NO.2. The primer sequences used are shown in Table 1, and the site-directed mutagenesis method and cloning method refer to the literature (Exploiting the activity-stability trade-offof glucose oxidase from Aspergillus niger using a simple approach to calculate thermostability of mutants. Jiang, et al., 2021).

[0033] Table 1 Error-prone PCR primers for glucose oxidase AeGoxA

[0034]

[0035] Example 2 Preparation of thermostable glucose oxidase mutants

[0036] The linear recombinant expression vector obtained by PCR in Example 1 was directly transformed into DMT competent cells, and colony PCR was used for verification to obtain the nucleotide sequence encoding the glucose oxidase mutant AeGoxA_A12S obtained by the mutant. The recombinant plasmid was linearized and transformed into Pichia pastoris GS115 to obtain the recombinant yeast strain GS115 / AeGoxA_A12S.

[0037] The GS115 strain containing the recombinant plasmid was inoculated into a 10 mL test tube containing 2 mL of BMGY medium, and cultured in a shaking incubator at 30°C and 220 rpm for 48 h. The culture solution was centrifuged at 3000 g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 2 mL of BMMY medium containing 0.5% methanol, and induced again at 30°C and 220 rpm for 48 h. The supernatant was used for enzyme activity detection, and a mutant AeGoxA_A12S (amino acid sequence as shown in SEQ ID NO.2, nucleotide sequence as shown in SEQ ID NO.4) with improved thermal stability compared to the wild enzyme was screened.

[0038] The wild-type GS115 / AeGoxA and three mutants were scaled up for fermentation. The seed culture was first inoculated into YPD medium, and then inoculated into a 1L Erlenmeyer flask containing 300mL BMGY medium at a 1% inoculum. The flask was placed at 30°C and shaken at 220rpm for 48h. The culture was then centrifuged at 3000g for 5min, the supernatant was discarded, and the precipitate was resuspended in 100mL BMMY medium containing 0.5% methanol, and then induced again at 30°C and 220rpm. 0.5mL of methanol was added every 12h to keep the methanol concentration in the bacterial solution at 0.5%, and the supernatant was taken for enzyme activity detection. Finally, the supernatant was concentrated to 20mL for enzyme property determination and comparison. The anion exchange method was used to purify the protein. After the expressed glucose oxidase was purified, polyacrylamide gel electrophoresis ( Figure 1 ) showed that the molecular weight of the wild-type and mutant glucose oxidase mutants in the fermentation supernatant was approximately 80-90 kDa, where M is Marker, 1 and 2 are AeGoxA and AeGoxA_A12S, respectively.

[0039] Example 3 Comparative analysis of enzymatic properties of recombinant thermostable glucose oxidase mutants and wild type

[0040] 1. Determination by o-dianisidine method

[0041] The specific method is as follows: Under standard conditions (pH 6.0, 30°C), 3 mL of the reaction system includes 2.5 mL of o-dianisidine buffer, 300 μL of substrate, 100 μL of peroxidase (90 U / mL), 100 μL of diluted enzyme solution, react for 3 minutes, and add 2 mL of 2MH2SO4 to terminate the reaction. The OD value is measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme required to decompose 1 μmoL of substrate to generate hydrogen peroxide per minute under standard conditions.

[0042] 2. Determination of properties of recombinant thermostable glucose oxidase mutants and wild type

[0043] 1. Method for determining the optimal temperature of recombinant thermostable glucose oxidase mutants and wild type

[0044] The optimum temperature of the recombinant thermostable glucose oxidase mutant and the wild-type glucose oxidase was determined by carrying out the enzymatic reaction in a 0.1 mol / L citric acid-disodium hydrogen phosphate buffer (pH 6) buffer system at different temperatures (0°C-75°C).

[0045] The results are as follows Figure 2 As shown, the optimum temperature of the recombinant wild-type glucose oxidase is 40°C, the optimum temperature of the thermostable glucose oxidase mutant is 45°C, and the relative enzyme activity under high temperature (60-75°C) conditions is significantly improved compared with the wild enzyme.

[0046] 2. Method for determining the optimal pH of recombinant thermostable glucose oxidase mutants and wild type

[0047] The glucose oxidase mutant and wild-type glucose oxidase of Example 2 were subjected to enzymatic reaction at different pH values ​​(1.0-12.0) to determine their optimum pH values. The glucose oxidase activity of the substrate β-D-glucose was determined at 30° C. using 0.1 mol / L citric acid-sodium hydrogen phosphate buffer at different pH values ​​(1.0-12.0).

[0048] The results are as follows Figure 3 As shown, the optimal reaction pH of the wild-type glucose oxidase and the glucose oxidase mutants is 6.0.

