Laccase mutant LacF-25 as well as expression strain and application thereof

Abstract

The invention discloses a laccase mutant LacF-25 as well as an expression strain and application thereof. According to the present invention, Trametes hirsuta AH28-2 laccase is adopted as a starting enzyme, and molecular docking is performed to analyze the position structure so as to obtain the mutant gene; after induced expression of engineering bacteria containing the mutant gene, the laccase mutant enzyme LacF-25 with improved toxin oxidative conversion capability is obtained. Under the conditions that the pH is 7 and the temperature is 50 DEG C, the oxidative conversion capacity of the mutant LacF-25 on AFB1 is improved to 1.14 times that of a starting enzyme. When ABTS is used as a substrate, the mutant enzyme LacF-25 is stably increased to 5-9 times that of a starting enzyme under the condition of 35-50 DEG C, 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 specifically 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 has the ability to catalyze the oxidation of a variety of refractory organic pollutants, including chlorophenols, polycyclic aromatic hydrocarbons, dyes, chloroform, benzene and its derivatives, trinitrotoluene (TNT), etc. Due to its advantages such as a wide range of catalytic substrates, low storage requirements, and no secondary pollution caused by oxidized substrates, the development and research of laccase has always been a hot topic for scholars at home and abroad. Laccase has important technical background and application value in environmental protection, papermaking, textile and other industries due to its unique catalytic properties and broad application prospects.

[0003] Aflatoxins (AFs) are highly toxic secondary metabolites produced by certain strains of Aspergillus flavus. There are more than 20 types of aflatoxins, including B1, B2, G1, G2, M1, M2, etc., all of which are polycyclic aromatic hydrocarbons. Among them, aflatoxin B1 (AFB 1) is the most toxic aflatoxin to a variety of organisms, carcinogenic, teratogenic and mutagenic. It is commonly found in various products, including grains, oilseeds and feed. The main toxin in AFB 1 is derived from the coumarin part, which has a furan-furan part and a lactone ring as the key active site. Its toxicity acts on multiple organs and systems, but mainly affects the liver, mainly inducing liver cancer. Because of 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, laccases used in toxin oxidation and transformation are mostly natural laccases produced by fungal fermentation. In the actual toxin oxidation and transformation system, natural laccases have low activity and are more easily inactivated. Therefore, it is necessary to transform natural laccases to screen mutants with improved activity and stability. Summary of the invention

[0005] In view of the problems existing in the above-mentioned prior art, the present invention provides a laccase mutant LacF-25 and its expression strain and application. The present invention uses Trametes hirsuta AH28-2 laccase LacF as the starting enzyme, and obtains the mutant gene through strategies such as structural simulation, molecular docking, and site-directed mutagenesis. After the expression bacteria containing the mutant gene are induced to express, the laccase mutant LacF-25 with improved stability is obtained. The half-life of the mutant enzyme at 50°C is increased to 35 h, which is 9 times that of the starting enzyme. The mutant enzyme LacF-25 has potential application value in the oxidation and conversion of aflatoxin: under the same conditions, LacF-25 oxidizes and converts 84.2% of aflatoxin, while the starting enzyme LacF-25 can only oxidize and convert 73.6% of aflatoxin, and the oxidation conversion rate of the mutant enzyme is increased to 1.14 times that of 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: the leucine at position 185 is mutated to glycine, the proline at position 227 is mutated to alanine, the asparagine at position 228 is mutated to phenylalanine, the asparagine at position 284 is mutated to glutamic acid, and the 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] The laccase LacF of Trametes hirsuta AH28-2 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 the conservation analysis, and mutations that can promote the enzyme catalytic reaction were designed accordingly. The laccase LacF-25 with potentially improved detoxification performance was determined, the mutant gene was constructed and connected to the vector pPic9k, the linearized plasmid pPic9k was electrotransformed into the expression host Pichia pastoris GS115, the mutant gene was integrated into the Pichia pastoris alcohol oxidase genome, and 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 fermenting the engineered strain.

