A thermostable glucose oxidase mutant with high antibacterial activity and its application

Abstract

The present invention discloses a thermostable glucose oxidase mutant with high antibacterial activity and its application, belonging to the field of genetic engineering. The amino acid sequence of the glucose oxidase mutant GOX1_P514K is shown in SEQ ID NO.2. The present invention uses glucose oxidase GOX1 derived from Aspergillus as the parent, and through site-directed mutagenesis, mutates the 514th position of the sequence shown in SEQ ID NO.1 from proline (Pro) to lysine (Lys), thereby obtaining a high-specific-activity thermostable mutant GOX1_P514K. This mutant is significantly superior to wild-type glucose oxidase GOX1 in terms of catalytic activity and thermal stability. At the same time, the mutant has high specific activity and strong antibacterial properties, showing great application prospects.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, in particular to a heat-resistant glucose oxidase mutant with high antibacterial property and application thereof. Background Art

[0002] Glucose oxidase (GOX, EC 1.1.3.4) is a flavonoid protein containing a tightly noncovalently bound coenzyme, flavin adenine dinucleotide (FAD; Wang, Sun, Qiao, Ouyang, & Na, 2017). In the catalytic reaction, FAD acts as a redox carrier, while GOX uses molecular oxygen as an electron acceptor to catalyze the oxidation of β-d-glucose to d-gluco-δ-lactone and hydrogen peroxide. Due to these unique properties, GOX-based applications are widely used in the chemical, food, beverage, pharmaceutical, and clinical industries. These applications include glucose sensors for clinical and environmental monitoring, oxidants in the food industry, and gluconic acid production. Enzymes used in these processes must meet at least three important criteria: high specificity, catalytic efficiency, and stability. Consequently, a wide range of research efforts are underway to identify or develop commercially viable enzymes, including protein engineering approaches to improve enzyme stability and catalytic activity.

[0003] Improving enzyme thermal stability through directed evolution and rational design is feasible. Non-rational design strategies do not require an understanding of the structural and functional characteristics of proteins, but they do require a high-throughput, directed screening method for mutants. This approach is labor-intensive and consumes significant manpower and capital. With the increasing maturity of protein structure and function identification technologies, research on protein structure has become increasingly in-depth, and strategies for molecular modification of proteins based on rational design have emerged. The development of this protein engineering technology is also inseparable from an understanding of enzyme stability. However, the mechanisms of thermal stability are complex. Although many effective approaches have been adopted, such as disulfide bonds, hydrogen bonds, and hydrophobic interactions, the current improvement results, while significant, still do not meet industrial needs.

[0004] In recent years, computer-aided design technology has been applied to the modification of the catalytic properties of enzymes and has achieved remarkable results. Protein structure is the combined effect of these factors in the enzyme molecule, and any change in these forces may lead to changes in stability. Among them, protein free unfolding energy (ΔG) is one of the important parameters for measuring the thermodynamic stability of proteins, and it is also the main indicator for measuring protein thermal stability. The effect of mutations on protein thermal stability can be determined by calculating the change in ΔG value (ΔΔG) after protein mutation, which can be predicted with the help of computer simulation. Molecular dynamics simulations have been used to identify valuable residues that may cause significant differences in ΔG. For example, using this strategy, the thermal stability of methylparathiolate hydrolase from Ochrobactrum and lipase B from Candida antarctica was significantly improved, and half of the protein molecules of the mutant enzyme were denatured (T m ) increased by 11.7°C and 3.6°C, respectively. However, most current modifications aimed at improving enzyme thermostability result in decreased rather than increased activity. Therefore, when improving enzyme stability through protein engineering, maintaining enzyme activity is crucial. Summary of the Invention

[0005] The present invention aims to provide a thermostable glucose oxidase mutant with high antibacterial activity and its application to address the aforementioned problems of the prior art. Through computer-aided simulation design and target scoring, the present invention obtained a thermostable mutant, GOX1_P514K, with high specific activity and strong antibacterial properties, showing great application prospects.

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

[0007] The present invention provides a thermostable glucose oxidase mutant GOX1_P514K with high antibacterial activity. The amino acid sequence of the glucose oxidase mutant GOX1_P514K is shown in SEQ ID NO.2.

[0008] The present invention also provides a nucleic acid encoding the glucose oxidase mutant GOX1_P514K, the sequence of which is shown in SEQ ID NO.4.

[0009] The present invention also provides a biological material comprising the nucleic acid.

[0010] Furthermore, the biological material includes a recombinant vector or a recombinant bacterium.

