A laccase mutant and its application in the oxidative conversion of daidzein to prepare polyflavonoids

Abstract

This invention discloses a laccase mutant and its application in the oxidative conversion of daidzein to prepare polyflavonoids. This invention uses *Coprinus comatus* (Grey-capped Coprinus) as a starting material. Coprinopsis cinerea Using laccase Lcc5 as the starting enzyme, molecular docking was used to analyze its positional structure and design mutations. The mutant gene was synthesized, and a recombinant expression engineered bacterium containing the mutant gene was constructed. After induction of expression in the engineered bacterium containing the mutant gene, the Lcc5 mutant Lcc5-T485E was obtained through isolation and purification. This mutant showed improved specific enzyme activity and ethanol tolerance, increasing the conversion rate of daidzein by approximately 1.26 times under the same enzyme activity. The antioxidant activity, enzyme-inhibiting activity, and antibacterial activity of the obtained transformation product were also significantly enhanced. This mutant enzyme has potential application value in the oxidative conversion of daidzein to improve its biological value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a laccase mutant and its application in the oxidative conversion of daidzein to prepare polyflavonoids. Background Technology

[0002] Lacase, a copper-containing polyphenol oxidase also known as phenolase, is a member of the blue polycopper oxidase family (MCO). Lacase utilizes the strong redox properties of copper ions to effectively oxidize phenolic and aromatic compounds, while simultaneously reducing molecular oxygen to water. Lacase has a very broad substrate catalytic range and can directly oxidize and degrade various toxic phenolic substances and some harmful aromatic compounds. Furthermore, laccase can generate free radicals by extracting electrons from substrates, thereby coupling small molecules. Therefore, lacase has numerous applications in industrial production, such as food processing, wastewater treatment, biosensors, and biotransformation.

[0003] Daidzein, primarily derived from legumes, has a structural skeleton consisting of two benzene rings (A and B rings) connected by a heterocyclic pyran ring (C ring) to form the parent nucleus. As a natural polyphenol compound, daidzein possesses various biological activities, including antioxidant, antibacterial, and anti-absorption activities. Currently, most drug development is based on natural small molecule compounds; therefore, enhancing the activity of existing small molecule compounds through processing, modification, and biotransformation is of great significance.

[0004] Generally, oligomers exhibit increased activity compared to their monomers. To date, only one oligomer of daidzein has been reported, which is a diflavonoid compound formed by the polymerization of two daidzein monomers catalyzed by oxidases such as horseradish peroxidase and laccase. Therefore, natural laccase can be modified to screen for mutants with high daidzein conversion rates, and these mutants can be used to oxidize daidzein to obtain poly(daidzein), a conversion product with enhanced bioactivity compared to the monomer. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a laccase mutant and its application in the oxidative conversion of daidzein to prepare polyflavonoids. This invention uses *Coprinus comatus* (Grey-capped Coprinus) as a mutant. C. cinereaUsing laccase Lcc5 as the starting enzyme, a mutant gene was obtained through strategies such as structural simulation, molecular docking, and site-directed mutagenesis. After induction expression in bacteria containing the mutant gene, the mutant Lcc5-T485E, exhibiting enhanced activity in daidzein conversion, was obtained. The mutant enzyme Lcc5-T485E has potential application value in the oxidative conversion of daidzein: under enzyme activity conditions of 40 U / mL (measured by ABTS), Lcc5-T485E can oxidize and convert 88% of daidzein within 1 h, while the starting enzyme Lcc5 can only oxidize and convert 70% of daidzein, increasing the conversion rate of the mutant enzyme to 1.26 times that of the starting enzyme.

[0006] The laccase mutant of this invention, abbreviated as Lcc5-T485E, has the amino acid sequence shown in SEQ ID NO:1. Compared with the starting enzyme Lcc5, the amino acid sequence changes are as follows: the threonine at position 485 is mutated to glutamic acid.

[0007] The laccase mutant enzyme of the present invention may further include a combination of nonsense mutations or synonymous mutations in its amino acid sequence.

