A laccase mutant and its application in converting zearalenone into detoxified zearalenone
By designing molecular docking and performing site-directed mutagenesis on the laccase Lcc5 of Coprinus comatus, a laccase mutant Lcc5-V189R was prepared, which solved the problem of low conversion efficiency of natural laccase, achieved efficient conversion of zearalenone, and improved the detoxification effect.
Patent Information
- Application Number
- CN202611464060.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-20
- Publication Date
- 2026-11-06
AI Technical Summary
Existing naturally sourced laccases have limited conversion efficiency for zearalenone, especially under actual treatment conditions, making it difficult to effectively reduce zearalenone contamination in grains and feed.
By designing molecular docking and performing site-directed mutagenesis on the laccase Lcc5 of Coprinus comatus, a laccase mutant Lcc5-V189R was prepared. The binding region of Lcc5 to zearalenone was modified to improve the conversion efficiency.
The mutant enzyme Lcc5-V189R increased the conversion rate of zearalenone to 1.8 times that of the starting enzyme under the same protein content conditions, significantly improving the conversion efficiency and shortening the apparent conversion half-life to 0.6 hours, thus significantly improving the detoxification effect.
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Figure CN123042965A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a laccase mutant and its application in the conversion of detoxified zearalenone. Background Technology
[0002] Laccases are a class of oxidoreductases containing multiple copper ions, widely found in fungi, plants, and bacteria. They can use molecular oxygen as an electron acceptor to catalyze the oxidation of phenols, aromatic amines, and other organic compounds, while simultaneously reducing oxygen to water. Due to their broad substrate range, mild reaction conditions, and good environmental compatibility, laccases have broad application potential in food processing, environmental pollutant treatment, and biotransformation. In recent years, the ability of laccases to transform certain mycotoxins has gradually attracted attention, providing a new technical approach for controlling mycotoxin contamination in grains and feed.
[0003] Zearalenone is a fungal toxin mainly produced by Fusarium wilt, widely found in corn, wheat and their processed products, and feed ingredients. Zearalenone exhibits estrogen-like activity; after ingestion, it can interfere with the animal endocrine system, affecting reproductive function and adversely impacting livestock production and food safety. Due to its widespread contamination in grains and feed, developing treatment technologies to effectively reduce zearalenone levels is of great significance. Existing physical and chemical treatment methods may be limited in practical applications by factors such as treatment conditions, nutrient loss, or residual risk. Therefore, enzymatic conversion with relatively mild reaction conditions has become an important research direction for controlling zearalenone contamination.
[0004] Zearalenone molecules contain phenolic hydroxyl groups and other structures, possessing the potential for conversion by laccase catalysis. However, different laccases exhibit significant differences in their conversion capabilities for zearalenone, and the conversion efficiency of naturally derived laccases is generally limited, especially under conditions close to actual grain and feed processing, where their application effectiveness still needs improvement. Therefore, developing laccase mutants with high zearalenone conversion performance has practical application value for the biological detoxification of contaminated grains and feeds. Rational design is a common strategy for modifying and improving enzyme performance. By speculating on the binding mode and possible catalytic mechanism between laccase and zearalenone, and by directionally modifying key amino acids in the substrate-binding region, it is hoped that the interaction between enzyme and substrate can be improved and the conversion efficiency increased. Summary of the Invention
[0005] To address the above problems, this invention provides a laccase mutant and its application in the conversion of detoxified zearalenone. 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 molecular docking design and site-directed mutagenesis. After inducing expression in bacteria containing the mutant gene, the mutant laccase Lcc5-V189R with improved transformation efficiency was obtained through screening. The mutant enzyme Lcc5-V189R has potential application value in the transformation of virus-free zearalenone: under the same protein content conditions, Lcc5-V189R transforms 94% of zearalenone, while the starting enzyme Lcc5 can only transform 51% of zearalenone, increasing the transformation rate of the mutant enzyme to 1.8 times that of the starting enzyme.
[0006] The laccase mutant of this invention, Lcc5-V189R, has the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence changes compared to the starting enzyme Lcc5 are as follows: valine at position 189 is mutated to arginine.
[0007] The nucleic acid molecule encoding the laccase mutant Lcc5-V189R includes nucleotide sequences encoding the same amino acid sequence due to genetic code degeneracy.
[0008] The encoding gene of the laccase mutant Lcc5-V189R 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 engineered strain expressing the laccase protein of the present invention contains the gene encoding the laccase protein.
