Fungus laccase mutant Lcc5-G, expression strain and application thereof

Abstract

The application discloses a kind of fungal laccase mutant Lcc5-G and its expression strain and application.The application is based on the fungal laccase from Coprinopsis cinerea heterologous expression since, by site-directed mutagenesis, obtain mutant gene.After fungal induction expression containing mutant plasmid, obtain laccase mutant enzyme Lcc5-G with improved specific activity, stability and AFB1 removal efficiency.When ABTS is used as substrate, the specific activity of the mutant is improved by 3 times, and the stability of the mutant enzyme is improved to 5 times of the original enzyme at 45 DEG C and pH 7.The mutant has potential application value in oxidizing aflatoxin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a fungal laccase mutant Lcc5-G and an expression strain and application thereof. BACKGROUND

[0002] Fungal laccase (EC 1.10.3.2, p-diphenol oxidase) belongs to the copper blue oxidase and exists in the form of monomeric glycoprotein. The main reaction is oxidation-reduction reaction, which can catalyze the oxidation of various phenols and non-phenols to generate the corresponding benzoquinone, accompanied by the transfer of electrons to reduce the molecules into water, and no other by-products are generated in the reaction process. The laccase substrate is wide, including phenols and their derivatives, aromatic amines and their derivatives. The laccase has important application values in the biological elimination of toxic compounds, the paper industry, the food industry, biofuels, biosensors, industrial dye decolorization and the wood industry.

[0003] Aflatoxin (AF) is a secondary metabolite produced by toxic strains of Aspergillus flavus and parasitic Aspergillus, and is a strong toxic substance. AF often exists in soil, animals and plants, various nuts, especially peanuts and walnuts. AF is a highly toxic substance, has strong toxicity to humans, and has obvious damage to the liver, can cause liver parenchymal necrosis, bile duct epithelial hyperplasia, liver fat infiltration and liver hemorrhage and other lesions. AF is a strong carcinogen, which can cause the loss of immune function of the human body or animals, and among all AF, AFB1 is the most toxic, which has great health risks to human health. SUMMARY

[0004] The present application provides a fungal laccase mutant Lcc5-G and an expression strain and application thereof. The present application takes the fungal laccase Lcc5 of C. cinerea as the starting enzyme, and obtains a single-point mutant gene by combining molecular docking, molecular dynamics simulation, and the active pocket of the combination of the protein and the substrate aflatoxin. After the induction expression of the engineering bacteria containing the mutant gene, the laccase mutant enzyme Lcc5-G with improved specific enzyme activity and stability is obtained. When ABTS is used as the substrate, the specific enzyme activity of the mutant Lcc5-G is improved by 3 times. At the same time of improving the specific enzyme activity, the half-life of the mutant enzyme is improved by 5 times at pH 7 and 45 DEG C. The mutant enzyme has potential application value in the oxidation conversion of aflatoxin. The experimental results show that under the same conditions, the mutant enzyme oxidizes and converts 98% of aflatoxin, and the starting enzyme Lcc5 oxidizes and converts 88% of aflatoxin. The mutant has potential application value in the efficient oxidation conversion of aflatoxin.

[0005] The fungal laccase mutant Lcc5-G of the present application is obtained by mutating glycine at the 164th position in the amino acid sequence of the laccase into glutamic acid, and the amino acid sequence is shown in SEQ ID NO: 1.

[0006] The application discloses a coding gene of a fungal laccase mutant Lcc5-G, and the nucleotide sequence of the coding gene is shown as SEQ ID NO: 2.

[0007] The application discloses a mutant gene, which is a coding gene of a laccase mutant shown as SEQ ID NO: 2.

[0008] The application discloses an expression strain of a laccase mutant, which contains the mutant gene.

[0009] The application discloses an expression strain of a laccase mutant, which is classified as Pichiapastoris GS115 / pPic9k(+)-lcc5-G, has been preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20232440, the preservation time is December 4, 2023, and the preservation address is Wuhan, China.