[0008] The coding gene for the laccase mutant of the present invention has the nucleotide sequence shown in SEQ ID NO:2.

[0009] The mutant plasmid of the present invention contains the coding gene of the laccase mutant as described in SEQ ID NO:2.

[0010] The expression strain of the laccase mutant enzyme of the present invention contains the mutant plasmid.

[0011] The strains expressed are classified as follows: Pichia pastoris GS115 / pPic9k(+)Lcc5-T485E It has been deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20242012, on September 20, 2024, at Wuhan University, Wuhan, China.

[0012] The method for constructing a laccase mutant expression strain of the present invention includes the following steps:

[0013] Cover the ghost umbrella with ash ( C. cinerea Using laccase Lcc5 as the starting enzyme, its structure was predicted through homology modeling. Molecular docking simulation with daidzein was performed to deduce key reaction sites and catalytic mechanisms on the enzyme, and based on this, mutants that promote enzyme-catalyzed reactions were designed. The laccase Lcc5-T485E, which may enhance daidzein activity, was identified. A mutant gene was constructed and ligated into the vector pPic9k. The linearized plasmid pPic9k was electroporated into the expression host Pichia pastoris. Pichia pastoris The GS115 mutant gene was integrated into the Pichia pastoris alcohol oxidase genome, and after plate screening, an engineered strain containing the mutant gene of this invention was obtained.

[0014] The laccase mutant of the present invention can be obtained by fermentation of the expressed strain.

[0015] The application of the laccase mutant of this invention in the preparation of oligomeric compounds by the oxidative conversion of soybean aglycones.

[0016] The reaction was carried out in a constant temperature water bath shaker at pH 8.0, 40℃, and 150 r / min for 1 h. The oxidation conversion rate of 40U (measured by ABTS) of the mutant enzyme Lcc5-T485E for 0.5 mg of daidzein was 88%, while the oxidation conversion rate of the starting enzyme Lcc5 was 70%. The oxidation conversion rate of the mutant enzyme was 26% higher than that of the starting enzyme.

[0017] The application of the oligomeric compounds obtained in this invention in the preparation of antioxidant agents.

[0018] The application of the oligomeric compounds obtained in this invention in the preparation of α-glucosidase inhibitors.

[0019] The application of the oligomeric compounds obtained in this invention in the preparation of antibacterial agents.

[0020] This invention uses the laccase mutant Lcc5-T485E to transform daidzein to obtain the product, which is then purified by rotary evaporation, reconstitution centrifugation, rotary evaporation, reconstitution centrifugation, and freeze-drying. The transformed product exhibits better biological activity than the daidzein monomer: the results of three common methods for detecting antioxidant activity (DPPH method, ABTS method, and FRAP method) all show that the antioxidant activity of the transformed product is significantly improved compared to daidzein. Figure 5 As shown. α-glucosidase is considered one of the key factors in postprandial hyperglycemia, and inhibiting its activity is one of the important treatment strategies for type 2 diabetes. The conversion product, compared to daidzein, significantly reduces the enzyme activity of α-glucosidase, such as... Figure 6 As shown in the figure. Antibacterial experiments showed that the transformation product had a more significant inhibitory activity against Staphylococcus aureus compared to daidzein, such as... Figure 7 As shown.

[0021] This invention measured and compared the temperature adaptability, specific enzyme activity, and ethanol adaptability of the mutant enzyme and the original enzyme. The results showed that, compared to the original enzyme, the optimal temperature of the mutant was higher. Furthermore, the specific enzyme activity of the mutant enzyme was also increased, approximately 1.92 times that of the original enzyme. Compared to the original enzyme, the ethanol adaptability of the mutant was also improved to some extent; the ethanol half-inhibition concentration of Lcc5 was approximately 15%-20%, while the ethanol half-inhibition concentration of the mutant increased to 20%-25%. Attached Figure Description

[0022] Figure 1The images show the SDS-PAGE spectra of the purified starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of this invention. Each lane represents the marker, the purified Lcc5 enzyme (1), and the purified Lcc5-T485E enzyme of this invention (2).