[0049] 3. pH stability determination method of wild-type glucose oxidase and mutants

[0050] The glucose oxidase mutant and wild-type glucose oxidase were diluted with 0.1 mol / L citric acid-disodium hydrogen phosphate buffer at different pH values ​​(1.0-12.0) 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 30°C. The enzyme activity without treatment was set as 100% of the control.

[0051] The results are as follows Figure 4 As shown, the pH stability of the glucose oxidase mutant AeGoxA_A12S in acidic and alkaline environments is better than that of the recombinant wild-type glucose oxidase.

[0052] 4. Determination of thermal stability of wild-type glucose oxidase and mutants

[0053] 40-80℃T 50 Value: After the mutant and wild type were treated at different temperatures of 40-80℃ for 30min, the residual enzyme activity of each was detected.

[0054] Half-life at 75℃ (t 1 / 2 ): The mutant and the wild type were treated at 75°C for different time periods, up to 60 min, and their respective residual enzyme activities were detected.

[0055] 40-80℃T 50 The results of the value determination are as follows Figure 5 As shown, the T of mutant AeGoxA_A12S 50 The half-life value is 72℃, which is 12℃ higher than the wild type (60℃); the half-life measurement results at 75℃ are as follows Figure 6 As shown, the half-life (t 1 / 2 ) were 27 min, which was 3.5 times higher than that of the wild enzyme AeGoxA (6 min).

[0056] 5. Kinetic parameter determination method of recombinant thermostable glucose oxidase mutant and wild type

[0057] The detection method refers to the literature (Improving the thermostability and catalytic efficiency of glucose oxidase from Aspergillus niger by molecular evolution. Tu, et al., 2019).

[0058] Different concentrations of glucose solution (3.125mM-1000mM) were prepared as substrates using pH 6.0, 0.1mol / L citric acid-sodium hydrogen phosphate buffer, and the enzyme activity was measured at 30°C and 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 and mutants of the recombinant thermostable glucose oxidase. m Value and V max .

[0059] The catalytic efficiency k of the recombinant thermostable glucose oxidase mutant AeGoxA_A12S with glucose as substrate under standard conditions (30°C, pH 6) cat / K m 32.9mM -1 s -1 , compared with wild-type AeGoxA (16.2 mM -1 s -1 ) was increased by 1 times; the specific activity of the mutant AeGoxA_A12S was 84.28 U / mg, which was 1.5 times higher than that of the wild type (33.67 U / mg) (see Table 2).

[0060] Table 2 Comparison of specific activities and kinetic parameters of recombinant thermostable glucose oxidase mutants and wild type

[0061]

[0062] Example 4 Analysis of antibacterial activity of recombinant thermostable glucose oxidase mutants and wild type

[0063] The Staphylococcus aureus culture solution cultured at 37°C for 12 h was diluted to a concentration of 2 × 10 7 CFU·mL -1 The glucose oxidase mutant enzyme solution of 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, and then the OD was measured. 600 IC values ​​were calculated using GraphPad Prism 5.01 software. 50 (half inhibitory concentration) to evaluate the antibacterial activity; erythromycin and ampicillin were mixed with an equal amount of bacterial solution, and the OD was measured after culturing at 37°C for 24 h. 600 IC values ​​were calculated using GraphPad Prism 5.01 software. 50 (half inhibitory concentration) to evaluate the antibacterial activity.

[0064] The results are as follows Figure 7 As shown, the IC of the recombinant thermostable glucose oxidase mutant AeGoxA_A12S 50 The IC values ​​of AeGoxA, erythromycin and ampicillin are 16 mg / L. 50 They were 27 mg / L, 13 mg / L and 18 mg / L respectively, indicating that the mutant AeGoxA_A12S had antibacterial activity against Staphylococcus aureus and exhibited comparable antibacterial properties to erythromycin and ampicillin.

Claims

1. A thermostable glucose oxidase mutant, characterized in that: The method comprises taking glucose oxidase AeGoxA as the parent, mutating Ala12 to Ser12 to obtain a mutant named AeGoxA_A12S; the amino acid sequence of the AeGoxA_A12S is shown in SEQ ID NO.

2.

2. The nucleotide sequence encoding the glucose oxidase mutant according to claim 1, characterized in that: The nucleotide sequence of AeGoxA_A12S is shown in SEQ ID NO.

4.

3. A recombinant vector, characterized in that: The recombinant vector comprises the nucleotide sequence according to claim 2.

4. A recombinant strain, characterized in that: Contains the recombinant vector according to claim 3.

5. Use of the recombinant strain according to claim 4 in antibacterial activities.

6. Use of the recombinant strain according to claim 4 in the preparation of antibacterial drugs.

7. An antibacterial drug, characterized in that: The active ingredient is obtained from the recombinant strain according to claim 4.

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