A laccase mutant LacF-25 and its expression strain and application

Abstract

The present invention discloses a laccase mutant LacF-25, its expression strain, and application. The present invention uses Trametes hirsuta AH28-2 laccase as the starting enzyme and obtains a mutant gene through molecular docking and positional structure analysis. After inducing expression of the engineered bacteria containing the mutant gene, a laccase mutant enzyme LacF-25 with improved ability to oxidize and convert toxins is obtained. The mutant LacF-25 has an increased ability to oxidize and convert AFB1 to 1.14 times that of the starting enzyme at pH 7 and 50°C. When using ABTS as the substrate, the mutant enzyme LacF-25 stably increases to 5-9 times that of the starting enzyme at 35-50°C. The mutant enzyme has potential application value in the oxidation and conversion of aflatoxins.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a laccase mutant LacF-25 and an expression strain and application thereof. Background Art

[0002] Laccase is a copper-containing polyphenol oxidase that catalyzes the oxidation of a variety of recalcitrant organic pollutants, including chlorophenols, polycyclic aromatic hydrocarbons, dyes, chloroform, benzene derivatives and their derivatives, and trinitrotoluene (TNT). Due to its broad substrate range, low storage requirements, and lack of secondary pollution, the development and research of laccases has been a hot topic for researchers both domestically and internationally. Due to its unique catalytic properties and broad application prospects, laccases hold significant technical and application value in industries such as environmental protection, papermaking, and textiles.

[0003] Aflatoxins (AFs) are highly toxic secondary metabolites produced by certain strains of Aspergillus flavus. There are over 20 aflatoxins, including B1, B2, G1, G2, M1, and M2, all of which are polycyclic aromatic hydrocarbons. Aflatoxin B1 (AFB 1) is the most toxic aflatoxin to a wide range of organisms, being carcinogenic, teratogenic, and mutagenic. It is commonly found in a variety of products, including grains, oilseeds, and feed. The primary toxin in AFB 1 is derived from a coumarin moiety, which has a furan-furan moiety and a lactone ring as its key active site. Its toxic effects are felt across multiple organs and systems, primarily affecting the liver, where it can induce liver cancer. Due to its acute toxicity to the liver and kidneys of humans and animals, the International Agency for Research on Cancer classifies this compound as a Class I carcinogen.

[0004] Currently, the laccases used in toxin oxidation and conversion are mostly natural laccases produced by fungal fermentation. In the actual toxin oxidation and conversion system, natural laccases have low activity and are easily inactivated. Therefore, it is necessary to modify natural laccases to screen mutants with improved activity and stability. Summary of the Invention

[0005] To address the problems of the prior art, the present invention provides a laccase mutant, LacF-25, its expression strain, and applications. Using the laccase LacF from Trametes hirsuta AH28-2 as the starting enzyme, the present invention obtains the mutant gene through structural modeling, molecular docking, and site-directed mutagenesis. After induction of expression in the expression strain containing the mutant gene, the laccase mutant LacF-25 with improved stability is obtained. The half-life of the mutant enzyme at 50°C is increased to 35 hours, nine times that of the starting enzyme. The mutant LacF-25 has potential application in the oxidation and conversion of aflatoxins: under identical conditions, LacF-25 oxidizes 84.2% of aflatoxins, while the starting enzyme LacF-25 only converts 73.6%. This represents a 1.14-fold increase in the conversion rate of the mutant enzyme compared to the starting enzyme.

[0006] The laccase mutant LacF-25 of the present invention has an amino acid sequence as shown in SEQ ID NO: 1. Compared with the amino acid sequence of the original enzyme LacF, the altered positions are as follows: leucine at position 185 is mutated to glycine, proline at position 227 is mutated to alanine, asparagine at position 228 is mutated to phenylalanine, asparagine at position 284 is mutated to glutamic acid, and threonine at position 410 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 LacF-25 of the present invention is shown in SEQ ID NO: 2.

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

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

[0011] Furthermore, the expression strain of the laccase mutant LacF-25 of the present invention is classified and named Pichia pastorisGS115 / pPic9k(+)LacF-25, and has been sent to the China Center for Type Culture Collection for preservation, with the preservation number CCTCC NO: M20242854, the preservation date is December 18, 2024, and the preservation address is: Wuhan University, Wuhan, China.