[0023] Figure 2 The results show the optimal temperature determination of the starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of this invention.

[0024] Figure 3 The relative activities of the starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of this invention are compared under conditions of ethanol concentrations of 5%, 10%, 15%, 20%, 25%, and 30%.

[0025] Figure 4 Experiments were conducted to convert daidzein into daidzein using the starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of this invention.

[0026] Figure 5 The results show the antioxidant effects of daidzein and its transformation products. Figure a shows the results of the DPPH assay, figure b shows the results of the ABTS assay, and figure c shows the results of the FRAP assay.

[0027] Figure 6 The results of experiments on the inhibition of α-glucosidase activity by daidzein and its transformation products are presented.

[0028] Figure 7 The results show the inhibitory activity of daidzein and its transformation products against Staphylococcus aureus. Figure a shows the inhibitory activity of daidzein against Staphylococcus aureus, and Figure b shows the inhibitory activity of the transformation products against Staphylococcus aureus.

[0029] Figure 8-19 LC-MS results (ESI, positive ion mode) for the conversion product. Detailed Implementation

[0030] Unless otherwise specified, the implementation methods in the following embodiments are all conventional methods.

[0031] (i) Construction of engineered strains containing the laccase mutant gene of the present invention

[0032] 1. Design of laccase gene mutants

[0033] This invention uses gray-covered ginkgo umbrellas C. cinereaLcc5, obtained from heterologous expression of strain FA2222, was used as the starting enzyme. Homology modeling was performed using an online website (https: / / swissmodel.expasy.org / ). Lcc5 was then simulated for docking with daidzein using AutoDock Vina. The docking results showed that in the optimal binding conformation, the enzyme interacts with daidzein through hydrogen bonds formed by multiple amino acids, including 485T. This amino acid can serve as a candidate site for modifying laccase to enhance the interaction between the enzyme and daidzein, thereby improving the enzyme's conversion performance. Based on the amino acid conservation analysis of WebLogo, a mutant was designed and named Lcc5-T485E.

[0034] The amino acid sequence of the laccase mutant enzyme described in this invention is as shown in SEQ ID NO:1. Compared with the starting enzyme Lcc5, the following changes are made: the threonine at position 485 is mutated to glutamic acid.

[0035] 2. Construction of laccase mutant genetically engineered strains

[0036] The laccase mutant gene from step 1 was constructed and ligated into the expression vector pPic9k, with Pichia pastoris as the expression host. Pichia pastoris, An engineered strain containing the mutant gene of this invention was obtained.

[0037] strains of the present invention Pichia pastoris GS115 / pPic9k(+)Lcc5-T485E It has been deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20242012, on September 20, 2024, at Wuhan University, Wuhan, China.

[0038] (II) Expression and protein purification of genetically engineered bacteria containing laccase mutants

[0039] The expression strain obtained in (I) Pichia pastoris GS115 / pPic9k(+)Lcc5-T485E Inoculate into test tubes containing 5 mL of BMGY, incubate overnight at 28°C and 200 rpm on a shaker, then transfer to Erlenmeyer flasks containing 50 mL of BMGY medium and incubate until OD (outlet size) is reached. 600 When the enzyme activity reaches 2.0, centrifuge at 3000 rpm for 20 min at 4℃, and resuspend the bacterial cells in BMM liquid. Inoculate the resuspended cells into a 500 mL Erlenmeyer flask with a baffle (containing 200 mL of BMM liquid medium). Samples are taken every 24 hours, and an equal volume of methanol is added to each sample. The samples are then induced to grow at 28℃. Changes in enzyme activity are monitored. After culturing in BMM medium for 7 days, the bacterial culture is collected to purify the target protein. The crude enzyme solution after centrifugation and dialysis is filtered through a 0.22 μm filter to remove impurities and air bubbles from the supernatant. The purified protein is then loaded onto a DEAE-Spharose FastFlow anion exchange column. SDS-PAGE analysis confirms the protein reaches the purity required for enzymatic testing. Figure 1 ).

