A laccase and its mutants and their applications
By performing site-directed mutagenesis on fungal laccase PnLac1 and heterologous expression in Pichia pastoris, the problems of insufficient activity and stability of existing laccases were solved, and the degradation efficiency of organic dyes and lignin was significantly improved.
Patent Information
- Application Number
- CN202510439677.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-09
- Publication Date
- 2025-12-02
- Estimated Expiration
- 2045-04-09
AI Technical Summary
Existing laccases have shortcomings in terms of enzyme activity and stability, especially in their low efficiency when degrading organic dyes and organic polymers such as lignin.
By performing site-directed mutagenesis on the fungal laccase PnLac1, specifically by mutating aspartic acid at position 166 to alanine (D166A), histidine at position 348 to phenylalanine (H348F), methionine at position 403 to phenylalanine (M403F), or leucine at position 467 to methionine (L467M), and then heterologously expressing and purifying the mutant in Pichia pastoris, a recombinant strain was formed.
The enzyme activity of laccase was improved. The enzyme activities of laccase mutants H348F and L467M against ABTS were increased by 1.89 times and 1.65 times, respectively, while the enzyme activities of D166A and M403F against guaiacol were increased by 2.07 times and 3.72 times, respectively, which significantly improved the degradation efficiency of organic dyes and lignin.
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Figure CN120210141B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a laccase, its mutants, and their applications, belonging to the field of biotechnology. Background Technology
[0002] Laccase (EC 1.10.3.2), also known as polyphenol oxidase, belongs to the polycopper oxidase superfamily. It can oxidize a variety of aromatic compounds, including phenols, polyphenols, and aniline, using molecular oxygen as the electron acceptor to produce water as the only byproduct. Due to its broad substrate spectrum and green catalytic properties, laccase is widely used in organic synthesis, biosensor construction, fuel cell development, biomass value-added, and exogenous biodegradation.
[0003] Laccases are widely distributed in nature and can be classified into insect laccases, plant laccases, bacterial laccases, and fungal laccases according to their origin. Currently, laccases are known to oxidize more than 250 substrates, and their enormous application potential has led to dedicated research, development, and industrial applications.
[0004] Most reported laccases are derived from bacteria. Fungal genomes also contain abundant laccase genes. Compared to bacteria, fungal laccases are extracellular enzymes, making protein purification simpler, and they exhibit higher enzyme activity and stability. Laccase-secreting fungi are mainly found in higher fungi such as basidiomycetes, ascomycetes, and deuteromycetes. The enzymatic properties and laccase-producing capacity of laccases vary considerably among different fungal species. Summary of the Invention
[0005] To address the problems existing in the prior art, this invention provides a laccase, its mutants, and its applications.
[0006] The technical solution of this invention is as follows:
[0007] A novel laccase, PnLac1, has the amino acid sequence shown in SEQ ID NO.2 and the nucleotide sequence of its encoding gene shown in SEQ ID NO.1.
[0008] A laccase PnLac1 mutant, wherein the mutant is a site-directed mutation of aspartic acid at position 166, histidine at position 348, methionine at position 403, or leucine at position 467 of laccase PnLac1.
[0009] According to a preferred embodiment of the present invention, the mutant is laccase mutant D166A, whose amino acid sequence is shown in SEQ ID NO.4 and whose nucleotide sequence encoding the gene is shown in SEQ ID NO.3; wherein the aspartic acid at position 166 of the laccase amino acid sequence is mutated to alanine.
[0010] According to a preferred embodiment of the present invention, the mutant is a laccase mutant H348F, whose amino acid sequence is shown in SEQ ID NO. 6 and whose nucleotide sequence encoding the gene is shown in SEQ ID NO. 5; wherein the histidine at position 348 of the laccase amino acid sequence is mutated to phenylalanine.
[0011] According to a preferred embodiment of the present invention, the mutant is a laccase mutant M403F, whose amino acid sequence is shown in SEQ ID NO. 8 and whose nucleotide sequence encoding the gene is shown in SEQ ID NO. 7; wherein the methionine at position 403 of the laccase amino acid sequence is mutated to phenylalanine.
[0012] According to a preferred embodiment of the present invention, the mutant is a laccase mutant L467M, whose amino acid sequence is shown in SEQ ID NO. 10 and whose nucleotide sequence encoding the gene is shown in SEQ ID NO. 9; wherein the 467th leucine in the laccase amino acid sequence is mutated to methionine.
[0013] A recombinant vector is a plasmid vector into which the encoding genes of the above-mentioned laccase PnLac1, laccase mutant D166A, laccase mutant H348F, laccase mutant M403F or laccase mutant L467M are inserted.
[0014] According to a preferred embodiment of the present invention, the plasmid vector is pPICZαA.
[0015] A recombinant strain is obtained by transforming the above-mentioned recombinant vector into a host cell.
[0016] According to a preferred embodiment of the present invention, the host cell is Pichia pastoris.
[0017] Applications of the above-mentioned laccase PnLac1, laccase mutant D166A, laccase mutant H348F, laccase mutant M403F or laccase mutant L467M in biodegradation.
[0018] According to a preferred embodiment of the present invention, the biodegradation refers to the use of ABTS as a medium to degrade organic dyes such as crystal violet or to degrade organic polymers such as lignin.
[0019] Experimental procedures not described in detail in this invention can be performed according to conventional experimental procedures in this technical field.