[0010] The application discloses a construction method of an expression strain of a laccase mutant, and the method comprises the following steps:

[0011] The application discloses a construction method of an expression strain of a laccase mutant, and the method comprises the following steps: 2+ The application discloses a construction method of an expression strain of a laccase mutant, and the method comprises the following steps:

[0012] The application discloses a construction method of an expression strain of a laccase mutant, and the method comprises the following steps:

[0013] The application discloses a construction method of an expression strain of a laccase mutant, and the method comprises the following steps:The application discloses a construction method of an expression strain of a laccase mutant, and the method comprises the following steps:

[0014] The present application determines and compares the specific enzyme activity, optimum temperature, stability and the like of the mutant protein and the original starting enzyme protein. The determination results show that the specific enzyme activity of the mutant is increased by 3 times when ABTS is used as the substrate. At the same time of the activity increase, the stability of the mutant is also improved, and the half-life of the mutant is increased by 5 times at 45℃, pH 7.0. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The SDS-PAGE patterns of the purified starting enzyme Lcc5 and the mutant enzyme Lcc5-G of the present application are shown in the following figure: each lane is Marker, Lcc5 pure enzyme and mutant Lcc5-G pure enzyme, respectively.

[0016] Figure 2 Fig. a is the determination result of the optimum pH of the starting enzyme Lcc5; Fig. b is the determination result of the optimum pH of the mutant Lcc5-G.

[0017] Figure 3 Fig. a is the temperature stability of the starting enzyme Lcc5 at 50℃, 45℃, 40℃, 35℃, pH 7.0; Fig. b is the temperature stability of the mutant Lcc5-G at 50℃, 45℃, 40℃, 35℃, pH 7.0.

[0018] Figure 4 The starting enzyme Lcc5 and the mutant Lcc5-G are used for aflatoxin oxidation conversion experiment. DETAILED DESCRIPTION

[0019] The methods for implementing the following embodiments are conventional methods unless otherwise specified.

[0020] (I) Construction of expression strain containing laccase mutant gene of the present application

[0021] 1. Obtaining of laccase gene mutant

[0022] The present application takes Lcc5 obtained by heterologous expression of enzyme extracted from C. cinerea as the starting enzyme, and performs molecular docking and molecular dynamics simulation on the starting enzyme and aflatoxin, to obtain a complex in which aflatoxin is located at the active site of Lcc5. According to the root mean square deviation (RMSD) and the root mean square fluctuation (RMSF), amino acids in Lcc5 that fluctuate greatly at the active site are found. According to the analysis of amino acid conservation by WebLogo, the analysis of hydrogen bond binding energy, and the analysis of the spatial position of aflatoxin, it is finally determined that glycine at position 164 may have a relatively large influence on the oxidation conversion of laccase to aflatoxin. The glycine at position G164 is mutated into glutamic acid which is easy to promote the ring opening of aflatoxin.

[0023] 2. Construction of laccase mutant gene engineering strain

[0024] The laccase mutant gene in step 1 is subjected to single-point mutant construction and is connected to the expression vector pPic9k, and the expression host is Pichia pastoris, to obtain an engineering strain containing the mutant gene of the application.

[0025] The strain Pichia pastoris GS115 / pPic9k(+)-lcc5-G of the application has been deposited with the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M 20232440, the deposit date is December 04, 2023, and the deposit address is Wuhan, China, Wuhan University.

[0026] (II) Expression of the laccase mutant gene engineering strain and protein purification

[0027] The engineering strain Pichia pastoris GS115 / pPic9k(+)-Lcc5-G obtained in (I) is inoculated into a test tube containing 5 mL of BMGY medium, and after 24 h of culture at 28°C and 200 rpm, it is transferred to a flask containing 50 mL of BMGY medium, and cultured to OD 600 When the OD reaches 1.3-1.5, centrifuge at 4°C and 4000 rpm for 10 min, and resuspend the bacterial body with BMM liquid; resuspend the bacterial body in a 1000 mL flask (containing 400 mL of BMM liquid medium), sample every 24 h, take one sample and add an equal amount of methanol, and continue to induce culture at 28°C. By detecting the change of sample enzyme activity, the bacterial liquid is collected after 7 days of culture in BMM medium to purify the target protein. After centrifugal dialysis, the crude enzyme solution is removed from the supernatant by using a 0.22 μm filter membrane to remove impurities and bubbles, and is loaded onto a DEAE-Spharose Fast Flow anion exchange column. The obtained protein is detected by SDS-PAGE to reach the purity required for enzyme property detection.

