A laccase mutant Lcc5-I479T, its expression strain, and its applications

Abstract

This invention discloses a laccase mutant, Lcc5-I479T, its expression strain, and its applications. Using laccase from *Coprinopsis cinerea* as the starting enzyme, this invention obtained the mutant gene through molecular docking and positional analysis. After induction expression in engineered bacteria containing the mutant gene, the stable laccase mutant enzyme Lcc5-I479T was obtained. Using ABTS as a substrate, the mutant enzyme Lcc5-I479T showed a stable increase in activity to 8-20 times that of the starting enzyme at 35-50℃. This mutant enzyme has potential application value in the oxidative transformation of aflatoxin.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a laccase mutant Lcc5-I479T, its expression strain, and its applications. Background Technology

[0002] Laccase, also known as phenolase or polyphenol oxidase, is a copper-containing polyphenol oxidase and the most important member of the blue polycopper oxidase family (MCO). It utilizes the unique redox properties of copper ions to oxidize phenolic and aromatic compounds, while simultaneously reducing molecular oxygen to water. Because laccase has a very broad substrate catalytic range and can directly act on many toxic phenolic substances and some harmful aromatic substances, and because it can also oxidize and decompose some phenolic substances that cannot be directly acted on, it 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 (AFTs) primarily originate from various molds such as Aspergillus, Aspergillus parasiticus, Aspergillus rubrum, Aspergillus lilacinus, and Aspergillus pseudolilacinus, and are commonly found in the fields, during storage, and processing of grains such as peanuts, oilseeds, and corn. Due to the presence of difuran rings and oxanaphenones, they exhibit strong carcinogenic and toxic properties. Cytochrome P450 enzymes in the liver can convert AFB1 into its main carcinogenic metabolite, AFB1-8,9-epoxide (AFBO), or a less mutagenic form. AFBO can bind to genetic material, causing mutations that lead to toxicity and a range of cancers.

[0004] Currently, most laccases used in toxin oxidation and conversion are natural laccases produced by fungal fermentation. In actual toxin oxidation and conversion systems, natural laccases are easily inactivated. Therefore, it is necessary to modify natural laccases to screen for mutants with higher stability. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a laccase mutant Lcc5-I479T, its expression strain, and its applications. This invention uses laccase Lcc5 from *C. cinerea* as the starting enzyme and obtains the mutant gene through strategies such as structural simulation, molecular docking, and site-directed mutagenesis. After induction expression in the expression strain containing the mutant gene, the stable laccase mutant enzyme Lcc5-I479T is obtained. The half-life of the mutant enzyme at 45℃ is increased to 84 h, which is 20 times that of the starting enzyme. The mutant enzyme Lcc5-I479T has potential application value in the oxidative conversion of aflatoxin: under the same conditions, Lcc5-I479T oxidizes and converts 76% of aflatoxin, while the starting enzyme Lcc5 can only oxidize and convert 58% of aflatoxin, increasing the oxidation conversion rate of the mutant enzyme to 1.31 times that of the starting enzyme.

[0006] The laccase mutant of the present invention, Lcc5-I479T, 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: isoleucine at position 457 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 encoding gene of the laccase mutant Lcc5-I479T of the present invention has the nucleotide sequence shown in SEQ ID NO:2.

[0009] The mutant gene of this invention is a gene encoding a laccase mutant as described in SEQ ID NO:2.

[0010] The engineered strain of the laccase mutant of the present invention contains the mutant gene.

[0011] The expression strain of the laccase mutant Lcc5-I479T of this invention, classified as Pichia pastorisGS115 / pPic9k(+)Lcc5-I479T, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20232460, on December 5, 2023, at Wuhan University, Wuhan, China.

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

[0013] Using laccase Lcc5 from *C. cinerea* as the starting enzyme, its structure was predicted through homology modeling. Molecular docking simulation with aflatoxin AFB1 was performed to deduce key sites on the enzyme involved in the reaction and its catalytic mechanism. Based on this, mutations that could promote the enzyme's catalytic reaction were designed. The laccase Lcc5-I479T, which was identified as potentially enhancing detoxification performance, was constructed and ligated into the vector pPic9k. The linearized plasmid pPic9k was electroporated into the expression host *Pichia pastoris* GS115. The mutant gene was integrated into the *Pichia pastoris* alcohol oxidase genome. After plate selection, engineered strains containing the mutant gene of this invention were obtained.

[0014] The laccase mutant enzyme of this invention can be obtained by fermentation of the engineered strain.

[0015] The application of the laccase mutant Lcc5-I479T in this invention involves using it for the oxidative conversion of aflatoxin AFB1. Under constant temperature water bath shaking conditions at pH 7, 45℃, and 150 rpm for 24 h, the mutant enzyme Lcc5-I479T achieved an oxidative conversion rate of 76% for aflatoxin AFB1, compared to 58% for the original enzyme Lcc5. The mutant enzyme's oxidative conversion rate was 31% higher than that of the original enzyme. This mutant enzyme has potential application value in the oxidative conversion of aflatoxin AFB1.

