Preparation method and application of sporocyst laccase and recombinant laccase protein of sporocyst laccase

Abstract

The invention belongs to the technical field of degrading enzymes, and particularly relates to sporocyst laccase and a preparation method and application of recombinant laccase protein of the sporocyst laccase. The sporophyte laccase is derived from sporophyte Dactylosporangium, and a laccase gene is obtained from the sporophyte Dactylosporangium; recombinant plasmids and recombinant strains of the laccase are successfully constructed through an escherichia coli vector and host bacteria, and recombinant laccase protein is obtained; the recombinant laccase protein disclosed by the invention has the capability of degrading indole and 3-methylindole with different concentrations, and can degrade the indole and the 3-methylindole at the same time, so that the recombinant laccase protein has important significance for solving the pollution problem of indole and skatole.

Description

Technical Field

[0001] The present invention belongs to the technical field of degrading enzymes, and particularly relates to a preparation method and application of cystomycete laccase and its recombinant laccase protein. Background Art

[0002] With the vigorous development of large-scale and intensive livestock and poultry farming, while meat, eggs, and milk, the by-products of farming, meet people's needs, the impact of a large amount of discharged fecal sewage and odor substances on environmental pollution and residents' lives has gradually emerged, seriously restricting the sustainable development of the livestock industry. The odor substances emitted by livestock and poultry mainly include volatile fatty acids, sulfur-containing compounds, nitrogen-containing compounds, and aromatic compounds, etc. Among them, indole and 3-methylindole (skatole), as the end products of anaerobic metabolism of L-tryptophan in animals, belong to typical nitrogen heterocyclic aromatic compounds and are one of the main odor substances in livestock and poultry fecal sewage, with a strong fecal odor. In addition, the accumulation of high-concentration indole in animals can cause diseases such as anemia, hemolytic disease, hemoglobinuric nephropathy, and glomerulosclerosis; damage the normal functions of the intestine and fallopian tube; accelerate tumor formation, and has serious teratogenic and mutagenic properties. Skatole is considered to be the third most harmful odor substance emitted by livestock and poultry after NH3 and H2S, which can induce acute pulmonary edema and emphysema in ruminants and human lung diseases; attract mosquitoes to lay eggs and reproduce, promoting the spread of vector-borne diseases; cause the generation of mutton smell in pork and reduce the meat quality. In recent years, the pollution of indole and skatole emitted by livestock and poultry has been reported in many places around the world. It is urgent to take effective measures to solve the problems of indole and skatole pollution.

[0003] At present, the methods for reducing the pollution of nitrogen heterocyclic compounds such as indole mainly include physical and chemical methods and biodegradation methods. Physical and chemical methods, such as adsorption and photocatalytic degradation, although have good removal effects on indole and skatole, the problems such as high cost, high energy consumption, and secondary pollution undoubtedly limit their application scope. And the biodegradation method using microorganisms has the characteristics of economy, high efficiency, and environmental friendliness in the repair of indole and its derivative pollution systems, and is widely used in the treatment of environmental pollutants. The microorganisms that can degrade indole and skatole reported currently mainly include Cupriavidus, Pseudomonas, Acinetobacter, and Burkholderia, etc. However, past research has mostly focused on the isolation and characterization of indole and skatole-degrading bacteria, and the development work on functional genes and enzymes of indole and skatole-degrading bacteria still needs to be further strengthened. Summary of the Invention

[0004] Aiming at the above problems, the purpose of the present invention is to provide a preparation method and application of cystomycete laccase and its recombinant laccase protein.

[0005] The technical content of the present invention is as follows:

[0006] The present invention provides a cystobacter laccase, whose nucleic acid sequence is shown in SEQ ID NO.1 of the sequence listing, and the amino acid sequence is shown in SEQ ID NO.2 of the sequence listing;

[0007] The cystobacter laccase is derived from Dactylosporangium;

[0008] The present invention also provides a gene encoding cystobacter laccase, and the nucleic acid sequence encoded by the gene is shown in SEQ ID NO.1 of the sequence listing, and the amino acid sequence is shown in SEQ ID NO.2 of the sequence listing.

[0009] The present invention also provides a recombinant plasmid and a recombinant strain containing the above-mentioned cystobacter laccase gene.

[0010] The present invention also provides a method for preparing a recombinant laccase protein, which comprises the following steps:

[0011] 1) Synthesize the laccase gene, connect it with an expression vector, and construct a recombinant plasmid;

[0012] The expression vector includes one of the Escherichia coli expression vectors pET-28a(+), pET32(a), and pET22(b);

[0013] 2) Transfer the recombinant plasmid into competent cells, obtain an expression plasmid, perform sequencing verification, and obtain a cloned plasmid with the correct gene sequence;

[0014] The competent cells include one of Escherichia coli DH5α, DH10B, and TOP10;

[0015] 3) Transfer the cloned plasmid with the gene sequence into a host bacterium to obtain a recombinant strain;

[0016] 4) Express and purify the recombinant strain to obtain the recombinant laccase protein;

[0017] The host bacterium includes one of Escherichia coli BL21(DE3), E.coli Rosetta(DE3), and Rosetta-gamiB(DE3).

