Laccase mutant and application thereof in preparation of polyflavone by oxidative conversion of daidzein

Abstract

The invention discloses a laccase mutant and application of the laccase mutant in preparation of polyflavone by oxidative conversion of daidzein. According to the invention, laccase Lcc5 of coprinus cinerea is used as a starting enzyme, and through molecular docking, a position structure is analyzed, and mutation is designed. Synthesizing a mutant gene, and constructing a recombinant expression engineering bacterium of the mutant gene. After engineering bacteria containing mutant genes are subjected to induced expression, the Lcc5 mutant Lcc5-T485E is obtained through separation and purification, the specific enzyme activity and ethanol tolerance of the mutant are improved, and the conversion rate of daidzein is increased to about 1.26 times under the same enzyme activity; the antioxidant activity, the enzyme inhibition activity and the antibacterial activity of the obtained conversion product are also remarkably improved. The mutant enzyme has potential application value in oxidative conversion of daidzein and improvement of biological value of daidzein.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a laccase mutant and application thereof in oxidatively transforming daidzein to prepare polyflavonoids. Background Art

[0002] Laccase, as a copper-containing polyphenol oxidase, also known as phenolase, is a member of the blue multi-copper oxidase family (MCO). Laccase can effectively oxidize phenols and aromatic compounds with the help of the strong redox ability of copper ions, and reduce molecular oxygen to water. Laccase can catalyze a wide range of substrates and can directly act on a variety of phenolic toxic substances and some aromatic harmful substances for oxidative degradation. In addition, laccase can also generate free radicals by extracting electrons from the substrate and then coupling small molecules. Therefore, laccase has many applications in industrial production, such as food processing, sewage treatment, biosensors and biotransformation.

[0003] Daidzein is mainly derived from leguminous plants. Its structural skeleton consists of two benzene rings (A and B rings) connected by a heterocyclic pyran ring (C ring) to form a parent nucleus. As a natural polyphenol compound, daidzein has a variety of biological activities including antioxidant activity, antibacterial activity, and anti-Ab aggregation activity. Currently, most drug development is based on natural small molecule compounds, so it is of great significance to improve the activity of existing small molecule compounds by processing, modifying and biotransforming them.

[0004] Generally speaking, the activity of oligomers is improved to a certain extent compared with their monomers. So far, only one oligomer of soybean isoflavone aglycone has been reported, which is a biflavonoid compound formed by the polymerization of two molecules of daidzein monomers catalyzed by oxidases such as horseradish peroxidase and laccase. Therefore, natural laccase can be modified to screen mutants with higher conversion rates to daidzein, and the mutants can be used to oxidize daidzein to obtain the conversion product poly (Daidzein) with improved biological activity compared with the monomer. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a laccase mutant and its application in the oxidation conversion of daidzein to prepare polyflavonoids. C. cinerea) was used as the starting enzyme, and the mutant gene was obtained through structural simulation, molecular docking, site-directed mutagenesis and other strategies. After the expression bacteria containing the mutant gene were induced to express, the mutant Lcc5-T485E with improved daidzein conversion activity was obtained. The mutant enzyme Lcc5-T485E has potential application value in the oxidation conversion of daidzein: under the condition of 40U / mL enzyme activity (measured by ABTS), Lcc5-T485E can oxidatively convert 88% of daidzein within 1 hour, while the starting enzyme Lcc5 can only oxidatively convert 70% of daidzein, and the conversion rate of the mutant enzyme is increased to 1.26 times that of the starting enzyme.

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

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

[0008] The nucleotide sequence of the gene encoding the laccase mutant of the present invention is 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 expression strains were classified as Pichia pastoris GS115 / pPic9k(+)Lcc5-T485E , has been sent to the China Center for Type Culture Collection for preservation, the preservation number is CCTCC NO: M 20242012, the preservation date is September 20, 2024, and the preservation address is Wuhan University, Wuhan, China.

[0012] The method for constructing a laccase mutant enzyme expression strain of the present invention comprises the following steps: Coprinus cinerea C. cinerea ) was used as the starting enzyme, its structure was predicted by homology modeling, and molecular docking simulation with soybean aglycone was performed to infer the key sites involved in the reaction on the enzyme and its catalytic mechanism, and mutations that can promote the enzyme catalytic reaction were designed based on this. The laccase Lcc5-T485E that may improve the activity of soybean aglycone was determined, the mutant gene was constructed and connected to the vector pPic9k, and the linearized plasmid pPic9k was electroporated into the expression host Pichia pastoris Pichia pastoris GS115, the mutant gene is integrated into the Pichia pastoris alcohol oxidase genome, and after plate screening, an engineered strain containing the mutant gene of the present invention is obtained.