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

[0013] Using the laccase LacF from Trametes hirsuta AH28-2 as a starting enzyme, their structure was predicted through homology modeling. Molecular docking simulations with aflatoxin AFB1 were performed to infer key sites involved in the enzyme reaction and conduct conservation analysis. Based on this information, mutations that could enhance the enzyme's catalytic reaction were designed. Laccase LacF-25, which had the potential to improve detoxification performance, was identified. A mutant gene was constructed and ligated into the vector pPic9k. The linearized plasmid pPic9k was then electroporated into the expression host, Pichia pastoris GS115. The mutant gene was integrated into the Pichia pastoris alcohol oxidase genome. After plate screening, an engineered strain containing the mutant gene of the present invention was obtained.

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

[0015] The laccase mutant LacF-25 of the present invention is used for the oxidative conversion of aflatoxin AFB1. When reacted in a constant-temperature water bath shaker at pH 7, 50°C, and 150 rpm for 48 hours, the mutant LacF-25 achieved an 84.2% oxidative conversion rate for aflatoxin AFB1, compared to 73.6% for the original LacF enzyme. This 14% improvement in oxidative conversion rate is a potential application for the oxidative conversion of aflatoxin AFB1.

[0016] The present invention measured and compared the temperature adaptability and temperature stability of the mutant enzyme and the original enzyme. The results showed that the mutant's optimal temperature remained unchanged compared to the original enzyme. However, the mutant enzyme's stability was improved. At 50°C and pH 7, the mutant enzyme's half-life was nine times that of the original enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The SDS-PAGE profiles of the purified starting enzyme LacF and the mutant enzyme LacF-25 of the present invention are shown in FIG. 1 : each lane represents a marker, pure LacF enzyme, and pure LacF-25 enzyme of the present invention, respectively.

[0018] Figure 2 These are the results of determining the optimum temperatures of the starting enzyme LacF and the mutant enzyme LacF-25 of the present invention.

[0019] Figure 3 Figure a shows the stability of the starting enzyme LacF at 35°C, 40°C, 45°C, and 50°C; Figure b shows the stability of the mutant enzyme LacF-25 of the present invention at 35°C, 40°C, 45°C, and 50°C.

[0020] Figure 4This is an experiment on the oxidation conversion of aflatoxin AFB1 by the starting enzyme LacF and the mutant enzyme LacF-25 of the present invention. DETAILED DESCRIPTION

[0021] The implementation methods in the following examples are all conventional methods unless otherwise specified.

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

[0023] 1. Obtaining laccase gene mutant enzyme

[0024] The present invention used LacF, heterologously expressed from Trametes hirsuta AH28-2, as the starting enzyme. Homology modeling was performed using the online website (https: / / swissmodel.expasy.org / ). Subsequently, AutoDock Vina docking simulations were performed between LacF and aflatoxin AFB1. The docking results showed that in the optimal binding conformation, the enzyme forms a hydrogen bond with the toxin via an amino acid. This amino acid is a potential candidate site for modifying laccase to enhance the interaction between the enzyme and the toxin, thereby improving the enzyme's detoxification performance. Based on amino acid conservation analysis using WebLogo, a mutant was designed using the Funclib online website and named LacF-25.

[0025] The amino acid sequence of the laccase mutant enzyme of the present invention is shown in SEQ ID NO: 1. Compared with the original enzyme LacF-25, the altered positions are as follows: leucine at position 185 is mutated to glycine, proline at position 227 is mutated to alanine, asparagine at position 228 is mutated to phenylalanine, asparagine at position 284 is mutated to glutamic acid, and threonine at position 410 is mutated to glutamic acid.

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

[0027] The laccase mutant gene in step 1 is constructed and ligated into the expression vector pPic9k, and the expression host is Pichia pastoris to obtain an engineered strain containing the mutant gene of the present invention.

[0028] The expression strain of the laccase mutant LacF-25 of the present invention is classified and named Pichia pastoris GS115 / pPic9k(+)LacF-25, and has been sent to the China Center for Type Culture Collection for preservation with the accession number CCTCC NO: M20242854. The preservation date is December 18, 2024, and the preservation address is Wuhan University, Wuhan, China.