[0011] The method for constructing an engineered strain expressing the laccase protein of the present invention includes the following steps:
[0012] 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 zearalenone was performed to deduce key sites and mechanisms involved in catalytic transformation on the enzyme, and based on this, mutants that could promote enzyme-catalyzed transformation were designed. A laccase Lcc5-V189R mutant with potentially improved transformation efficiency was identified, synthesized, and ligated into the vector pPIC9K. The linearized plasmid pPIC9K was electroporated into the expression host Pichia pastoris. Pichia pastoris After plate screening, GS115 was obtained as an engineered strain containing the mutant gene of this invention.
[0013] The engineered strain is classified and named Pichia pastorisGS115 / pPIC9K(+)Lcc5-V189R has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20262018, deposit date September 3, 2026, and address: Wuhan University, Wuhan, China.
[0014] The laccase mutant of the present invention can be obtained by fermentation and purification of the expressed strain.
[0015] Application of the laccase mutant of this invention in the transformation of virus-free zearalenone (ZEN):
[0016] The reaction was carried out in a constant temperature water bath at pH 7, 45℃, and 150 rpm for 12 h. In a 1 mL system, 0.01 mg of the mutant enzyme Lcc5-V189R achieved a 94% conversion rate of 10 μg / mL zearalenone (ZEN), while the conversion rate of the same amount of the starting enzyme Lcc5 was 51%, representing a 0.8-fold increase in conversion rate. Based on the mechanism design, the mutant V189R showed improved ZEN conversion ability under the same protein content conditions, indicating its potential application in the oxidative conversion of zearalenone (ZEN).
[0017] This invention measured and compared the reaction process of laccase mutant Lcc5-V189R and starting enzyme Lcc5 in the conversion of zearalenone. The results showed that the apparent conversion half-life of zearalenone by Lcc5-V189R was 0.6 h, which was significantly shorter than the 2.8 h of the starting enzyme Lcc5, indicating that this mutation significantly improved the conversion efficiency of laccase to zearalenone.
[0018] This invention also directly applies the laccase protein to corn flour containing zearalenone, including artificially contaminated and naturally contaminated corn flour, to transform these toxins. After a 12-hour reaction in a constant-temperature water bath at pH 7, 50°C, and 150 rpm, 50 μg of Lcc5 and Lcc5-V189R achieved 17.3% and 38.3% conversion rates of zearalenone in artificially contaminated corn flour, respectively. When the enzyme amount was increased to 100 μg, Lcc5 achieved a ZEN conversion rate of 19.7%, while V189R achieved a conversion rate of 57.2%, with the mutant enzyme efficiency being 2.9 times that of the wild type. This laccase mutant has high application value in transforming zearalenone in detoxified contaminated foods and feeds. Attached Figure Description
[0019] Figure 1 SDS-PAGE images of the purified starting enzyme Lcc5 and the mutant enzyme Lcc5-V189R of this invention. Each lane represents a marker, 1: purified Lcc5 enzyme, 2: purified Lcc5-V189R enzyme of this invention.
[0020] Figure 2 The optimal temperature (Figure A) and optimal pH (Figure B) for the starting enzyme Lcc5 and the mutant enzyme Lcc5-V189R of this invention are shown in the figure.
[0021] Figure 3 The oxidation conversion rate of zearalenone by the starting enzyme Lcc5 and the mutant enzyme Lcc5-V189R of the present invention is compared under the same conditions.
[0022] Figure 4 This is a comparison of the apparent transformation half-life of ZEN by the starting enzyme Lcc5 and the laccase mutant Lcc5-V189R of this invention at 45 °C and pH 7.0.
[0023] Figure 5 This is a comparison graph showing the conversion effects of the starting enzyme Lcc5 and the laccase mutant Lcc5-V189R of this invention on zearalenone in artificially contaminated corn flour.
[0024] Figure 6 The graph shows a comparison of the conversion effects of the starting enzyme Lcc5 and the laccase mutant Lcc5-V189R of this invention on zearalenone in naturally contaminated corn flour under different solid-liquid ratio conditions. Detailed Implementation
[0025] The technical solution of the present invention will be further analyzed and explained through specific embodiments below.
[0026] Unless otherwise specified, the implementation methods in the following embodiments are all conventional methods.