[0013] The laccase mutant of the present invention can be obtained by fermenting the expression strain.

[0014] The invention discloses an application of the laccase mutant in preparing oligomer compounds by oxidative conversion of soybean aglycone.

[0015] After reacting in a constant temperature water bath shaker at pH 8.0, 40℃, and 150 r / min for 1 h, the oxidation conversion rate of 0.5 mg daidzein by 40U (measured by ABTS) of the mutant enzyme Lcc5-T485E 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.

[0016] Application of the oligomer compound obtained by the invention in the preparation of antioxidant preparations.

[0017] The oligomer compound obtained by the invention is used in the preparation of an α-glucosidase inhibitor.

[0018] The oligomer compound obtained by the invention is used in preparing antibacterial preparations.

[0019] The present invention uses the laccase mutant Lcc5-T485E to transform daidzein to obtain a product, and then purifies the product by rotary evaporation, reconstitution centrifugation, rotary evaporation, reconstitution centrifugation and freeze drying. The transformation product has better biological activity than daidzein monomer: the test results of three common methods for detecting antioxidant activity (DPPH method, ABTS method and FRAP method) all show that the antioxidant activity of the transformation product is significantly improved compared with daidzein, such as Figure 5 As shown. a-glucosidase is considered to be one of the key factors for postprandial blood sugar increase. Inhibiting its activity is one of the important treatment strategies for type 2 diabetes. The conversion product significantly reduces the enzymatic activity of a-glucosidase compared to daidzein, such as Figure 6 As shown in the antibacterial experiment, the transformation product has a more significant inhibitory activity against Staphylococcus aureus than daidzein. Figure 7 shown.

[0020] The present invention measures and compares the temperature adaptability, specific enzyme activity and ethanol adaptability of the mutant enzyme and the starting enzyme. The results show that compared with the starting enzyme, the optimum temperature of the mutant is higher. The specific enzyme activity of the mutant enzyme is also improved, about 1.92 times that of the starting enzyme. Compared with the starting enzyme, the ethanol adaptability of the mutant is also improved to a certain extent. The ethanol half-inhibitory concentration of Lcc5 is about 15%-20%, while the ethanol half-inhibitory concentration of the mutant is increased to 20%-25%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1The SDS-PAGE profiles of the purified starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of the present invention are shown in Figure 1. The lanes are respectively a marker, pure Lcc5 enzyme (1), and pure Lcc5-T485E enzyme of the present invention (2).

[0022] Figure 2 The optimum temperature determination results of the starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of the present invention are shown in FIG.

[0023] Figure 3 The relative activities of the starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of the present invention under ethanol concentrations of 5%, 10%, 15%, 20%, 25%, and 30%.

[0024] Figure 4 This is an experiment on the conversion of daidzein by the starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of the present invention.

[0025] Figure 5 The results of the antioxidant test of daidzein and its transformation products are shown in Figure a, which are the results of the DPPH method, the results of the ABTS method, and the results of the FRAP method.

[0026] Figure 6 These are the experimental results of daidzein and its conversion products inhibiting the activity of a-glucosidase.

[0027] Figure 7 The results of the experimental study on the inhibitory activity of daidzein and its transformation products on Staphylococcus aureus are shown in Figure a, and the results of the experimental study on the inhibitory activity of daidzein and its transformation products on Staphylococcus aureus are shown in Figure b.

[0028] Figure 8-19 LC-MS results of the conversion products (ESI, positive ion mode). DETAILED DESCRIPTION

[0029] The implementation methods in the following embodiments are all conventional methods unless otherwise specified.

[0030] (I) Construction of an engineered strain containing the laccase mutant gene of the present invention 1. Design of laccase gene mutants Coprinus cinerea C. cinereaLcc5 obtained by heterologous expression of FA2222 strain was used as the starting enzyme, and homology modeling was performed through the online website (https: / / swissmodel.expasy.org / ). Then Lcc5 and daidzein were simulated and docked using AutoDock Vina. The docking results showed that in the optimal binding conformation, the enzyme formed hydrogen bond interactions with daidzein through multiple amino acids including 485T, which can be used as a candidate site for modifying laccase to enhance the interaction between the enzyme and daidzein and thus improve the conversion performance of the enzyme. Then, based on the amino acid conservation analysis of WebLogo, a mutant was designed and named Lcc5-T485E.

[0031] The amino acid sequence of the laccase mutant enzyme of the present invention is shown in SEQ ID NO: 1, and the changed positions compared with the starting enzyme Lcc5 are as follows: the threonine at position 485 is mutated to glutamic acid.

