A laccase mutant and its application

Abstract

The present invention relates to a laccase mutant and its application, belonging to the field of genetic engineering. The present invention obtains a mutant laccase Tth-MCO (G266D / D106E / V442G / V448T) by subjecting a wild-type laccase Tth-MCO to site-directed mutagenesis. The mutant plasmid pET-28a(+)-Tth-MCO (G266D / D106E / V442G / V448T) is transformed into Escherichia coli BL21(DE3) to obtain recombinantly expressed recombinant engineered bacteria E. coli BL21(DE3)-Tth-MCO (G266D / D106E / V442G / V448T). The laccase mutant of the present invention has good alkali resistance and higher enzyme activity than the wild-type at pH 8.5-10.0.

Description

Technical Field

[0001] The present invention belongs to the field of gene engineering technology, and in particular relates to a laccase mutant and application thereof. Background Art

[0002] Laccase (EC1.10.3.2) is a copper-containing polyphenol oxidase that can oxidize a variety of phenolic and non-phenolic aromatic substrates and reduce them to water. It has broad substrate specificity and eco-friendliness (using molecular oxygen in the air as the final electron acceptor and only releasing water as a by-product). It is considered to be a biological green tool with broad application prospects. Laccase has excellent substrate broadness.

[0003] As a green biocatalyst, laccase offers attractive advantages in treating resistant environmental pollutants and dye wastewater. It can oxidize and degrade many harmful substances and is widely used in the food industry, dye decolorization, pulping, bleaching, and papermaking wastewater treatment. It also has great potential for bioremediation of toxic compounds and carcinogens.

[0004] The main sources of laccase are fungal laccase and bacterial laccase. Fungal laccase is generally 60-70kDa, with severe glycosylation modification, the optimal reaction temperature is generally 30℃-60℃, and the pH tolerance range is between 3.5-7.0; while bacterial laccase has significant advantages such as a wide substrate spectrum, no need for glycosylation modification, good thermal stability, and a wide pH tolerance range, and has attracted widespread attention.

[0005] Laccase (Tth-MCO) from the archaeon Thermus thermophilus HB27 has an optimal reaction temperature of 92°C and a half-life of 14 hours at 80°C. It catalyzes the oxidation of aromatic phenols and methoxyphenols, diamines, aminophenols, and hydroxyindoles, but Tth-MCO protein has difficulty maintaining activity in alkaline environments. Summary of the Invention

[0006] In order to overcome the problems existing in the background technology, the present invention provides a laccase mutant that can still maintain activity in an alkaline environment, and the enzyme activity between pH 8.5-10.0 is higher than that of the wild type. It can still catalyze the oxidation reactions of aromatic phenols and methoxyphenols, diamines, aminophenols and hydroxyindoles under alkaline conditions, and can be used in brewing, agriculture or sewage treatment industries.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions:

[0008] The first object of the present invention is to provide a laccase mutant, the amino acid sequence of the laccase mutant is shown in SEQ ID NO.4.

[0009] Furthermore, the nucleotide sequence of the gene encoding the laccase mutant is shown in SEQ ID NO.3.

[0010] The present invention also provides a recombinant plasmid comprising the above encoding gene.

[0011] Furthermore, the plasmid is selected from pET-28a(+).

[0012] The present invention also provides a recombinant engineered bacterium comprising the above encoding gene.

[0013] Furthermore, the bacterial strain is selected from BL21 (DE3).

[0014] The invention relates to an application of the laccase mutant in catalyzing the oxidation of aromatic phenols and methoxyphenols, diamines, aminophenols and hydroxyindole.

[0015] Beneficial effects of the present invention:

[0016] The laccase mutant of the present invention can still maintain high activity in an alkaline environment, improve the ability of laccase to catalyze the oxidation reaction of aromatic phenols and methoxyphenols, diamines, aminophenols and hydroxyindoles in an alkaline environment, and can be used in winemaking, agriculture or sewage treatment industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The enzyme activities of the wild-type Tth-MCO and the mutant laccase Tth-MCO (G266D / D106E / V442G / V448T) of the present invention are shown. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and beneficial effects of the present invention more clear, the technical solution of the present invention will be described in detail below. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without making creative efforts shall fall within the scope of protection of the present invention.