[0011] Furthermore, the backbone vector of the recombinant vector is a pPIC9k plasmid.

[0012] Furthermore, the basic strain of the recombinant bacteria is Pichia pastoris GS115.

[0013] The present invention also provides use of the glucose oxidase mutant GOX1_P514K or the biomaterial in preparing an antibacterial agent or a food preservative.

[0014] Furthermore, the antibacterial agent is used to inhibit Staphylococcus aureus.

[0015] The present invention also provides an antibacterial agent, the active ingredient of which includes the glucose oxidase mutant GOX1_P514K.

[0016] The present invention also provides a method for preparing the antibacterial agent, comprising the step of obtaining the glucose oxidase mutant GOX1_P514K using the above-mentioned biological material.

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

[0018] The present invention uses glucose oxidase GOX1 from Aspergillus as the parent, and through site-directed mutagenesis, mutates the 514th position of the sequence shown in SEQ ID NO.1 from Pro to Lys to obtain a mutant. The specific method is to construct a recombinant strain containing the mutant, and screen out a glucose oxidase mutant GOX1_P514K with high specific activity after induction culture. In terms of catalytic activity, the specific activity of the mutant GOX1_P514K is 110.8 U / mg, which is 2.3 times higher than that of the wild type (33.7 U / mg); in terms of stability, the T of the mutant GOX1_P514K is 110.8 U / mg, which is 2.3 times higher than that of the wild type (33.7 U / mg); in terms of stability, the T of the mutant GOX1_P514K is 110.8 U / mg, which is 2.3 times higher than that of the wild type (33.7 U / mg). 50 The half-life of mutant GOX1_P514K at 75℃ (t 1 / 2 ) is 25 min, which is 19 min longer than that of the wild enzyme GOX1 (6 min); the optimal pH and temperature are basically the same as those of the wild type.

[0019] The present invention uses computer-aided simulation design and modified target scoring to obtain a high-specific-activity heat-resistant mutant GOX1_P514K. This mutant has high specific activity and strong antibacterial properties, showing great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1Polyacrylamide gel electrophoresis of glucose oxidase GOX1 and its mutant GOX1_P514K;

[0022] Figure 2 The results of the optimal pH determination of glucose oxidase GOX1 and its mutant GOX1_P514K are shown;

[0023] Figure 3 The results of pH stability test of glucose oxidase GOX1 and its mutant GOX1_P514K are shown;

[0024] Figure 4 The results of the optimal temperature determination of glucose oxidase GOX1 and its mutant GOX1_P514K are shown;

[0025] Figure 5 T is the T of glucose oxidase GOX1 and its mutant GOX1_P514K 50 Measurement results;

[0026] Figure 6 is the half-life of glucose oxidase GOX1 and its mutant GOX1_P514K at 75°C 1 / 2 Measurement results;

[0027] Figure 7 These are the results of antibacterial activity assays of glucose oxidase GOX1 and its mutant GOX1_P514K. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The test materials used in the following examples are:

[0034] 1. Strains and Vectors: The expression host was Pichia pastoris GS115 (purchased from Invitrogen), and the expression vector was pPIC9k plasmid (purchased from Invitrogen), both of which were maintained in the inventor's laboratory.

[0035] 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 other reagents were domestic analytical grade (all purchased from Sinopharm Group);

[0036] 3. Culture medium:

[0037] (1) YPD medium: 1% yeast extract, 2% peptone, 2% glucose;

[0038] (2) MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin;

[0039] (3) BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (v / v), 1.34% YNB, 0.00004% Biotin;

[0040] (4) LB medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;

[0041] (5) MM solid medium: 1.5% agarose, 1.34% YNB, 0.00004% Biotin, 0.5% methanol;

[0042] (6) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 0.5% methanol (v / v);

[0043] The percentages in the above culture media are all by mass.

[0044] Example 1 Synthesis of Glucose Oxidase Mutant Gene and Acquisition of Recombinant Plasmid

[0045] To improve the catalytic performance of glucose oxidase from Aspergillus, molecular dynamics simulation and computer-aided design were used to analyze key differential amino acids, score the modified targets, and screen for mutants. The wild-type glucose oxidase gene GOX1 was inserted into the pPIC9k plasmid to construct the recombinant expression vector pic9k-GOX1. Using the recombinant expression vector pic9k-GOX1 as a template, site-directed mutagenesis was performed to mutate position 514 of the sequence shown in SEQ ID NO.1 from proline (Pro) to lysine (Lys). Primer design is shown in Table 1. The mutation and cloning methods are based on the literature (Enhanced Thermostability of Glucose Oxidase through Computer-Aided Molecular Design. Ning, et al., 2018).

