A laccase mutant and its application in the oxidative transformation of aflatoxin

Abstract

This invention discloses a laccase mutant and its application in the oxidative transformation of aflatoxin. This invention uses *Coprinus comatus* (Grey-capped Coprinus) as an example. Coprinopsis cinerea Using laccase as the starting enzyme, structure-activity relationship analysis was conducted through molecular docking, and mutations were designed. After inducing expression in engineered bacteria containing the mutant gene, a mutant laccase Lcc5-T485E with improved detoxification efficiency was obtained through screening. When using the same protein content, the mutant Lcc5-T485E showed a significant improvement in AFB1 conversion efficiency compared to Lcc5, indicating its potential application value in the oxidative conversion of aflatoxin.

Description

Technical Field

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

[0002] Lacase, a copper-containing polyphenol oxidase also known as phenolase, is a member of the blue polycopper oxidase (MCO) family. It effectively oxidizes phenolic and aromatic compounds using the strong redox properties of copper ions, while simultaneously reducing molecular oxygen to water. Lacase has a very broad substrate catalytic range and can directly act on many toxic phenolic substances and some harmful aromatic compounds. For some phenolic substances that cannot be directly acted on, laccase can effectively detoxify them by adding common mediators such as vanillic acid, vanillin, and syringic acid. Therefore, laccase has many applications in industrial production, such as food processing, wastewater treatment, biosensors, and biological detoxification.

[0003] Aflatoxins (AFT) are primarily derived from the Aspergillus genus. Due to the presence of a difuran ring and oxanaphenone in their structure, aflatoxin B1 (AFB1) is the most toxic of all its structural analogs, exhibiting strong carcinogenicity and toxicity. Within liver cells, cytochrome P450 enzymes can convert AFB1 into its main carcinogenic metabolite, AFBO (AFB1-8,9-epoxide). AFBO can bind to genetic material, inducing mutations, thereby leading to toxic effects and the development of various cancers.

[0004] Currently, most laccases used in toxin oxidation and conversion are natural laccases produced by fungal fermentation. However, in actual toxin oxidation and conversion systems, the enzyme activity of natural laccases is insufficient for practical applications. Therefore, it is necessary to modify natural laccases to screen for mutants with higher enzyme activity. 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 transformation of aflatoxin. This invention uses *Coprinus comatus* (Grey-capped Coprinus) as an example. C. cinerea Using laccase Lcc5 as the starting enzyme, a mutant gene was obtained through strategies such as structural mimicry, molecular docking, and site-directed mutagenesis. After induction of expression in bacteria containing the mutant gene, the laccase mutant Lcc5-T485E with enhanced enzyme activity was obtained. The mutant enzyme Lcc5-T485E has potential application value in the oxidative conversion of aflatoxin: under the same protein content conditions, Lcc5-T485E oxidizes and converts 96% of aflatoxin, while the starting enzyme Lcc5 can only oxidize and convert 80% of aflatoxin, increasing the oxidation conversion rate of the mutant enzyme to 1.2 times that of the starting enzyme.

[0006] The laccase mutant of the present invention, 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 Lcc5-T485E of the present invention may further include a combination of nonsense mutations or synonymous mutations in its amino acid sequence.

[0008] The encoding gene of the laccase mutant Lcc5-T485E 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 expression strain of the laccase mutant enzyme of this invention is classified and named 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 aflatoxin AFB1 was performed to deduce key reaction sites and mechanisms on the enzyme, and based on this, mutants that could promote enzyme-catalyzed reactions were designed. A laccase Lcc5-T485E with potentially enhanced detoxification performance was identified. The mutant gene was synthesized 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 Lcc5-T485E of the present invention can be obtained by fermentation of the expressed strain.

[0015] The application of laccase mutant enzyme in the oxidative transformation of aflatoxin AFB1 is described in this invention.

[0016] The enzyme activity and protein concentration of laccase were determined, and the specific enzyme activity was calculated. The specific enzyme activity of the starting enzyme Lcc5 was 946.4 U / mg, and that of Lcc5-T485E was 2128.3 U / mg. The specific enzyme activity of Lcc5-T485E was significantly increased by 1.25 times compared to the starting enzyme Lcc5. After reacting in a constant temperature water bath at pH 7, 45℃, and 150 r / min for 12 h, in a 1 ml system, 0.01 mg of the mutant enzyme Lcc5-T485E achieved a 96% oxidation conversion rate of 1 μg / ml aflatoxin AFB1, while the same amount of the starting enzyme Lcc5 achieved a 80% oxidation conversion rate. The oxidation conversion rate of the mutant enzyme was 20% higher than that of the starting enzyme. The mutant T485E, designed based on the oxidation mechanism, showed a significant increase in specific enzyme activity and improved ability to convert AFB1 under the same enzyme activity and protein content conditions. This mutant enzyme has potential application value in the oxidative conversion of aflatoxin AFB1.

[0017] This invention measured and compared the temperature adaptability of the mutant enzyme and the starting enzyme. The results showed that the optimal temperature of the mutant was higher than that of the starting enzyme.

[0018] This invention measured and compared the ethanol adaptability of the mutant enzyme and the starting enzyme. The relative activities of the starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of this invention were measured at ethanol concentrations of 5%, 10%, 15%, 20%, 25%, and 30%. The results showed that, compared with the starting enzyme, the mutant's ethanol adaptability was also improved to a certain 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

[0019] Figure 1 SDS-PAGE images of the purified starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of this invention: each lane represents a marker, 1: pure Lcc5 enzyme, 2: pure Lcc5-T485E enzyme of this invention.

[0020] Figure 2 The results show the optimal temperature determination of the starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of this invention.