[0019] Some experimental materials and reagents used in this invention:

[0020] Strains and vectors: The PET-28a(+) expression vector was obtained from Jiangsu Suzhou Hongxun Biotechnology Co., Ltd. BL21(DE3) competent Escherichia coli cells were commercially available.

[0021] LB medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, add distilled water to 1000 mL, and adjust the pH to 6.5-7.5. For solid medium, add 2.0% (w / v) agar.

[0022] PCR reaction system: 5 μL of 10x high-fidelity buffer containing MgSO4, 1 μL of 10 mM dNTP mixture, 1 μL of upstream primer solution, 1 μL of downstream primer solution, 1 μL of template plasmid solution, 1.2 μL of high-fidelity DNA polymerase, 2.5 U / μl, and 9.8 μL of ddH2O3.

[0023] The PCR program was set as follows: initial denaturation at 98°C for 3 min, denaturation at 98°C for 30 s, annealing at 56°C for 1 min, extension at 68°C for 14 min, 22 cycles, final extension at 68°C for 10 min, and storage at 12°C.

[0024] Example 1 Construction and transformation of wild-type Tth-MCO expression vector

[0025] The amino acid sequence of Tth-MCO, shown as SEQ ID NO. 2, was downloaded from GenBank (referenced from the NCBI gene sequence database, gene sequence accession number: AAS81712.1). Suzhou Hongxun Biotechnology Co., Ltd. was commissioned to convert the wild-type Tth-MCO amino acid sequence into the DNA sequence SEQ ID NO. 1 suitable for expression in E. coli. This gene sequence was synthesized and inserted into the pET-28a(+) vector. The NdeI and XhoI restriction sites were selected to construct the pET-28a(+)-Tth-MCO recombinant plasmid. The pET-28a(+)-Tth-MCO recombinant plasmid was transformed into E. coli BL21(DE3), plated onto solid LB culture plates containing 50 mg / L kanamycin, and incubated at 37°C for 16 hours. Positive colonies were selected, representing wild-type E. coli BL21(DE3)-Tth-MCO.

[0026] Example 2 Site-directed mutagenesis of Tth-MCO (G266D)

[0027] The recombinant strain containing the recombinant plasmid pET-28a(+)-Tth-MCO obtained in Experimental Example 1 was inoculated into LB medium (containing 50 μg / mL kanamycin) at a concentration of 0.1%, cultured overnight at 37° C., and the plasmid was extracted using a kit.

[0028] The wild-type Tth-MCO enzyme was mutated using the whole-plasmid site-directed mutagenesis PCR method. The first round of site-directed mutagenesis PCR was performed using the plasmid pET-28a(+)-Tth-MCO as a template and G266D_F and G266D_R as a primer pair. The primers were designed as follows:

[0029] G266D_F: 5'-ACGAACGCGCGGAAGTGCTGGTGCGCCTG-3' (SEQ ID NO.5)

[0030] G266D_R: 5'-TTCCGCGCGTTCGTCCGGCGCCAGCAGCAGTT-3' (SEQ ID NO.6)

[0031] After the PCR is completed, 1 μL of DPn I enzyme is added and mixed thoroughly, and the template plasmid is digested by incubation at 37 ° C for 1 h. Then 5 μL of PCR reaction solution is added to the DH5α competent cells, and the cells are allowed to stand on ice for 30 min. Heat shock is performed in a 42 ° C water bath for 60 s, and the cells are immediately returned to ice for cooling for 3 min. 500 μL of anti-antibody LB medium is added to the clean bench for culture (37 ° C, 220 rpm, 60 min), the bacterial solution is centrifuged (12000 rpm, 5 min), 500 μL of the supernatant is discarded, and the remaining 100 μL is resuspended in the cells by pipetting with a pipette tip. 100 μL of bacterial solution is transferred to the LB solid medium containing Kana resistance in the clean bench with a pipette, and gently spread with a sterile coating stick. After the bacterial solution is absorbed, the plate is inverted and cultured at 37 ° C overnight. A single colony is picked from each plate and inoculated into 5 mL of 50 μg / L Kana After culturing in LB medium (37°C, 220 rpm, 6 h), the sample was sent for sequencing. After sequencing, the service company returned the correct mutant plasmid pET-28a(+)-Tth-MCO(G266D).