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

[0030] The expression strain Pichia pastoris GS115 / pPic9k(+)LacF-25 obtained in (I) was inoculated into a test tube containing 5 mL of BMGY, cultured overnight in a shaker at 28°C and 200 rpm, and then transferred to a flask containing 50 mL of BMGY medium and cultured until the OD 600 When the concentration reaches 2.0, centrifuge at 4°C and 3000 rpm for 20 minutes, and resuspend the bacteria in BMM liquid; transfer the resuspended bacteria to a 500 mL baffled triangular flask (containing 200 mL BMM liquid culture medium). Take samples every 24 hours, take a sample and add an equal amount of methanol, and induce culture at 28°C. Detect the changes in sample enzyme activity, culture in BMM culture medium for 6 days, collect the bacterial liquid, and purify the target protein. After centrifugation and dialysis, the crude enzyme solution is filtered with a 0.22 μm membrane to remove impurities and bubbles in the supernatant, and loaded onto a DEAE-SpharoseFastFlow anion exchange column. The obtained protein is tested by SDS-PAGE to achieve the purity of enzymatic properties ( Figure 1 ).

[0031] (III) Detection of Temperature Adaptability of the Mutant Enzyme Containing the Present Invention

[0032] Laccase activity assay: The total reaction system is 1 mL, including 33 μL ABTS (final concentration 15 mM) and 950 μL sodium tartrate buffer solution (pH 4.0). Incubate at 30°C for 5 min, then add 17 μL enzyme solution to the EP tube to make the reaction system 1 mL. After reacting at 30°C for 3 min, place on ice for 30 s, and measure the OD value using a spectrophotometer. 420 value.

[0033] The enzyme activity calculation formula is: enzyme activity (U / L) = 555.56 × dilution factor × OD 420

[0034] 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.

[0035] (IV) Stability testing of laccase-containing mutants at 50°C, 45°C, 40°C, and 35°C

[0036] Using ABTS as the substrate, the mutant enzyme was incubated at 50°C, 45°C, 40°C, and 35°C at pH 7.0. Samples were taken at regular intervals, and the residual enzyme activity after a certain period was calculated, with the initial enzyme activity as 100%. The formula is as follows: Remaining enzyme activity = (initial enzyme activity - lost enzyme activity) / initial enzyme activity × 100%. The results showed that the half-life of the mutant obtained in this invention at 45°C and pH 7.0 was 84 hours, 20 times that of the original enzyme.

[0037] (V) Application of the Laccase Mutant Enzyme Containing the Present Invention in Toxin Oxidation and Conversion Experiments

[0038] The detoxification reaction system was 1 mL, in which the enzyme amount was 10 μg, the final concentration of aflatoxin AFB1 was 1 μg / mL, and the citric acid disodium hydrogen phosphate buffer (pH 7.0) was added to 1000 μL, shaken and mixed, and placed in a constant temperature water bath shaker at 45°C and 150 r / min for 48 hours. 200 μL of the reaction system was taken and 200 μL of methanol was added 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 preparation for detection.

[0039] 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 was used.

[0040] 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 LacF-25 was 84.2%, and the oxidation conversion rate of the original enzyme LacF was 73.6%. The oxidation conversion rate of the mutant enzyme was 14% higher than that of the original enzyme.

Claims

1. A laccase mutant LacF-25, characterized in that Its amino acid sequence is shown in SEQ ID NO:

1.

2. The gene encoding the laccase mutant LacF-25 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 LacF-25 according to claim 1, characterized in that: The expression strain is classified and named Pichia pastoris GS115 / pPic9k(+)LacF-25, and has been sent to the China Center for Type Culture Collection for preservation. The preservation number is CCTCC NO: M 20242854, the preservation time is December 18, 2024, and the preservation address is: Wuhan University, Wuhan, China.

4. Use of the laccase mutant LacF-25 according to claim 1 in the oxidation and conversion of aflatoxin AFB1.

5. The use according to claim 4, characterized in that: During the oxidative transformation of aflatoxin AFB1, the system temperature is 45-55°C and the pH value is 7.

0.

6. The use according to claim 5, characterized in that: The system temperature was 50°C.

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