[0040] (III) Detection of temperature adaptability of the mutant enzyme of the present invention

[0041] Laccase activity assay: The total reaction volume was 1 mL, including 33 μL ABTS (final concentration 15 mM) and 950 μL sodium tartrate buffer solution (pH 4.0). Incubation was performed at 30°C for 5 min. Then, 17 μL of enzyme solution was added to an EP tube to bring the reaction volume to 1 mL. After reacting at 30°C for 3 min, the mixture was incubated on ice for 30 s, and the OD was measured using a spectrophotometer. 420 value.

[0042] The formula for calculating enzyme activity is: Enzyme activity (U / L) = 555.56 × Dilution factor × OD 420 .

[0043] Test results as follows Figure 2 The results show that the optimal temperature for obtaining the mutant enzyme in this invention is 70°C when using ABTS as a substrate.

[0044] (iv) Detection of ethanol tolerance to enzymes containing the mutant enzyme of this invention

[0045] Using ABTS as the substrate, the total reaction volume was 1 mL, including 33 μL of ABTS (final concentration 15 mM). Then, 50 μL, 100 μL, 150 μL, 200 μL, 250 μL, and 300 μL of anhydrous ethanol were added to bring the final ethanol concentrations to 5%, 10%, 15%, 20%, 25%, and 30%, respectively. The volume was then brought to 983 μL with sodium tartrate buffer (pH 4.0). The mixture was incubated at 30°C for 5 min, and then 17 μL of enzyme solution was added to an EP tube to bring the reaction volume to 1 mL. After reacting at 30°C for 3 min, the mixture was incubated on ice for 30 s, and the OD was measured using a spectrophotometer. 420 value.

[0046] The formula for calculating enzyme activity is: Enzyme activity (U / L) = 555.56 × Dilution factor × OD 420 .

[0047] With the enzyme activity of the ethanol blank treatment group as 100%, the enzyme activity residual rate in ethanol systems of different concentrations was calculated using the following formula: Enzyme activity residual rate = (Enzyme activity of blank treatment group - Lost enzyme activity) / Enzyme activity of blank treatment group × 100%.

[0048] The measurement results are as follows Figure 3 As shown, the ethanol half-inhibitory concentration of the mutant enzyme activity obtained by the present invention is between 20% and 25%, which is higher than the ethanol half-inhibitory concentration of the starting enzyme, which is between 15% and 20%.

[0049] (v) Detection of enzyme activity containing the mutant enzyme of the present invention

[0050] Laccase activity assay: The total reaction volume was 1 mL, including 33 mL ABTS (final concentration 15 mM) and 950 mL sodium tartrate buffer solution (pH 4.0). Incubation was performed at 30°C for 5 min. Then, 17 mL of enzyme solution was added to an EP tube to bring the reaction volume to 1 mL. After reacting at 30°C for 3 min, the mixture was incubated on ice for 30 s, and the OD was measured using a spectrophotometer. 420 value.

[0051] The formula for calculating enzyme activity is: Enzyme activity (U / L) = 555.56 × Dilution factor × OD 420 .

[0052] Protein concentration assay: Add 20 mL of the protein solution to be tested and 200 mL of Brandford solution to a 96-well plate and mix thoroughly. Analyze the protein concentration using a microplate reader at OD500. 595 The absorbance of the mixture was measured at a specific wavelength. Based on the measured absorbance data, the concentration of laccase protein was further calculated.

[0053] Formula for calculating enzyme activity: enzyme activity / protein concentration.

[0054] The test results are shown in Table 1 below. The specific enzyme activity of the mutant enzyme obtained in this invention is increased by 92% compared with the original enzyme.