[0032] Example 3 Site-directed mutagenesis of Tth-MCO (G266D / D106E)

[0033] The second round of site-directed mutagenesis was performed using pET-28a(+)-Tth-MCO(G266D) as the template plasmid and G266D / D106E_F and G266D / D106E_R as primer pairs. The primers were designed as follows:

[0034] G266D / D106E_F:5'-AGATCCGTTTCTGGAAATTCCGCCG-3'(SEQ ID NO.7)

[0035] G266D / D106E_R:5'-CCAGAAACGGATCTTCCACTTTCGGGCTA-3'(SEQ ID NO.8)

[0036] The remaining steps were the same as in Example 2 to obtain the mutant plasmid pET-28a(+)-Tth-MCO (G266D / D106E).

[0037] Example 4 Site-directed mutagenesis of Tth-MCO (G266D / D106E / V442G)

[0038] The third round of site-directed mutagenesis was performed using pET-28a(+)-Tth-MCO(G266D / D106E) as the template plasmid and G266D / D106E / V442G_F and G266D / D106E / V442G_R as primer pairs. The primers were designed as follows:

[0039] G266D / D106E / V442G_F:

[0040] 5'-GGTTTCATTGCCATATTGTGGAACATGAAG-3'(SEQ ID NO.9)

[0041] G266D / D106E / V442G_R:

[0042] 5'-GGCAATGAAACCCGGTGCGGCCTTTTTC-3'(SEQ ID NO.10)

[0043] The remaining steps were the same as in Example 2 to obtain the mutant plasmid pET-28a(+)-Tth-MCO (G266D / D106E / V442G).

[0044] Example 4 Tth-MCO (G266D / D106E / V442G / V448T) site-directed mutagenesis

[0045] The fourth round of site-directed mutagenesis was performed using pET-28a(+)-Tth-MCO(G266D / D106E / V442G) as the template plasmid and G266D / D106E / V442G / V448T_F and G266D / D106E / V442G / V448T_R as primer pairs. The primers were designed as follows:

[0046] G266D / D106E / V442G / V448T_F:

[0047] 5'-GTTGTTTCATTGCCATATTACTGAACATGAAGATCGCGGCATG-3'(SEQ ID NO.11)

[0048] G266D / D106E / V442G / V448T_R:

[0049] 5'-CATGCCGCGATCTTCATGTTCAGTAATATGGCAATGAAACAC-3'(SEQ ID NO.12)

[0050] The remaining steps were as in Example 2 to obtain the mutant plasmid pET-28a(+)-Tth-MCO (G266D / D106E / V442G / V448T). The amino acid sequence of the mutant laccase Tth-MCO (G266D / D106E / V442G / V448T) is shown in SEQ ID NO. 4, and the nucleotide sequence is shown in SEQ ID NO. 3.

[0051] Example 5 Construction of recombinant engineering bacteria

[0052] The mutant plasmid pET-28a(+)-Tth-MCO(G266D / D106E / V442G / V448T) was transformed into Escherichia coli BL21(DE3) to obtain recombinant expression engineered bacteria E. coli BL21(DE3)-Tth-MCO(G266D / D106E / V442G / V448T). The recombinant bacteria were plated on solid LB culture plates containing 50 mg / L kanamycin resistance and cultured at 37°C for 16 h. Positive colonies were picked, namely the recombinant engineered bacteria E. coli BL21(DE3)-Tth-MCO(G266D / D106E / V442G / V448T).

[0053] Example 6 Preparation of bacterial slurries of wild-type Tth-MCO and mutant Tth-MCO (G266D / D106E / V442G / V448T) The recombinant engineered bacteria E. coli BL21(DE3)-Tth-MCO and E. coli BL21(DE3)-Tth-MCO (G266D / D106E / V442G / V448T) were respectively inoculated into 5 mL of LB medium containing kanamycin in a shake tube and cultured on a shaker at 37°C, 220 rpm, and shaken for 16 h for activation.