[0018] The present invention also provides the recombinant laccase protein obtained by the above preparation method.

[0019] The present invention also provides the application of the above-mentioned cystobacter laccase and recombinant laccase protein in degrading livestock and poultry odor substances, and the livestock and poultry odor substances include indole and 3-methylindole;

[0020] The degradation includes simultaneous degradation.

[0021] The beneficial effects of the present invention are as follows:

[0022] The laccase of the present invention is derived from Dactylosporangium, and the laccase gene is obtained therefrom; and its recombinant plasmid and recombinant strain are successfully constructed through an Escherichia coli vector and host bacteria, and the recombinant laccase protein is obtained.

[0023] The recombinant laccase protein of the present invention has the ability to degrade indole and 3-methylindole at different concentrations and can degrade them simultaneously, which is of great significance for solving the pollution problems of indole and skatole. Brief Description of the Drawings

[0024] Figure 1 It is the SDS-PAGE identification result of the recombinant laccase protein;

[0025] Figure 2 It is the degradation result diagram of the recombinant laccase protein for indole and 3-methylindole at different concentrations. Detailed Description of the Invention

[0026] The present invention will be further described in detail below through specific implementation cases and the description of the drawings. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art fall within the scope defined by the appended claims of this application.

[0027] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents on the conventional market.

[0028] Example

[0029] Preparation of a Degrading Enzyme Recombinant Protein

[0030] 1) Synthesis of the degrading enzyme gene and construction of its recombinant expression vector

[0031] The gene information of Dactylosporangium sp. NPDC050588 is obtained from the public database (https: / / npdc.rc.ufl.edu / home), and the gene encoding Multicopperoxidase of laccase is mined from the genome. Its nucleic acid sequence is shown in SEQ ID NO.1, with a length of 1158bp. The gene is named CotA, and 1-141bp is the signal peptide sequence. After removing the signal peptide sequence part, it is named CotA6.

[0032] According to the gene information, the synthesis of CotA was completed by Anhui General Systems Biology Co., Ltd. Specific primers were designed (forward primer SEQ ID NO.3: 5’-ATGAATTCCAGACCGGGATGTTCGCCGAGCG; reverse primer SEQ ID NO.4: 5’-ATCTCGAGGTTGTGCACGTGGGCGTCGTCGG). Using the DNA of the cystomycete Dactylospora ngium sp. NPDC050588 as a template, PCR amplification was carried out. The amplification conditions were as follows: ① Pre-denaturation at 95°C for 5 min; ② 95°C for 30 s, 58°C for 30 s, 72°C for 1 min, with 30 cycles; ③ 72°C for 5 min. Restriction enzyme cleavage sites EcoRI (5’-GAATTC-3’) and XhoI (5’-CTCGAG-3’) were added to both ends of the gene. The vector pET-28a(+)- and the gene fragment were digested with EcoRI and XhoI respectively, and then ligated with T4 ligase to obtain the recombinant plasmid pET-28a(+)-CotA;

[0033] The recombinant plasmid was transformed into DH5α competent cells. After ice-bathing for 30 min, heat shock was performed in a 42°C water bath for 90 s. After ice-bathing for 2 min, 500 μL of LB liquid medium was added, and the cells were cultured at 37°C and 200 r / min for 1 h. After centrifugation of the culture, it was spread on an LB solid plate containing 50 μL / mL of sodium ampicillin. After culturing at 37°C for 16 h, single colonies were selected. The cloned plasmid was extracted by digestion and verified by sequencing. The cloned plasmid was then transformed into Escherichia coli BL21(DE3) to construct a recombinant strain;

[0034] 2) Expression and purification of recombinant laccase protein

[0035] The recombinant strain was inoculated into an LB liquid medium containing sodium ampicillin (0.1 mg / mL) and activated in a shaker at 37°C and 170 r / min for 24 h. The activated bacterial solution was transferred with an inoculation amount of 1% to an OD 600nm ≈0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG) with a final concentration of 0.5 mmol / L and a sterile CuSO4 solution with a final concentration of 0.25 mmol / L were added, and the cells were cultured at 16°C and 150 r / min for 18 - 20 h;

[0036] The bacterial solution was taken and centrifuged at 4°C and 4000 r / min for 10 min to collect the bacteria. The bacteria were resuspended in 25 mL of (50 mmol / L, pH 7.2) phosphate buffer, sonicated in an ice-water bath for 15 min, and then centrifuged at 4°C and 10000 r / min for 10 min to collect the supernatant;

[0037] The supernatant was added to a nickel column for purification. Impurity proteins were eluted with 10 mmol / L imidazole, and the recombinant laccase protein was eluted with 300 mmol / L imidazole.