[0055]

[0056] (vi) Application of the laccase mutant enzyme of the present invention in the oxidative conversion experiment of daidzein

[0057] The conversion reaction system consisted of 1 mL of enzyme (40 U, ​​determined using ABTS as substrate), citrate-disodium hydrogen phosphate buffer (pH 8.0) to 1 mL, and daidzein to a final concentration of 0.5 mg / mL. The mixture was shaken and incubated in a constant temperature water bath at 40 °C and 150 r / min for 1 h. 500 μL of the reaction system was taken and 500 μL of ethanol was added to terminate the reaction. The mixture was then vortexed for 30 s. The ethanol containing daidzein was filtered through a 0.22 μm organic and aqueous syringe filter for instrumental analysis.

[0058] HPLC detection conditions: mobile phase: containing 0.1% acetonitrile and water; column: Agilent XDB-C18 column (250 mm length, 4.6 mm inner diameter, 5 μm particle size, Sun Fire); flow rate: 0.5 mL / min; column temperature: 30℃; detection wavelength: 330 nm.

[0059] HPLC elution system:

[0060]

[0061] Experimental results are as follows Figure 4 The results showed that, under pH 8.0 and 40℃ conditions, using daidzein as a substrate, the oxidation conversion rate of the mutant enzyme Lcc5-T485E was 88%, while that of the starting enzyme Lcc5 was 70%. The oxidation conversion rate of the mutant enzyme was 26% higher than that of the starting enzyme.

[0062] (vii) Enhanced antioxidant activity of the conversion product of this invention

[0063] This activity was measured using three methods: DPPH method, ABTS method, and FRAP method.

[0064] DPPH Method: Prepare a 1 mg / mL solution of the transformation product and daidzein. Weigh 2 mg of DPPH and dissolve it in 48 mL of anhydrous ethanol. Store in the dark and use within 5 hours. Take 1 mL of the DPPH solution prepared above, dilute it with anhydrous ethanol to an absorbance of approximately 0.8, and obtain the working solution. Add 1 mL of the DPPH working solution to 0.5 mL of the test solution, mix well, and let stand in the dark for 30 min. Measure the absorbance at 517 nm, zeroing with anhydrous ethanol.

[0065] Antioxidant rate = {1-(A1-A2) / A3}×100%.

[0066] A1: DPPH + test solution.

[0067] A2: Anhydrous ethanol + test solution.

[0068] A3: DPPH + anhydrous ethanol.

[0069] Test results as follows Figure 5 As shown in Figure a, using the DPPH method, the antioxidant rate of the conversion product was approximately 26.64% under the same conditions, while the antioxidant rate of the daidzein monomer was approximately 12.72%. The antioxidant rate of the conversion product was 2.09 times higher than that of the daidzein monomer.

[0070] ABTS Method: Prepare a 1 mg / mL solution of the conversion product and daidzein; dissolve 3 mg of ABTS diammonium salt in 0.735 mL of distilled water to prepare an ABTS diammonium salt stock solution (7.4 mmol / L). Dissolve 10 mg of potassium persulfate in 14.3 mL of distilled water to prepare a potassium persulfate stock solution (2.6 mmol / L). Mix 0.2 mL of the ABTS diammonium salt stock solution and 0.2 mL of the potassium persulfate stock solution, incubate at room temperature in the dark for 12 hours, and dilute the mixture with anhydrous ethanol to an absorbance of 0.70 ± 0.02. This solution is the ABTS solution. +Free radical working solution. Take 950 ml of working solution, add 50 ml of the test solution, mix well, and let stand in the dark for 30 min. Measure the absorbance at 734 nm, zeroing with anhydrous ethanol.

[0071] Antioxidant activity = { (A0-A1-A2) / A0}×100%.

[0072] A0: Working solution + anhydrous ethanol.

[0073] A1: Working solution + test solution.

[0074] A2: Anhydrous ethanol + test solution.

[0075] Test results as follows Figure 5 As shown in Figure b, using the ABTS method, the antioxidant rate of the conversion product was approximately 77.46% under the same conditions, while the antioxidant rate of the daidzein monomer was approximately 42.44%. The antioxidant rate of the conversion product was 1.83 times higher than that of the daidzein monomer.