[0027] (i) Construction of engineered strains containing the laccase mutant gene of the present invention
[0028] 1. Design of laccase gene mutants
[0029] This invention uses gray-covered ginkgo umbrellas C. cinerea Lcc5, obtained from heterologous expression of strain FA2222, served as the starting enzyme. Homology modeling was performed using an online website (https: / / swissmodel.expasy.org / ). The Lcc5 structural model was then simulated with ZEN using AutoDock Vina. Based on the preferred docking conformation, the spatial location, side chain properties, and substrate interactions of amino acid residues near the ZEN binding region were analyzed. Multiple sites, including V189, were screened as candidate sites for modifying laccase to promote enzyme-toxin interaction and thus enhance the enzyme's transformation and detoxification performance. Considering that the imidazole group on the histidine (His) side chain and the guanidinium group on the arginine (Arg) side chain can act as proton acceptors to promote deprotonation of substrates and water molecules, thereby improving laccase catalytic conversion efficiency, these sites were replaced with His or Arg, respectively, forming mutants such as V189R that may promote ZEN transformation.
[0030] The amino acid sequence of the laccase mutant of the present invention is shown in SEQ ID NO:1. Compared with the starting enzyme Lcc5, the altered sites are as follows: valine at position 189 is mutated to arginine.
[0031] 2. Construction of engineered laccase mutant strains
[0032] The laccase mutant gene from step 1 was prepared and ligated into the expression vector pPIC9K, then transformed into the expression host Pichia pastoris. Pichia pastoris An engineered strain containing the laccase gene of this invention was obtained. Pichia pastoris GS115 / pPIC9K(+)Lcc5-V189R.
[0033] The engineering strains of this invention are classified and named as follows: Pichia pastoris GS115 / pPIC9K(+)Lcc5-V189R has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20262018, deposit date September 3, 2026, and address: Wuhan University, Wuhan, China.
[0034] (II) Expression and protein purification of engineered laccase mutant bacteria
[0035] The expression strain obtained in (I) Pichia pastoris GS115 / pPIC9K(+)Lcc5-V189R was inoculated into a test tube containing 5 mL of BMGY and cultured overnight at 28°C and 200 rpm on a shaker. Then, it was transferred to an Erlenmeyer flask containing 50 mL of BMGY and cultured until OD... 600 When the enzyme activity reaches 2.0-6.0, centrifuge at 5000 rpm for 5 min at 4℃, and resuspend the cells in BMM liquid. Transfer the resuspended cells to a 1 L Erlenmeyer flask with baffle (containing 400 mL of BMM liquid medium). Take samples every 24 h, add 0.5% methanol to each sample, and induce culture at 28℃. Detect enzyme activity changes in the samples. Culture in BMM medium for 7 days and collect the bacterial suspension. After centrifugation and dialysis, remove impurities and air bubbles from the supernatant using a 0.22 μm filter membrane, load the solution onto a DEAE-Spharose Fast Flow anion exchange column, and analyze the obtained protein using SDS-PAGE. The protein must reach the purity required for enzymatic property detection (>95%). Figure 1 ).
[0036] (III) Detection of the optimal temperature and optimal pH of the laccase mutant of the present invention
[0037] Optimal temperature detection: The reaction system was 1 mL, containing 0.01 mg of enzyme, a final ZEN concentration of 10 μg / mL, and pH 7.0 buffer was added to 1 mL. The reaction was carried out at 20, 25, 30, 35, 40, 45, 50 and 55 °C for 12 h, and then an equal volume of methanol was added to terminate the reaction.
[0038] Optimal pH determination: Buffer solutions with pH values of 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5 were prepared, with a reaction volume of 1 mL containing 0.01 mg of enzyme and 10 μg / mL ZEN. After reacting at 45℃ and 150 rpm for 12 h, an equal volume of methanol was added to terminate the reaction.
[0039] Test results as follows Figure 2 As shown, the optimal temperature for the transformation of zearalenone by the laccase mutant of the present invention is 45°C, and the optimal pH is 7.0.
[0040] (iv) Conversion efficiency of the laccase mutant Lcc5-V189R of the present invention for pure zearalenone
[0041] The detoxification reaction system was 1 mL, containing 950 μL of citrate-disodium hydrogen phosphate buffer, 0.01 mg of enzyme, and 10 μg of zearalenone. The mixture was shaken and placed in a constant temperature water bath at 50 °C and 150 rpm. Samples were taken at intervals, and the reaction was terminated by adding an equal volume of methanol. Then, the mixture was vortexed for 30 s. After shaking, the mixture was centrifuged at 12000 rpm for 10 min using a refrigerated centrifuge. The supernatant was collected and filtered through a 0.22 μm organic syringe filter to remove the methanol containing zearalenone for HPLC detection. An Agilent 5 TC-C18(2) column (250 mm in length, 4.6 mm in inner diameter, and 5 μm in particle size, Agilent) was selected. HPLC detection conditions for zearalenone: Detection wavelength of fluorescence detector: λ ex =270nm, λ em =440nm; Mobile phase: Acetonitrile: Methanol: Water (46:8:46).
