Laccase mutant and application thereof in oxidative conversion of aflatoxin

Abstract

The invention discloses a laccase mutant and application thereof in oxidative conversion of aflatoxin. According to the invention, laccase of coprinus cinerea is used as a starting enzyme, and through molecular docking, a structure-function relationship is analyzed, and mutation is designed. After induced expression of engineering bacteria containing mutant genes, mutant laccase Lcc5-T485E with improved detoxification efficiency is obtained through screening. When the enzyme with the same protein mass is used, the AFB1 conversion efficiency of the mutant Lcc5-T485E is obviously improved compared with that of Lcc5, and the mutant enzyme has potential application value in oxidative conversion of aflatoxin.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a laccase mutant and application thereof in oxidative transformation of aflatoxin. 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). It can effectively oxidize phenols and aromatic compounds with the help of the strong redox ability of copper ions, and reduce molecular oxygen to water. Since laccase can catalyze a wide range of substrates and can directly act on a variety of phenolic toxic substances and some aromatic harmful substances, and for some phenolic substances that cannot be directly acted on, laccase can also effectively detoxify them after adding some common mediators such as vanillic acid, vanillin, and syringic acid. Therefore, laccase has many applications in industrial production, such as food processing, sewage treatment, biosensors, and biological detoxification.

[0003] Aflatoxins (AFT) are mainly derived from Aspergillus. Because of the presence of a difuran ring and oxa-naphthoquinone in its structure, aflatoxin B1 (AFB1) is the most toxic among all its structural analogs, and has strong carcinogenicity and toxicity. In liver cells, cytochrome P450 enzymes can convert AFB1 into the main carcinogenic metabolite AFBO (AFB1-8,9-epoxide), which can bind to genetic material and induce mutations, leading to toxic effects and the occurrence of various cancers.

[0004] Currently, laccases used in toxin oxidation and transformation are mostly natural laccases produced by fungal fermentation. In the actual toxin oxidation and transformation system, the enzymatic activity of natural laccases is not sufficient for practical application. Therefore, it is necessary to transform natural laccases to screen mutants with higher enzymatic activity. Summary of the invention

[0005] In view of the problems existing in the above-mentioned prior art, the present invention provides a laccase mutant and its application in the oxidation and conversion of aflatoxin. C. cinerea ) as the starting enzyme, and the mutant gene was obtained through strategies such as structural simulation, molecular docking, and site-directed mutagenesis. After the expression bacteria containing the mutant gene were induced to express, the laccase mutant Lcc5-T485E with improved enzyme activity was obtained. The mutant enzyme Lcc5-T485E has potential application value in the oxidation and conversion of aflatoxin: under the same protein quality conditions, Lcc5-T485E oxidized and converted 96% of aflatoxin, while the starting enzyme Lcc5 could only oxidize and convert 80% of aflatoxin, and the oxidation conversion rate of the mutant enzyme was increased to 1.2 times that of the starting enzyme.

[0006] The amino acid sequence of the laccase mutant Lcc5-T485E of the present invention 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 Lcc5-T485E 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 Lcc5-T485E 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 strain of the laccase mutant enzyme of the present invention is classified and named 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 was performed with aflatoxin AFB1 to infer the key sites involved in the reaction on the enzyme and their mechanism, and mutations that can promote the enzyme catalytic reaction were designed based on this. The laccase Lcc5-T485E with potentially improved detoxification performance was identified, the mutant gene was synthesized 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 Lcc5-T485E of the present invention can be obtained by fermenting the expression strain.

[0014] The invention discloses an application of the laccase mutant enzyme in the oxidation and conversion of aflatoxin AFB1.

[0015] 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 946.4U / mg, and the specific enzyme activity of Lcc5-T485E was 2128.3U / mg. The specific enzyme activity of Lcc5-T485E was significantly improved by 1.25 times compared with the starting enzyme Lcc5. The reaction was carried out in a constant temperature water bath shaker at pH 7, 45℃, and 150 r / min for 12 h. In a 1ml system, the oxidation conversion rate of 0.01mg mutant enzyme Lcc5-T485E to 1ug / ml aflatoxin AFB1 was 96%, while the oxidation conversion rate of the same amount of starting enzyme Lcc5 was 80%. The oxidation conversion rate of the mutant enzyme was 20% higher than that of the starting enzyme. The mutant T485E designed according to the oxidation mechanism has a significantly improved specific enzyme activity, and the ability to convert AFB1 is improved under the same enzyme activity and the same protein quality conditions. The mutant enzyme has potential application value in the oxidation and conversion of aflatoxin AFB1.

[0016] The present invention measured and compared the temperature adaptability of the mutant enzyme and the original enzyme. The measurement results showed that the optimum temperature of the mutant was higher than that of the original enzyme.

[0017] The present invention measures and compares the ethanol adaptability of the mutant enzyme and the starting enzyme. The starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of the present invention were subjected to relative activity measurement under the conditions of ethanol concentrations of 5%, 10%, 15%, 20%, 25%, and 30%. The results showed that compared with the starting enzyme, the ethanol adaptability of the mutant was also improved to a certain extent. The ethanol half-inhibition concentration of Lcc5 was about 15%-20%, while the ethanol half-inhibition concentration of the mutant was increased to 20%-25%. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The SDS-PAGE diagram of the purified starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of the present invention: the lanes are Marker, 1: pure Lcc5 enzyme, 2: pure Lcc5-T485E enzyme of the present invention.

[0019] 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.