[0028] (III) Detection of laccase mutant enzyme activity

[0029] Laccase enzyme activity determination method: the total reaction system is 1 mL, 17 μL of ABTS (15 mM) and 950 μL of sodium tartrate buffer solution are added to a 2.0 mL EP tube, and incubated at 30°C for 5 min. 33 μL of enzyme solution is added to the EP tube to make the reaction system 1 mL. After 3 min of reaction at 30°C, stand on ice for 30 s, and measure the OD 420 value by spectrophotometer.

[0030] The enzyme activity calculation formula is: enzyme activity (U / L) = 555.6 x dilution factor x OD 420

[0031] (iv) Detection of the stability of the laccase mutant at 50℃, 45℃, 40℃, 35℃

[0032] The mutant enzyme was incubated at 50℃, 45℃, 40℃, 35℃, pH 7.0 with ABTS as the substrate, and samples were taken every 0.5 h. The residual rate of enzyme activity after a certain time was calculated with the initial enzyme activity as 100%, according to the formula: residual rate of enzyme activity = (initial enzyme activity - lost enzyme activity) / initial enzyme activity x 100%. The results showed that the half-life of the mutant obtained in the application was 20 h at 45℃, pH 7.0, which was 5 times that of the starting enzyme.

[0033] (v) Application of the laccase mutant in toxin oxidative conversion experiments

[0034] The detoxification reaction system was 1 mL, in which the enzyme amount was 20 U, i.e. the enzyme activity was 20 U / mL, 950 μL of citric acid disodium hydrogen phosphate buffer was added, the final concentration of aflatoxin was 1 μg / mL, and the mixture was shaken and mixed, and then placed in a constant temperature water bath shaker at 45℃ and 150 r / min for reaction for 12 h. After 12 h, 200 μL of the reaction system was taken and 800 uL of methanol was added to terminate the reaction. Then, the aflatoxin-containing methanol was filtered with a 0.22 μm organic needle filter for machine detection. The control was a mixture of 950 μL of buffer and 50 μL of aflatoxin standard working solution, and three parallel controls and experimental groups were set.

[0035] HPLC detection conditions:

[0036] Aflatoxin: mobile phase: methanol-water = 45:55 (V / V) at a flow rate of 1 mL / min; column temperature: 25℃; detector setting: fluorescence detection excitation wavelength: 360 nm; emission wavelength: 440 nm. After the column, an optical chemical derivatization device was connected.

[0037] The results showed that under the same conditions, the oxidation conversion rate of the mutant Lcc5-G was 98% with aflatoxin as the substrate, and the oxidation conversion rate of the starting enzyme Lcc5 was 88%, and the oxidation conversion efficiency of the mutant was significantly improved compared with the starting enzyme.

Claims

1. A fungal laccase mutant Lcc5-G, characterized in that... Its amino acid sequence is shown in SEQ ID NO:

1.

2. The encoding gene of the fungal laccase mutant Lcc5-G according to claim 1, characterized in that... Its nucleotide sequence is shown in SEQ ID NO:

2.

3. The expression strain of the fungal laccase mutant Lcc5-G according to claim 1, characterized in that: The expressed strain is classified as Pichiapastoris GS115 / pPic9k(+)-lcc5-G and has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20232440, on December 4, 2023, at Wuhan University, Wuhan, China.

4. The application of the fungal laccase mutant Lcc5-G according to claim 1, characterized in that: The fungal laccase mutant Lcc5-G was used for the oxidative transformation of aflatoxin.

5. The application according to claim 4, characterized in that: During the oxidative transformation of aflatoxin, the system temperature was 45℃ and the pH value was 7.0.

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