[0046] Table 1 Primers for site-directed mutagenesis of glucose oxidase GOX1

[0047]

[0048] The amino acid sequence of wild-type glucose oxidase is shown in SEQ ID NO.1:

[0049] LPHYIRSNGIEASLLTDPKDVAGRTVDYIIAGGGLNGLTTAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVELATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDNVAAYSLQAERARAPNAKQIAAGHYFNASCHGLNGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGEPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAIGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSVLEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYAEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVNHNVAYSELFLDTAGVASFDVWDLLPFTRGYVHILDKDPYLRLFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLSAWVEYIPYSFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASM。

[0050] The sequence of the wild-type glucose oxidase gene GOX1 is as SEQ ID NO.3:

[0051]

[0052] Example 2 Preparation of recombinant high specific activity glucose oxidase mutant

[0053] 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 / GOX1_P514K.

[0054] 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, GOX1_P514K, was identified, which exhibited increased specific activity compared to the wild-type DMT-competent strain.

[0055] The amino acid sequence of the mutant GOX1_P514K is shown in SEQ ID NO.2:

[0056] LPHYIRSNGIEASLLTDPKDVAGRTVDYIIAGGGLNGLTTAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVELATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDNVAAYSLQAERARAPNAKQIAAGHYFNASCHGLNGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGEPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAIGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSVLEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYAEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVNHNVAYSELFLDTAGVASFDVWDLLPFTRGYVHILDKDPYLRLFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLSAWVEYIKYSFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASM。

[0057] The gene sequence of the mutant GOX1_P514K is shown in SEQ ID NO.4:

[0058]

[0059] The recombinant yeast strain GS115 / GOX1 (the target site in the recombinant plasmid was not mutated) and the recombinant yeast strain GS115 / GOX1_P514K were amplified into a fermentation system. First, the seed culture was inoculated into YPD medium, and the inoculum volume was 1% in a 1L Erlenmeyer flask containing 300mL of BMGY medium. The culture was placed at 30°C and shaken at 220rpm for 48h. The culture was then centrifuged at 3000×g for 5min, the supernatant was discarded, and the precipitate was resuspended in 100mL of BMMY medium containing 0.5% methanol and induced again at 30°C and 220rpm. 0.5mL of methanol was added every 12h to maintain the methanol volume concentration in the bacterial solution at 0.5%. At the same time, 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 ), where M is a marker, 1 and 2 are wild-type GOX1 and mutant GOX1_P514K, respectively.

[0060] Example 3 Comparative analysis of enzymatic properties of recombinant high specific activity glucose oxidase mutant and wild type

[0061] 1. Determination by o-dianisidine method

[0062] The specific method is as follows: Under standard conditions (pH 6.0, 30°C), a 3 mL reaction system consists of 2.5 mL of o-dianisidine buffer, 300 μL of substrate, 100 μL of peroxidase (90 U / mL), and 100 μL of diluted enzyme solution. The reaction is incubated for 3 minutes and terminated with 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.

[0063] 2. Determination of properties of recombinant high-specific-activity glucose oxidase mutants and wild-type

[0064] 1. Kinetic parameter determination method for recombinant high specific activity glucose oxidase mutant and wild type

[0065] 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).

[0066] Glucose solutions of varying concentrations (3.125 mM-1000 mM) were prepared in 0.1 mol / L citric acid-sodium hydrogen phosphate buffer at pH 6.0 as substrates, and enzyme activity was determined under standard conditions (30°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 .

[0067] Under standard conditions, with glucose as substrate, the specific activity of the glucose oxidase mutant GOX1_P514K was 110.8 U / mg, which was 2.3 times higher than that of the wild-type GOX1 (33.7 U / mg); the catalytic efficiency of GOX1_P514K was 21.7 mM -1 ·s -1 , compared with wild-type GOX1 (16.2 mM -1 ·s -1 ) was increased by 34%. The specific activities and kinetic parameters of the mutant and wild type are shown in Table 2.

[0068] Table 2 Comparison of specific activity and kinetic parameters between recombinant high specific activity glucose oxidase mutant and wild type

[0069]

[0070] 2. Optimal pH determination method for recombinant high specific activity glucose oxidase mutants and wild type

[0071] The glucose oxidase mutant and wild-type glucose oxidase from Example 2 were subjected to enzymatic reactions at various pH values (1.0-9.0) to determine their optimal pH. Glucose oxidase activity was determined at 30°C using 0.1 mol / L citric acid-sodium hydrogen phosphate buffer at various pH values (1.0-9.0) using the substrate β-D-glucose.