[0042] Test results as follows Figure 3 The results showed that, under the same conditions, using zearalenone as a substrate, the conversion rate of the mutant enzyme Lcc5-V189R was 94%, while the conversion rate of the starting enzyme Lcc5 was 51%, and the conversion rate of the mutant enzyme was 0.8 times higher than that of the starting enzyme.
[0043] (v) Time kinetics of the transformation of zearalenone by the laccase mutant Lcc5-V189R of the present invention
[0044] 10 μg of purified starting enzyme and mutant were added separately to 1 mL of reaction system containing 50 mmol / L citrate-phosphate buffer (pH 7.0) and 10 μg / mL ZEN, and reacted at 45 °C and 150 rpm. Samples were taken at 0, 1, 2, 3, 4, 6, 8, and 12 h, and the reaction was terminated by adding an equal volume of methanol. After centrifugation and filtration through a 0.22 μm organic phase filter membrane, the residual ZEN concentration was determined by HPLC, and the ZEN conversion rate over time was plotted. The results are as follows. Figure 4 The results show that the ZEN conversion half-life of the mutant enzyme Lcc5-V189R obtained in this invention is about 0.6 h, which is significantly shorter than the 2.8 h of the starting enzyme.
[0045] (vi) Application of the laccase mutant Lcc5-V189R of the present invention in the transformation and detoxification of corn flour contaminated with zearalenone.
[0046] ZEN was artificially added to corn flour to a concentration of 3 μg / g (ZEN / corn flour). The transformation system contained 0.20 g of artificially contaminated corn flour, and citrate-phosphate buffer (pH 7.0) was added at a solid-liquid ratio of 15%, followed by the addition of ethanol to a final volume fraction of 10%. The mixture was then sonicated for 30 min. Subsequently, 50 μg or 100 μg of purified laccase Lcc5 or the laccase mutant Lcc5-V189R were added, and the reaction was carried out at 45℃ and 150 rpm for 12 h with shaking. The protein addition amounts and reaction conditions for Lcc5 and Lcc5-V189R were consistent across all groups. After the reaction, the samples were freeze-dried, and zearalenone was extracted using an immunoaffinity column. The extracted zearalenone was then analyzed by HPLC.
[0047] 2.0 g of naturally contaminated corn flour with a ZEN content of 3 μg / g was added to citrate-phosphate buffer (pH 7.0) at solid-liquid ratios (m:V) of 1:6, 1:8, and 1:10, respectively. Purified laccase Lcc5 or the laccase mutant Lcc5-V189R was then added to bring the enzyme concentration to 200 μg / g corn flour. All reaction systems were incubated at 45℃ and 180 rpm for 24 h with shaking. The protein addition and reaction conditions for Lcc5 and Lcc5-V189R were kept consistent. After the reaction, the samples were freeze-dried, and zearalenone was extracted using an immunoaffinity column. The extracted zearalenone was then analyzed by HPLC.
[0048] In the experiment of artificially contaminated corn flour, the test results were as follows: Figure 5The results showed that the mutant enzyme V189R significantly improved the conversion efficiency of ZEN compared with the starting enzyme Lcc5. After 12 h of reaction, under the condition of 50 μg laccase, the conversion rate of ZEN by Lcc5 was 17.3%, while that of V189R was 38.3%; when the enzyme amount was increased to 100 μg, the conversion rate of ZEN by Lcc5 was 19.7%, while that of V189R was 57.2%, and the efficiency of the mutant enzyme was 2.9 times that of the wild type.
[0049] In experiments with naturally contaminated corn flour, the test results were as follows: Figure 6 The results showed that, similar to those observed in artificially contaminated corn flour, the mutant enzyme V189R significantly improved the conversion efficiency of ZEN compared to the starting enzyme Lcc5. At a solid-liquid ratio of 1:8, V189R achieved a maximum conversion rate of 56.5%, while Lcc5 achieved a conversion efficiency of 36.4%, meaning the mutant enzyme's efficiency was 1.6 times that of the wild type.
Claims
1. A laccase mutant, abbreviated as Lcc5-V189R, 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 engineered strain is classified and named Pichia pastoris GS115 / pPIC9K(+)Lcc5-V189R has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20262018, deposit date September 3, 2026, and address: Wuhan University, Wuhan, China.
4. The application of the laccase mutant of claim 1 in the oxidative transformation of detoxified zearalenone.
5. The application according to claim 4, characterized in that: The temperature of the oxidation conversion system is 20-55℃, and the pH value is 5.0-7.
5.
6. The application according to claim 5, characterized in that: The temperature of the oxidation conversion system was 45℃ and the pH was 7.