[0038] Desalting was performed using a desalting column C500090 purchased from Sangon Biotech Co., Ltd., and the specific method was referred to the instruction manual. The purified and desalted protein was identified for its molecular weight and purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results were as Figure 1 shown. The recombinant laccase protein could be expressed in Escherichia coli. Enrichment of the recombinant protein could be seen in the supernatant around 40 KDa. The protein molecular weight was consistent with the theoretical value deduced from the amino acid sequence. After affinity chromatography purification, a recombinant laccase protein with a purity greater than 85% was obtained.

[0039] Experimental Example

[0040] Determination of the degradation activity of recombinant laccase protein CotA

[0041] The total volume of the reaction system for detecting the degradation activities against indole and 3-methylindole was 200 μL, including 50 mmol / L phosphate buffer (pH 6), indole or 3-methylindole (final concentrations of 50, 100, 200 mg / L), mediator ABTS (final concentration of 5 mmol / L), and CotA laccase. The reaction was carried out at room temperature in the dark, and the degradation rates of the reaction system were measured at 3, 6, 12, 24 h, and 48 h respectively. The experimental group without adding the recombinant laccase protein was used as a control, and all experiments were set with 3 replicates.

[0042] Determination of indole and 3-methylindole concentrations: Detection was performed using high performance liquid chromatography-photoelectric reactor-fluorescencedetector (HPLC-PHR-FLD). 400 μL of methanol was added to the 200 μL reaction system, shaken well, and the mixture was filtered through a 0.22 μm organic filter membrane and injected into the injection vial. Chromatographic conditions: The chromatographic column was XBridgeTM C18 (4.6 mm × 250 mm, 5 μm), the mobile phase was acetonitrile∶water = 60∶40 (V / V), the flow rate was 1 mL / min, the column temperature was 35 °C, the elution time was 15 min, the fluorescence excitation wavelength was 270 nm, and the emission wavelength was 350 nm.

[0043] The results were as Figure 2As shown, the recombinant laccase protein has a certain degradation ability for indole and 3-methylindole at different concentrations, but as the substrate concentration increases, the degradation rate also continuously decreases. When the substrate concentration is 50 mg / L, the degradation rates of indole ( Figure 2 -a) and 3-methylindole ( Figure 2 -b) are both above 60% within 3 h, and reach 95.7% and 94.4% respectively within 48 h. When the substrate concentrations are 100 mg / L and 200 mg / L, the degradation rates of indole within 48 h are 83.1% and 65.9% respectively, and those of 3-methylindole are 88.1% and 85.7% respectively.

Claims

1. A cyst-forming fungus laccase, characterized in that, Its nucleic acid sequence is shown in SEQ ID NO.1 of the sequence listing, and its amino acid sequence is shown in SEQ ID NO.2 of the sequence listing.

2. The cysteine laccase according to claim 1, characterized in that, The cystomycete laccase is derived from the cystomycete Dactylosporangium.

3. A gene encoding the cysteine laccase according to claim 1, characterized in that, The nucleic acid sequence encoded by the gene is shown in SEQ ID NO.1 of the sequence listing, and its amino acid sequence is shown in SEQ ID NO.2 of the sequence listing.

4. A recombinant plasmid or recombinant strain containing the cystomycete laccase gene according to claim 3.

5. A method for preparing a recombinant laccase protein, characterized in that, Comprising the following steps: 1) Synthesize the laccase gene according to claim 2, ligate it with an expression vector to construct a recombinant plasmid; 2) Transfer the recombinant plasmid into competent cells, obtain the expression plasmid, perform sequencing verification, and obtain a cloned plasmid with the correct gene sequence; 3) Transfer the cloned plasmid with the gene sequence into a host bacterium to obtain a recombinant strain; 4) Express and purify the recombinant strain to obtain the recombinant laccase protein.

6. The preparation method of the recombinant laccase protein according to claim 5, wherein The expression vector in step 1) includes one of the Escherichia coli expression vectors pET-28a(+), pET32(a), and pET22(b).

7. The preparation method of the recombinant laccase protein according to claim 5, characterized in that, The competent cells include one of Escherichia coli DH5α, DH10B, and TOP10.

8. The preparation method of the recombinant laccase protein according to claim 5, characterized in that, The host bacterium includes one of Escherichia coli BL21(DE3), E.coli Rosetta(DE3), and Rosetta-gamiB(DE3).

9. A recombinant laccase protein obtained by the preparation method according to any one of claims 5 to 8.

10. Use of the cysteine laccase according to claim 1 and the recombinant laccase protein according to claim 9 in degrading livestock and poultry odor substances, characterized in that, The livestock and poultry odor substances include indole and 3-methylindole; The degradation includes simultaneous degradation.

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