[0020] Figure 3 The figures are the results of ethanol tolerance test of the starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of the present invention.

[0021] Figure 4 is the oxidation conversion rate of aflatoxin AFB1 by the starting enzyme Lcc5 and the mutant enzyme Lcc5-T485E of the present invention. DETAILED DESCRIPTION

[0022] The implementation methods in the following embodiments are all conventional methods unless otherwise specified.

[0023] (I) Construction of an engineered strain containing the laccase mutant gene of the present invention 1. Design of laccase gene mutants Coprinus cinerea C. cinerea Lcc5 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 aflatoxin AFB1 were simulated and docked using AutoDock Vina. The docking results showed that in the optimal binding conformation, the enzyme formed hydrogen bond interactions with the toxin through multiple amino acids including T485, and this amino acid can be used as a candidate site for modifying laccase to enhance the interaction between the enzyme and the toxin and thus enhance the detoxification performance of the enzyme. Based on the amino acid conservation analysis of WebLogo, a mutant was designed and named Lcc5-T485E.

[0024] 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.

[0025] 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.

[0026] The expression strain of the laccase mutant enzyme of the present invention is classified and named 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.

[0027] 2. Determination of the relative enzyme activity of genetically engineered bacteria containing laccase mutations 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.

[0028] The enzyme activity calculation formula is: enzyme activity (U / L) = 555.56 × dilution factor × OD420 .

[0029] Protein concentration detection: Add 20 μL of the protein solution to be detected and 200 μL of Brandford solution into a 96-well plate and mix well. OD 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.

[0030] The specific enzyme activity calculation formula is: enzyme activity / protein concentration.

[0031] The determination results are shown in Table 1. The specific enzyme activity of Lcc5 is 946.4 U / mg; the specific enzyme activity of Lcc5-T485E is 2128.3 U / mg.

[0032]

[0033] 3. Expression and protein purification of genetically engineered bacteria containing laccase mutations 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). Sampling is performed 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 0.22μm filter membrane to remove impurities and bubbles in the supernatant, and then loaded onto a DEAE-SpharoseFastFlow anion exchange column for purification. The obtained protein is tested by SDS-PAGE to reach the purity of enzymatic property detection ( Figure 1 ).

[0034] (IV) Detection of temperature adaptability of the laccase mutant enzyme containing 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.

[0035] The enzyme activity calculation formula is: enzyme activity (U / L) = 555.56 × dilution factor × OD 420 .

[0036] Test results such as Figure 2 As shown, when ABTS is used as substrate, the optimum temperature of the mutant enzyme obtained by the present invention is 70°C.

[0037] (V) Ethanol tolerance test of the laccase mutant enzyme containing the present invention In the system, ABTS was used as the substrate, including 33 μL ABTS (final concentration 15mM), and 50 μL, 100 μL, 150 μL, 200 μL, 250 μL, and 300 μL of 100% anhydrous ethanol were added respectively, so that the final concentration of ethanol in the system was 5%, 10%, 15%, 20%, 25%, and 30%, respectively. Sodium tartrate buffer solution (pH 4.0) was used to make up to 983 μL. Incubate at 30℃ for 5min, add 17 μL enzyme solution to the EP tube, and make the reaction system 1mL. After reacting at 30℃ for 3min, stand on ice for 30s, and measure the OD420 value with a spectrophotometer.

[0038] The enzyme activity calculation formula is: enzyme activity (U / L) = 555.56 × dilution factor × OD 420 .

[0039] 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%.

[0040] The test results show that the ethanol half-inhibitory concentration of the mutant enzyme activity obtained by the present invention is between 20% and 25%, compared with the ethanol half-inhibitory concentration of the original enzyme between 15% and 20%, indicating that its ethanol tolerance is improved.

[0041] (VI) Application of the Laccase Mutant Enzyme Containing the Present Invention in Toxin Oxidation and Conversion Experiments The detoxification reaction system was 1 mL, in which the enzyme amount was 0.01 mg, the citric acid disodium hydrogen phosphate buffer (pH 7.0) was added to 950 μL, and the final concentration of aflatoxin AFB1 was 1 μg / mL. The mixture was shaken and placed in a constant temperature water bath shaker at 45°C and 150 rpm for 12 h. 200 μL of the reaction system was added with 200 μL of methanol to terminate the reaction, and then vortexed for 30 s using a vortex shaker. The methanol containing aflatoxin AFB1 was filtered using a 0.22 μm organic needle filter for detection on the machine.

[0042] HPLC detection conditions: mobile phase: methanol-water = 45:55 (V / V), chromatographic column selected Agilent 5 TC-C18 (2) column (column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm, Sun Fire); flow rate set to 0.8 mL / min; column temperature set to 25°C; detector set to fluorescence detection excitation wavelength 360 nm; emission wavelength 440 nm. Post-column photochemical derivatization device.

[0043] Test results such as Figure 4 It showed that under the same conditions, with aflatoxin AFB1 as substrate, the oxidation conversion rate of the mutant enzyme Lcc5-T485E was 96%, and the oxidation conversion rate of the starting enzyme Lcc5 was 80%. The oxidation conversion rate of the mutant enzyme was 20% higher than that of the starting enzyme.

Claims

1. A laccase mutant, the amino acid sequence of which 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 oxidation and conversion of aflatoxin AFB1.

7. The use according to claim 6, characterized in that: During the oxidation conversion of aflatoxin, the system temperature was 45°C and the pH value was 7.0.

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