[0072] The results are as follows Figure 2 As shown, the optimal reaction pH of wild-type glucose oxidase and glucose oxidase mutants is 6.0.

[0073] 3. pH Stability Determination of Recombinant High Specific Activity Glucose Oxidase Mutants and Wild Type

[0074] 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 30°C. The untreated enzyme activity was set as 100% of the control.

[0075] The results are as follows Figure 3 As shown, the pH stability of the high specific activity glucose oxidase mutant GOX1_P514K in an environment of pH 1.0-9.0 is comparable to that of the recombinant wild-type glucose oxidase.

[0076] 4. Method for determining the optimal temperature of recombinant high specific activity glucose oxidase mutants and wild type

[0077] The optimal temperatures of the recombinant high specific activity glucose oxidase mutant and the wild-type glucose oxidase were determined by performing enzymatic reactions in a 0.1 mol / L citric acid-disodium hydrogen phosphate buffer (pH 6.0) system at different temperatures (0°C-75°C).

[0078] The results are as follows Figure 4 As shown, the optimum temperature of the recombinant wild-type glucose oxidase is 40°C, the optimum temperature of the high specific activity glucose oxidase mutant GOX1_P514K is 45°C, and the relative enzyme activity under high temperature (50-75°C) conditions is significantly improved compared with the wild enzyme.

[0079] 5. Thermal stability determination method of recombinant high specific activity glucose oxidase mutant and wild type

[0080] T at 40-80℃ 50 Value: After the mutant and wild-type enzymes were treated at different temperatures of 40-80℃ for 30min, the residual enzyme activities of each were detected.

[0081] Half-life at 75℃ (t 1 / 2 ):The mutant and wild-type enzymes were treated at 75℃ for different time periods, up to 60 min, and their residual enzyme activities were detected.

[0082] T at 40-80℃ 50 The results of the value determination are as follows Figure 5 As shown, the T of the high specific activity glucose oxidase mutant GOX1_P514K 50 The half-life value is 70℃, which is 10℃ higher than that of the wild type (60℃); the half-life determination results at 75℃ are as follows Figure 6 As shown, the half-life of mutant GOX1_P514K at 75°C (t 1 / 2) was 26 min, which was 20 min longer than that of the wild-type enzyme GOX1 (6 min), indicating that the thermal stability of the high-specific-activity glucose oxidase mutant GOX1_P514K was improved.

[0083] Example 4 Analysis of Antibacterial Activity of Recombinant High Specific Activity Glucose Oxidase Mutants and Wild Type

[0084] 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 enzyme solution (GOX1 and GOX1_P514K) 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) was used to evaluate the antibacterial activity of high-specific-activity glucose oxidase mutant enzymes.

[0085] The results are as follows Figure 7 As shown, the IC of mutant GOX1_P514K against Staphylococcus aureus 50 The value was 20 mg / L, and its antibacterial performance was significantly better than that of the wild type (IC 50 value is 27mg / L), and compared with ampicillin (IC 50 The value was 19 mg / L) and showed considerable antibacterial performance.

[0086] 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 thermostable glucose oxidase mutant GOX1_P514K with high antibacterial activity, characterized in that The amino acid sequence of the glucose oxidase mutant GOX1_P514K is shown in SEQ ID NO.

2.

2. The nucleic acid encoding the glucose oxidase mutant GOX1_P514K according to claim 1, characterized in that The sequence of the nucleic acid is shown in SEQ ID NO.

4.

3. A biological material comprising the nucleic acid according to claim 2, characterized in that The biological material is a recombinant vector or a recombinant bacterium.

4. The biomaterial according to claim 3, characterized in that The backbone vector of the recombinant vector is pPIC9k plasmid.

5. The biomaterial according to claim 3, characterized in that The basic strain of the recombinant bacteria is Pichia pastoris GS115.

6. Use of the glucose oxidase mutant GOX1_P514K according to claim 1 or the biomaterial according to any one of claims 3 to 5 in the preparation of an antibacterial agent or a food preservative.

7. The use according to claim 6, characterized in that The antibacterial agent is used to inhibit Staphylococcus aureus.

8. An antibacterial agent, characterized in that The active ingredient comprises the glucose oxidase mutant GOX1_P514K according to claim 1.

9. The method for preparing the antibacterial agent according to claim 8, wherein The method comprises the step of obtaining the glucose oxidase mutant GOX1_P514K by using the biological material according to any one of claims 3 to 5.

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