[0015] The application of the laccase mutant LacF-25 of the present invention is to use the laccase mutant LacF-25 for the oxidation conversion of aflatoxin AFB1. When reacted in a constant temperature water bath shaker at pH 7, 50°C, and 150 r / min for 48 hours, the oxidation conversion rate of aflatoxin AFB1 by the mutant enzyme LacF-25 is 84.2%, and the oxidation conversion rate of the starting enzyme LacF is 73.6%. The oxidation conversion rate of the mutant enzyme is 14% higher than that of the starting enzyme. The mutant enzyme has potential application value in the oxidation conversion of aflatoxin AFB1.

[0016] The present invention measured and compared the temperature adaptability and temperature stability of the mutant enzyme and the starting enzyme. The results showed that the optimum temperature of the mutant was unchanged compared with the starting enzyme. However, the stability of the mutant enzyme was improved. At 50°C and pH 7, the half-life of the mutant enzyme was increased to 9 times that of the starting 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 Figure 1. The lanes are respectively the Marker, the pure LacF enzyme, and the pure LacF-25 enzyme of the present invention.

[0018] Figure 2 The optimum temperature determination results of the starting enzyme LacF and the mutant enzyme LacF-25 of the present invention are shown in FIG.

[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 using the starting enzyme LacF and the mutant enzyme LacF-25 of the present invention. DETAILED DESCRIPTION

[0021] The implementation methods in the following embodiments 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 LacF obtained by heterologous expression of Trametes hirsuta AH28-2 of the present invention is the starting enzyme, and homology modeling is performed through an online website (https: / / swissmodel.expasy.org / ). Then, LacF and aflatoxin AFB1 are docked and simulated by AutoDock Vina. The docking results show that in the optimal binding conformation, the enzyme forms a hydrogen bond interaction with the toxin through an amino acid, and the 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, the Funclib online website designed a mutant and named it 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 starting enzyme LacF-25, the changed positions are as follows: the leucine at position 185 is mutated to glycine, the proline at position 227 is mutated to alanine, the asparagine at position 228 is mutated to phenylalanine, the asparagine at position 284 is mutated to glutamic acid, and the threonine at position 410 is mutated to glutamic acid.

[0026] 2. Construction of laccase mutant genetic engineering strain

[0027] The laccase mutant gene in step 1 is constructed and connected to 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 preservation number of CCTCC NO: M20242854, the preservation time of December 18, 2024, and the preservation address: Wuhan University, Wuhan, China.

[0029] 2. Expression of genetically engineered bacteria containing laccase mutations and protein purification

[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 at 28°C and 200 rpm in a shaking incubator, and then transferred to a flask containing 50 mL of BMGY medium and cultured 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). Samples are taken 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 6 days of culture in BMM culture medium to purify the target protein. The crude enzyme solution after centrifugation and dialysis is filtered through a 0.22 μm filter 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 reach the purity of enzymatic property detection ( Figure 1 ).

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

[0032] 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.

[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 mutants at 50°C, 45°C, 40°C, and 35°C

[0036] Using ABTS as substrate, the mutant enzyme was incubated at 50°C, 45°C, 40°C, 35°C, and pH 7.0. Samples were taken at regular intervals, and the initial enzyme activity was taken as 100%. The remaining enzyme activity after a certain period of time was calculated using the following formula: Remaining enzyme activity = (initial enzyme activity - lost enzyme activity) / initial enzyme activity × 100%. The results showed that the half-life of the mutant obtained by the present invention at 45°C and pH 7.0 was 84h, which was 20 times that of the starting 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 sodium hydrogen phosphate citric acid 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 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 machine 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.

[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 starting enzyme LacF was 73.6%. The oxidation conversion rate of the mutant enzyme was 14% higher than that of the starting enzyme.

Claims

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

1.

2. The coding gene of 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.

5. The use according to claim 4, characterized in that: During the oxidation conversion of aflatoxin, 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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