[0032] 2. Construction of laccase mutant genetic engineering strain The laccase mutant gene in step 1 was constructed and connected to the expression vector pPic9k, and the expression host was Pichia pastoris Pichia pastoris, An engineered strain containing the mutant gene of the present invention is obtained.

[0033] The strain of the present invention Pichia pastoris GS115 / pPic9k(+)Lcc5-T485E , has been sent to the China Center for Type Culture Collection for preservation, the preservation number is CCTCC NO: M 20242012, the preservation date is September 20, 2024, and the preservation address is Wuhan University, Wuhan, China.

[0034] 2. Expression of genetically engineered bacteria containing laccase mutations and protein purification The expression strain obtained in (a) Pichia pastoris GS115 / pPic9k(+)Lcc5-T485E Inoculate into a test tube containing 5 mL of BMGY, culture overnight in a shaker at 28°C and 200 rpm, then transfer to a flask containing 50 mL of BMGY medium and culture until OD 600 When it reaches 2.0, centrifuge at 4°C and 3000 rpm for 20 minutes, and use BMM liquid to resuspend the bacteria; the resuspended bacteria are transferred to a 500mL triangular flask with a baffle (containing 200 mL BMM liquid culture medium). Samples are taken every 24 hours, and an equal amount of methanol is added to the sample, and the culture is induced at 28°C. The changes in the enzyme activity of the sample are detected, and the bacterial liquid is collected after culturing in BMM culture medium for 7 days to purify the target protein. The crude enzyme solution after centrifugation and dialysis is filtered through a 0.22 μm filter membrane to remove impurities and bubbles in the supernatant, and loaded onto a DEAE-SpharoseFastFlow anion exchange column. The purified protein is tested by SDS-PAGE to achieve the purity of enzymatic property detection ( Figure 1 ).

[0035] (III) Detection of temperature adaptability of the mutant enzyme of the present invention Laccase enzyme activity assay method: The total reaction system is 1 mL, including 33 μL ABTS (final concentration 15 mM), 950 μL sodium tartrate buffer solution (pH 4.0), incubated at 30°C for 5 min, and 17 μL enzyme solution was added to the EP tube to make the reaction system 1 mL. After reacting at 30°C for 3 min, the mixture was placed on ice for 30 s and the OD was measured by spectrophotometer. 420 value.

[0036] The enzyme activity calculation formula is: enzyme activity (U / L) = 555.56 × dilution factor × OD 420 .

[0037] Test results such as Figure 2 It was shown that when ABTS was used as substrate, the optimum temperature of the mutant enzyme obtained in the present invention was 70°C.

[0038] (IV) Detection of ethanol tolerance of the mutant enzyme of the present invention With ABTS as substrate, the total reaction system is 1 mL, including 33 μL ABTS (final concentration 15 mM), and 50 μL, 100 μL, 150 μL, 200 μL, 250 μL, and 300 μL of anhydrous ethanol are added respectively, so that the final concentration of ethanol in the system is 5%, 10%, 15%, 20%, 25%, and 30%, respectively. Make up to 983 μL with sodium tartrate buffer solution (pH 4.0). Incubate at 30°C for 5 min, add 17 μL enzyme solution to the EP tube, and make the reaction system 1 mL. After reacting at 30°C for 3 min, let stand on ice for 30 s, and measure OD by spectrophotometer. 420 value.

[0039] The enzyme activity calculation formula is: enzyme activity (U / L) = 555.56 × dilution factor × OD 420 .

[0040] Taking the enzyme activity of the ethanol blank treatment group as 100%, the residual enzyme activity rate in different ethanol concentration systems was calculated using the following formula: residual enzyme activity rate = (blank treatment group enzyme activity - lost enzyme activity) / blank treatment group enzyme activity × 100%.

[0041] The results of the test 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 original enzyme between 15% and 20%.

[0042] (V) Detection of specific enzyme activity of the mutant enzyme of the present invention Laccase enzyme activity assay method: The total reaction system is 1 mL, including 33 mL ABTS (final concentration 15 mM), 950 mL sodium tartrate buffer solution (pH 4.0), incubated at 30°C for 5 min, and 17 mL enzyme solution was added to the EP tube to make the reaction system 1 mL. After reacting at 30°C for 3 min, the mixture was placed on ice for 30 s and the OD was measured by spectrophotometer. 420 value.

[0043] The enzyme activity calculation formula is: enzyme activity (U / L) = 555.56 × dilution factor × OD 420 .