[0021] Figure 3 The results show the ethanol tolerance assay results for the starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of this invention.

[0022] Figure 4 The oxidation conversion rate of aflatoxin AFB1 by the starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of this invention is given. Detailed Implementation

[0023] Unless otherwise specified, the implementation methods in the following embodiments are all conventional methods.

[0024] (i) Construction of engineered strains containing the laccase mutant gene of the present invention

[0025] 1. Design of laccase gene mutants

[0026] This invention uses gray-covered ginkgo umbrellas C. cinerea Lcc5, obtained from heterologous expression of strain FA2222, was used as the starting enzyme, and homology modeling was performed using an online website (https: / / swissmodel.expasy.org / ). Next, Lcc5 was simulated for docking with aflatoxin AFB1 using AutoDock Vina. The docking results showed that in the optimal binding conformation, the enzyme interacts with the toxin through hydrogen bonds formed by multiple amino acids, including T485. This amino acid can serve as a candidate site for modifying laccase to enhance enzyme-toxin interaction and thus improve the enzyme's detoxification performance. Based on the amino acid conservation analysis of WebLogo, a mutant was designed and named Lcc5-T485E.

[0027] 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.

[0028] 2. Construction of laccase mutant genetically engineered strains

[0029] 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.

[0030] The expression strain of the laccase mutant enzyme of this invention is classified and named 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.

[0031] (II) Determination of enzyme activity of genetically engineered bacteria containing laccase mutants

[0032] 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.

[0033] The formula for calculating enzyme activity is: Enzyme activity (U / L) = 555.56 × Dilution factor × OD 420 .

[0034] Protein concentration detection: Add 20 μL of the protein solution to be tested and 200 μL of Brandford solution to a 96-well plate and mix thoroughly. Use a microplate reader to analyze the protein concentration. OD 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.

[0035] Formula for calculating enzyme activity: enzyme activity / protein concentration.

[0036] The results are shown in Table 1. The enzyme activity of Lcc5 was 946.4 U / mg, and the enzyme activity of Lcc5-T485E was 2128.3 U / mg.

[0037]

[0038] (III) 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 medium and incubate overnight at 28°C and 200 rpm. Then transfer to Erlenmeyer flasks containing 50 mL of BMGY medium and incubate until... OD 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 h, 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 solution is then purified using a DEAE-Spharose FastFlow anion exchange column. The obtained protein is tested by SDS-PAGE to achieve the purity required for enzymatic characterization. Figure 1 ).

[0040] (iv) Detection of temperature adaptability of the laccase 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 As shown, when using ABTS as a substrate, the optimal temperature for the mutant enzyme obtained in this invention is 70°C.

[0044] (v) Detection of ethanol tolerance to laccase mutant enzyme of the present invention

[0045] In the system, ABTS was used as the substrate, including 33 μL of ABTS (final concentration 15 mM), followed by the addition of 50 μL, 100 μL, 150 μL, 200 μL, 250 μL, and 300 μL of 100% anhydrous ethanol, respectively, to achieve final ethanol concentrations of 5%, 10%, 15%, 20%, 25%, and 30%. The final volume was 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 OD420 value was measured using a spectrophotometer.

[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 test results show that the ethanol half-inhibitory concentration of the mutant enzyme activity obtained in this invention is between 20% and 25%, which is between 15% and 20% compared with the original enzyme, indicating that its ethanol tolerance has been improved.

[0049] (vi) Application of the laccase mutant enzyme of the present invention in the toxin oxidation-conversion experiment

[0050] The detoxification reaction system consisted of 1 mL of 0.01 mg enzyme, 950 μL of disodium citrate phosphate buffer (pH 7.0), and a final concentration of 1 μg / mL of aflatoxin AFB1. The mixture was shaken and incubated in a constant temperature water bath at 45°C and 150 rpm for 12 h. 200 μL of the reaction system was taken and 200 μL of methanol was added to terminate the reaction. The mixture was then vortexed for 30 s. The methanol containing aflatoxin AFB1 was filtered through a 0.22 μm organic syringe filter for instrumental analysis.

[0051] HPLC detection conditions: Mobile phase: methanol-water = 45:55 (V / V); chromatographic column: Agilent 5 TC-C18(2) column (250 mm length, 4.6 mm inner diameter, 5 μm particle size, Sun Fire); flow rate: 0.8 mL / min; column temperature: 25℃; detector settings: fluorescence detection excitation wavelength: 360 nm; emission wavelength: 440 nm. A photochemical derivatization device was then connected after the column.

[0052] Test results as follows Figure 4 The results showed that, under the same conditions, using aflatoxin AFB1 as a substrate, the oxidation conversion rate of the mutant enzyme Lcc5-T485E was 96%, while that of the starting enzyme Lcc5 was 80%, indicating that the oxidation conversion rate of the mutant enzyme was 20% higher than that of the starting enzyme.

Claims

1. A laccase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A gene encoding the laccase mutant of claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

2.

3. A plasmid, characterized in that, The plasmid contains the encoding gene as described in claim 2.

4. The expression strain of the laccase mutant according to claim 1, characterized in that: The expression strain contains the plasmid described in claim 3.

5. The expression strain of the laccase mutant according to claim 1, characterized in that: The taxonomic designation of the expression strain is Pichia pastoris GS115 / pPic9k(+)Lcc5-T485E, which has been deposited with the China Center for Type Culture Collection and has the accession number CCTCC NO: M 20242012, and was deposited on September 20, 2024, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

6. The application of the laccase mutant of claim 1 in the oxidative transformation of aflatoxin AFB1.

7. The application according to claim 6, characterized in that: During the oxidative transformation of aflatoxin AFB1, the system temperature was 45℃ and the pH value was 7.0.

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