[0076] FRAP method: Prepare a 1 mg / mL solution of transformation product and daidzein; detect the product using the method described in the FRAP kit (Solepro). First, establish the Fe concentration in the solution. 2+ A standard curve for concentration was obtained, and then the conversion product and the effect of daidzein on Fe were detected according to the method in the instruction manual. 3+ The ability to restore. The test results are as follows: Figure 5 As shown in Figure c, using the same mass concentration of the conversion product and daidzein solution, the conversion product caused the Fe in the solution to... 2+ It increased to 3.44 times that of the daidzein treatment group.

[0077] (viii) Increased anti-α-glucosidase activity of the product of this invention

[0078] α-Glucosidase and p-nitrophenyl-αD-glucopyranoside (pNPG) were purchased from Yuanye Company. First, a 100 U / mL enzyme solution was prepared according to the instructions and diluted to 1 U / mL with pH 7.0 citrate-disodium hydrogen phosphate buffer as the working solution. pNPG solutions with concentrations of 5 mM, 7.5 mM, 10 mM, 12.5 mM, 15 mM, 17.5 mM, 20 mM, 22.5 mM, and 25 mM were prepared. The transformation product and daidzein were dissolved in dimethyl sulfoxide (DMSO) solution to a concentration of 1 mg / mL. A pH 7.0, 200 mM PBS buffer was prepared. The reaction system consisted of 500 mL, with 25 mL each of the enzyme working solution, different concentrations of pNPG solutions, and the test solution, which were then brought to 500 mL with PBS. The mixture was placed in a 37°C water bath and reacted for 30 minutes. Then, 500 ml of 1 M Na2CO3 solution was added to terminate the reaction. The absorbance was measured at a wavelength of 405 nm.

[0079] The results are as follows Figure 6 As shown, daidzein inhibits the activity of α-glucosidase in the conversion of pNPG, while the inhibition by the conversion product is more pronounced. Compared to daidzein, the inhibitory effect of the conversion product is significantly enhanced.

[0080] (ix) Enhanced inhibitory activity against Staphylococcus aureus containing the transformation product of this invention

[0081] Staphylococcus aureus was obtained from previous laboratory preservation. First, Staphylococcus aureus was inoculated into LB agar for activation. After 12 hours of growth in shake flasks, it was diluted approximately 3000-fold with LB agar. The transformation product and daidzein were dissolved in DMSO at a concentration of 1 mg / mL, subsequently diluted to 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL, and 0.2 mg / mL, respectively. The bacterial culture and test samples were added to 96-well plates in a volume of 190 mL bacterial culture + 10 mL test solution, with the test sample concentrations in the system being 50 mg / mL, 40 mg / mL, 30 mg / mL, 20 mg / mL, and 10 mg / mL, respectively. The plates were continuously incubated at 37℃ and 220 rpm for 8 hours, with OD measured every 2 hours. 600 Absorbance value.

[0082] The results are as follows Figure 7 As shown, both daidzein and its transformation products slowed the increase in OD value of Staphylococcus aureus, with the transformation products showing a more significant inhibitory effect on the increase in OD value of Staphylococcus aureus.

[0083] (x) LC-MS detection results of products containing the transformation products of this invention

[0084] The elution system was as described in specific method (V), and the mass spectrometry conditions were selected as ESI, positive ion mode, with a detection range of 100-1500 m / z. The detection results are as follows: Figure 8-19 As shown, the results indicate that the transformation product underwent coupling, forming an oligomeric compound. Analysis suggests that the oligomeric product includes tetramer and dimer structures.

Claims

1. The application of a laccase mutant in the oxidative conversion of soybean aglycones, characterized in that: The amino acid sequence of the laccase mutant is shown in SEQ ID NO:1; The laccase mutant is obtained by fermentation of its expression strain, which is classified as Pichia pastoris GS115 / pPic9k(+)Lcc5-T485E, which has been deposited with the China Center for Type Culture Collection, with the accession number CCTCC NO: M20242012, on September 20, 2024, and at the address: Wuhan, China, Wuhan University.

2. The application according to claim 1, characterized in that: During the oxidative conversion of daidzein, the system temperature was 40℃ and the pH value was 8.0.

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