[0044] Protein concentration detection: Add 20 mL of the protein solution to be detected and 200 mL of Brandford solution into a 96-well plate and mix well. 595 The absorbance value of the mixed solution is measured at a wavelength, and the concentration of the laccase protein is further calculated based on the measured absorbance data.

[0045] The specific enzyme activity calculation formula is: enzyme activity / protein concentration.

[0046] The test results are shown in Table 1. The specific enzyme activity of the mutant enzyme obtained in the present invention is increased by 92% compared with the starting enzyme.

[0047]

[0048] (VI) Application of the Laccase Mutant Enzyme Containing the Present Invention in the Oxidative Conversion Experiment of Soybean Aglycone The conversion reaction system was 1 mL, in which the enzyme amount was 40 U (measured with ABTS as substrate), citric acid-sodium hydrogen phosphate buffer (pH 8.0) was added to 1 mL, and the final concentration of daidzein was 0.5 mg / mL. The mixture was shaken and placed in a constant temperature water bath shaker at 40°C and 150 r / min for 1 h. 500 μL of the reaction system was added with 500 μL of ethanol to terminate the reaction, and then vortexed for 30 s using a vortex oscillator. The ethanol containing daidzein was filtered using a 0.22 μm organic and aqueous needle filter for detection on the machine.

[0049] HPLC detection conditions: mobile phase: containing 0.1% acetonitrile and water, the chromatographic column selected is Agilent XDB-C18 column (column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm, Sun Fire); set the flow rate to 0.5 mL / min; column temperature to 30 °C; detection wavelength to 330 nm.

[0050] HPLC elution system:

[0051] The experimental results are as follows Figure 4It showed that under the conditions of pH 8.0 and 40℃, with soybean aglycone as substrate, the oxidation conversion rate of the mutant enzyme Lcc5-T485E was 88%, and 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.

[0052] (VII) Improvement of the antioxidant activity of the conversion product of the present invention The activity was measured by three methods: DPPH method, ABTS method and FRAP method.

[0053] DPPH method: Prepare 1mg / ml conversion product and daidzein solution, weigh 2mg of DPPH and dissolve it in 48mL of anhydrous ethanol. Store in dark and use up within 5 hours. Take 1mL of the DPPH solution prepared in the above steps, dilute it with anhydrous ethanol, and make its absorbance about 0.8 to obtain the working solution. Take 1ml of DPPH working solution and add 0.5ml of the test solution, mix well and place in dark for 30min. Detect the absorbance at 517nm and adjust it to zero with anhydrous ethanol.

[0054] Antioxidant rate = {1-(A1-A2) / A3}×100%.

[0055] A1: DPPH + test solution.

[0056] A2: Anhydrous ethanol + test solution.

[0057] A3: DPPH + anhydrous ethanol.

[0058] Test results such as Figure 5 As shown in Figure a, using the DPPH method, under the same conditions, the antioxidant rate of the conversion product was about 26.64%, and the antioxidant rate of daidzein monomer was about 12.72%. The antioxidant rate of the conversion product was 2.09 times higher than that of daidzein monomer.

[0059] ABTS method: Prepare 1 mg / mL conversion product and daidzein solution; take 3 mg of ABTS diammonium salt and add 0.735 mL of distilled water to prepare ABTS diammonium salt stock solution (7.4 mmol / L). Take 10 mg of potassium persulfate and add 14.3 mL of distilled water to prepare potassium persulfate stock solution (2.6 mmol / L). Mix 0.2 mL of ABTS diammonium salt stock solution and 0.2 mL of potassium persulfate stock solution, place at room temperature in a dark environment for 12 hours, and dilute the mixture with anhydrous ethanol to an absorbance of 0.70 ± 0.02. This solution is ABTS. + Free radical working solution. Take 950ml of working solution, add 50ml of test solution, mix well and place in dark place for 30min. Detect absorbance at 734nm and adjust to zero with anhydrous ethanol.

[0060] Antioxidant activity = { (A0-A1-A2) / A0} × 100%.

[0061] A0: working solution + anhydrous ethanol.

[0062] A1: working solution + test solution.

[0063] A2: Anhydrous ethanol + test solution.

[0064] Test results such as Figure 5 As shown in Figure b, using the ABTS method, under the same conditions, the antioxidant rate of the conversion product was about 77.46%, and the antioxidant rate of daidzein monomer was about 42.44%. The antioxidant rate of the conversion product was increased to 1.83 times that of the daidzein monomer.