[0054] The activated recombinant engineered bacteria E. coli BL21 (DE3) -Tth-MCO and recombinant engineered bacteria E. coli BL21 (DE3) -Tth-MCO (G266D / D106E / V442G / V448T) were inoculated into 5 mL LB medium containing kanamycin at a 1% inoculum size, and cultured on a shaker at 37 ° C., 220 rpm, and shaken for about 2 to 3 h (OD600 reached 0.6 to 1.0). After that, IPTG was added to a final concentration of 0.25 mM and continued to shake at 20 ° C., 160 rpm / min for about 20 h. After the end of the culture, the cell pellet was collected by centrifugation to obtain bacterial slurries containing the target protein Tth-MCO and the target protein Tth-MCO (G266D / D106E / V442G / V448T), respectively.

[0055] Example 7 Preparation of pure proteins of wild-type Tth-MCO and mutant Tth-MCO (G266D / D106E / V442G / V448T)

[0056] The E. coli BL21 (DE3) -Tth-MCO engineered bacterial sludge and the E. coli BL21 (DE3) -Tth-MCO (G266D / D106E / V442G / V448T) engineered bacterial sludge obtained in Example 6 were added with an appropriate amount of PBS buffer (pH 7.5) to fully disperse the sludge, and then the cells were disrupted by mechanical ultrasonic disruption in an ice salt bath. The expression of the target protein was preliminarily determined by protein gel electrophoresis, and then the precipitate was removed by centrifugation. A slightly excess amount of copper sulfate solution was added to the supernatant, and the reaction was stirred at room temperature for 1 h. After the reaction, it was filtered through a 0.22 μm filter head and purified by molecular exclusion chromatography SEC to obtain the target pure protein solution. Finally, the protein concentration was determined by the Bradford method.

[0057] Example 8 Determination of Enzyme Activity of Wild-Type Tth-MCO and Mutant Laccase Tth-MCO (G266D / D106E / V442G / V448T)

[0058] Place 750 μL of Britton-Robinson buffer and 200 μL of ABTS (5 mmol / L) in an enzyme activity tube. Preheat the tube at 37°C for 2 minutes, then add 50 μL of purified laccase solution. Allow the tube to react for 3 minutes, then immediately place the tube in an ice-water mixture to terminate the reaction. Read the plate using a microplate reader at ε420 nm. One unit (U) of laccase activity is defined as the amount of enzyme required to catalyze the oxidation of 1 μmol of substrate per minute under optimal conditions.

[0059] Optimal pH determination: The purified wild-type laccase Tth-MCO and mutant laccase Tth-MCO (G266D / D106E / V442G / V448T) were quantified and diluted to 0.25 mg / mL. The catalytic activity of the wild-type laccase Tth-MCO and mutant laccase Tth-MCO (G266D / D106E / V442G / V448T) was measured at 50°C in Bryant-Robinson buffer at different pH values ​​(pH 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0).

[0060] Here are the results:

[0061]

[0062] The wild-type Tth-MCO has a maximum enzyme activity of 120.3 U / L at pH 7.5.

[0063] The enzyme activity of the mutant Tth-MCO (G266D / D106E / V442G / V448T) reached a maximum of 115.6 U / L at pH 9.0. The enzyme activity of the mutant Tth-MCO (G266D / D106E / V442G / V448T) was higher than that of the wild-type Tth-MCO between pH 8.5 and 10.0.

[0064] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A laccase mutant, characterized in that The amino acid sequence of the laccase mutant is shown in SEQ ID NO.

4.

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

3.

3. A recombinant plasmid comprising the coding gene as claimed in claim 2.

4. The recombinant plasmid according to claim 3, wherein The plasmid is selected from pET-28a(+).

5. A recombinant engineered bacterium comprising the coding gene as claimed in claim 2.

6. Use of the recombinant engineered bacteria according to claim 5 in preparing laccase mutants.

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