[0016] This invention measured and compared the temperature adaptability and temperature stability of the mutant enzyme and the original enzyme. The results showed that the optimal temperature of the mutant was higher than that of the original enzyme. Furthermore, the stability of the mutant enzyme was also improved; at 45°C and pH 7, the half-life of the mutant enzyme was increased to 20 times that of the original enzyme. Attached Figure Description

[0017] Figure 1 SDS-PAGE images of the purified starting enzyme Lcc5 and the mutant enzyme Lcc5-I479T of this invention: each lane represents the marker, the purified Lcc5 enzyme, and the purified Lcc5-I479T enzyme of this invention, respectively.

[0018] Figure 2 The results show the optimal temperature determination of the starting enzyme Lcc5 and the mutant enzyme Lcc5-I479T of this invention.

[0019] Figure 3 Figure a shows the stability of the starting enzyme Lcc5 at 35℃, 40℃, 45℃, and 50℃; Figure b shows the stability of the mutant enzyme Lcc5-I479T of this invention at 35℃, 40℃, 45℃, and 50℃.

[0020] Figure 4This study investigated the oxidative transformation of aflatoxin AFB1 by the starting enzyme Lcc5 and the mutant enzyme Lcc5-I479T of this invention. Detailed Implementation

[0021] Unless otherwise specified, the implementation methods in the following embodiments are all conventional methods.

[0022] (I) Construction of engineered strains containing the laccase mutant gene of the present invention

[0023] 1. Obtaining enzymes by mutating the laccase gene

[0024] This invention uses Lcc5, obtained from heterologous expression of the *C. cinerea* FA2222 strain, as the starting enzyme, and performs homology modeling using an online website (https: / / swissmodel.expasy.org / ). Then, Lcc5 is subjected to AutoDockVina docking simulation with aflatoxin AFB1. The docking results show that in the optimal binding conformation, the enzyme interacts with the toxin through hydrogen bonds formed by amino acids. This amino acid can serve as a candidate site for modifying laccase to enhance the interaction between the enzyme and the toxin, thereby improving the enzyme's detoxification performance. Based on the amino acid conservation analysis of WebLogo, a mutant was designed and named Lcc5-I479T.

[0025] 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 have been made: isoleucine at position 457 has been mutated to glutamic acid.

[0026] 2. Construction of laccase mutant genetically engineered strains

[0027] The laccase mutant gene from step 1 was constructed and ligated into the expression vector pPic9k. The expression host was Pichia pastoris, resulting in an engineered strain containing the mutant gene of this invention.

[0028] The strain Pichia pastoris GS115 / pPic9k(+)Lcc5-I479T of this invention has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20232460, on December 5, 2023, at Wuhan University, Wuhan, China.

[0029] (II) Expression and protein purification of genetically engineered bacteria containing laccase mutants

[0030] The expression strain Pichia pastoris GS115 / pPic9k(+)Lcc5-I479T obtained in step (I) was inoculated into a test tube containing 5 mL of BMGY medium and cultured overnight at 28°C and 200 rpm. Then, it was transferred to an Erlenmeyer flask containing 50 mL of BMGY medium and cultured until OD500. 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. Induction culture is performed 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 loaded onto 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 ).

[0031] (III) Detection of temperature adaptability of the mutant enzyme of the present invention

[0032] Laccase activity assay: The total reaction volume was 1 mL, including 33 μL LABTS (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. 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] 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.

[0035] (iv) Stability testing of laccase mutants at 50℃, 45℃, 40℃, and 35℃

[0036] Using ABTS as a substrate, the mutant enzyme was incubated at 50℃, 45℃, 40℃, and 35℃ at pH 7.0. Samples were taken every 0.5 hours, and the residual enzyme activity was calculated after a certain period, with the initial enzyme activity as 100%. The formula is as follows: Residual enzyme activity = (Initial enzyme activity - Lost enzyme activity) / Initial enzyme activity × 100%. The results showed that the half-life of the mutant obtained by this invention under the conditions of 45℃ and pH 7.0 was 84 hours, which is 20 times that of the starting enzyme.

[0037] (V) Application of the laccase mutant enzyme of this invention in toxin oxidation and transformation experiments

[0038] The detoxification reaction system consisted of 1 mL of 2 U of enzyme, 950 μL of disodium citrate phosphate buffer (pH 7.0), and a final concentration of aflatoxin AFB1 of 0.2 μg / mL. The mixture was shaken and incubated in a constant temperature water bath at 45°C and 150 rpm for 24 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.

[0039] HPLC detection conditions: mobile phase: methanol-water = 45:55 (V / V); column selected: Agilent 5TC-C18(2) column (column length 250mm, column inner diameter 4.6mm, particle size 5μm, Sun Fire); flow rate set to 0.8mL / min; column temperature set to 25℃; detector set: fluorescence detection excitation wavelength 360nm; emission wavelength 440nm. A photochemical derivatization device was then connected after the column.

[0040] 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-I479T was 76%, while that of the starting enzyme Lcc5 was 58%, indicating that the oxidation conversion rate of the mutant enzyme was 31% 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. The coding gene of the laccase mutant according to claim 1, characterized in that... Its nucleotide sequence is shown in SEQ ID NO:

2.

3. The expression strain of the laccase mutant according to claim 1, characterized in that: The strains expressed are classified as follows: Pichia pastoris GS115 / pPic9k(+)Lcc5-I479T has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20232460, on December 5, 2023, at Wuhan University, Wuhan, China.

4. The application of the laccase mutant according to claim 1, characterized in that: The laccase mutant was used for the oxidative transformation of aflatoxin AFB1.

5. The application according to claim 4, 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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