[0065] FRAP method: Prepare 1 mg / mL solution of transformation product and daidzein; perform detection according to the method described in the instruction manual of FRAP kit (Solabo). 2+ The concentration standard curve was then used to detect the conversion product and the effect of daidzein on Fe according to the method in the manual. 3+ The test results are as follows: Figure 5 As shown in Figure c, using the same mass concentration of transformation product and soybean aglycone solution, the transformation product makes the Fe 2+ It increased to 3.44 times that of the daidzein-treated group.

[0066] (VIII) Improvement of the anti-α-glucosidase activity of the conversion product of the present invention α-Glucosidase and p-nitrophenyl-aD-pyranoglucoside (pNPG) were purchased from Yuanye Company. First, 100U / mL enzyme solution was prepared according to the instructions, and diluted to 1U / mL using pH7.0 citric acid-sodium hydrogen phosphate buffer as the working solution. 5mM, 7.5mM, 10mM, 12.5mM, 15mM, 17.5mM, 20mM, 22.5mM and 25mM pNPG solutions were prepared respectively. The conversion product and soybean aglycone were dissolved in dimethyl sulfoxide (DMSO) solution at a concentration of 1mg / mL. PBS buffer with pH 7.0 and 200mM was prepared. The reaction system was 500ml, of which the volume of the enzyme working solution, pNPG solutions of different concentrations and the solution to be tested was 25ml, and the volume was supplemented to 500ml with PBS solution. The mixture was placed in a 37°C water bath for 30 min, and then 500 ml of 1 M Na2CO3 solution was added to terminate the reaction. The absorbance was detected at a wavelength of 405 nm.

[0067] The results are as follows Figure 6As shown in the figure, it can be seen that daidzein inhibits the activity of a-glucosidase in converting pNPG, and the degree of inhibition of the conversion product is more obvious. Compared with daidzein, the inhibitory effect of the conversion product is significantly improved.

[0068] (IX) Improvement of the inhibitory activity of Staphylococcus aureus containing the transformation product of the present invention Staphylococcus aureus comes from the previous collection in the laboratory. First, Staphylococcus aureus was inoculated in LB for activation. After growing in a shake flask for 12 hours, it was diluted about 3000 times with LB. The transformation product and soybean aglycone were dissolved in DMSO at a concentration of 1 mg / mL, and then diluted to 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL, and 0.2 mg / mL, respectively. The bacterial solution and the sample to be tested were added to a 96-well plate with a volume of 190 ml bacterial solution + 10 ml test solution. The concentrations of the samples to be tested in the system were 50 mg / mL, 40 mg / mL, 30 mg / mL, 20 mg / mL, and 10 mg / mL, respectively. Place in a shaker at 37°C and 220 rpm for continuous culture for 8 hours, and detect OD every 2 hours. 600 Absorbance value.

[0069] The results are as follows Figure 7 As shown, both daidzein and its transformation product slowed down the growth of the OD value of Staphylococcus aureus, and the inhibition of the growth of the OD value of Staphylococcus aureus by the transformation product was more significant.

[0070] (X) LC-MS test results of the conversion product of the present invention The elution system is as described in the specific method (V), and the mass spectrometry conditions are ESI, positive ion mode, and the detection range is 100-1500m / z. Figure 8-19 As shown, the results showed that the conversion products were coupled to form oligomeric compounds. After analysis, it was speculated that the oligomeric products included tetrameric and dimer structures.

Claims

1. A laccase mutant, abbreviated as Lcc5-T485E, whose amino acid sequence is shown in SEQ ID NO:

1.

2. The coding gene of the laccase mutant according to claim 1, wherein the nucleotide sequence thereof is shown in SEQ ID NO:

2.

3. A mutant plasmid containing the coding gene as claimed in claim 2.

4. An expression strain of the laccase mutant according to claim 1, comprising the mutant plasmid according to claim 3.

5. The expression strain of the laccase mutant according to claim 1, characterized in that: The expression strains were classified as Pichia pastoris GS115 / pPic9k(+)Lcc5-T485E , has been sent to the China Center for Type Culture Collection for preservation, the preservation number is CCTCC NO: M 20242012, the preservation date is September 20, 2024, and the preservation address is Wuhan University, Wuhan, China.

6. Use of the laccase mutant according to claim 1 in the preparation of oligomeric compounds by oxidative conversion of soybean aglycones.

7. The use according to claim 6, characterized in that: During the oxidative transformation of daidzein, the system temperature was 40°C and the pH value was 8.

0.

8. Use of the oligomer compound prepared according to claim 6 or 7 in the preparation of antioxidant preparations.

9. Use of the oligomer compound prepared according to claim 6 or 7 in the preparation of α-glucosidase inhibitors.

10. Use of the oligomer compound prepared according to claim 6 or 7 in the preparation of an antibacterial preparation.

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