[0020] Beneficial effects
[0021] 1. This invention discovers a novel laccase, PnLac1, which has a high decolorization efficiency for crystal violet, with a degradation rate of 75% in 5 hours, and can be used to degrade organic dyes.
[0022] 2. This invention is based on the novel laccase PnLac1, and site-directed mutagenesis is performed at positions 166, 348, 403, or 467. Specifically, aspartic acid at position 166 is mutated to alanine (D166A), histidine at position 348 to phenylalanine (H348F), methionine at position 403 to phenylalanine (M403F), or leucine at position 467 to methionine (L467M), resulting in laccase mutants H348F, D166A, L467M, and M403F. Compared to wild-type laccase PnLac1, the laccase mutants exhibit higher enzyme activity. When using ABTS as a substrate, the enzyme activities of laccase mutants H348F and L467M were increased by 1.89 times and 1.65 times, respectively, compared with wild-type PnLac1, showing potential for using ABTS as a medium to degrade stubborn substances. When using guaiacol as a substrate, the enzyme activities of laccase mutants D166A and M403F were increased by 2.07 times and 3.72 times, respectively, compared with wild-type PnLac1, and can be used to degrade lignin and other organic polymers, showing high application prospects in the field of lignin. Attached Figure Description
[0023] Figure 1 The image shows the results of polyacrylamide gel electrophoresis (SDS-PAGE) of wild-type laccase PnLac1.
[0024] In the figure, lane 1 is Maker, and lanes 2 and 3 are both wild-type laccase PnLac1.
[0025] Figure 2 This is a spectral scan of wild-type laccase PnLac1.
[0026] Figure 3 The results show the optimal reaction pH for the in vitro reaction of wild-type laccase PnLac1.
[0027] Figure 4 A diagram illustrating the process of degrading crystal violet using ABTS as a medium for wild-type laccase PnLac1.
[0028] Figure 5 The graph shows the decolorization rate of wild-type laccase PnLac1 in the presence and absence of a medium, as a function of time, representing the decolorization rate of crystal violet and bromophenol blue.
[0029] Figure 6 The relative enzyme activities of wild-type laccase PnLac1 and its mutants are given when ABTS is used as a substrate.
[0030] Figure 7 The relative enzyme activities of wild-type laccase PnLac1 and its mutants are given when guaiacol is used as a substrate. Detailed Implementation
[0031] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are all methods known to those skilled in the art.
[0032] Example 1: Heterologous expression of novel laccase PnLac1
[0033] 1. During a bioinformatics database search using the BLASTp algorithm, the inventors discovered that the amino acid sequence of a gene in *Phellopilus nigrolimitatus* shared significant homology with the amino acid sequence of laccase. Therefore, the inventors speculated that the protein product expressed by this gene might have laccase activity and named this gene PnLac1. The inventors then heterologously expressed the protein in *Pichia pastoris*, naming the expressed protein product PnLac1, whose amino acid sequence is shown in SEQ ID NO.2, and whose nucleotide sequence encoding the gene is shown in SEQ ID NO.1.
[0034] The heterologous expression and purification of laccase PnLac1 are detailed below:
[0035] The PnLac1 gene was artificially synthesized by Qingke Biotechnology Co., Ltd. according to the nucleotide sequence shown in SEQ ID NO.1, and cloned into the pPICZαA vector to obtain the recombinant vector pPICZαA-PnLac1. Then, the recombinant vector pPICZαA-PnLac1 was transformed into Pichia pastoris X-33 competent cells by electroporation. The cells were cultured on LB solid medium containing bleomycin (50 μg / mL) for 12 h, and transformants were screened to obtain positive transformants.
[0036] Single colonies of laccase PnLac1 positive transformants were picked and inoculated into long glass tubes containing 2 mL of YPG liquid medium. The tubes were incubated at 200 rpm and 30 °C at an angle for 30 h to obtain seed culture. The seed culture was then inoculated into 125 mL of BMGY liquid medium at a volume ratio of 1% and incubated at 130 rpm and 30 °C for 20 h until OD600 = 3. The cells were then collected by centrifugation at 3500 rpm for 20 min at 4 °C.
[0037] The bacterial cells were resuspended in 150 mL of BMMY liquid medium containing 0.5% v / v methanol and cultured at 30°C and 140 rpm. Methanol was added every 24 h to maintain a final methanol concentration of 1%, and the total culture time was 72 h. After the culture was completed, the culture medium was collected by centrifugation at 8000 rpm for 20 min. The culture medium was concentrated using a 30 kDa ultrafiltration module, which concentrated it approximately 10 times. Finally, the recombinant protein was purified using a nickel ion affinity chromatography column. The purified protein was then desalted by ultrafiltration using a 50 kDa threshold tube to obtain the target protein laccase PnLac1.
[0038] The purified laccase PnLac1 was identified by polyacrylamide gel electrophoresis (SDS-PAGE), and the results are as follows: Figure 1 As shown.
[0039] Depend on Figure 1 It can be seen that due to the excessive glycosylation of the Pichia pastoris expression system, the molecular weight of laccase PnLac1 in the SDS-PAGE results is around 100 kDa, indicating that the heterologous expression of laccase PnLac1 was successful.
[0040] Example 2
[0041] The laccase PnLac1 prepared in Example 1 was diluted with 20 mM citrate-phosphate buffer (pH 7) to a protein concentration of 1 mg / ml. PBS buffer was used as a blank solution. The laccase solution and blank solution were scanned across the entire wavelength range of 200–800 nm using a UV spectrophotometer. The results are as follows: Figure 2 As shown.
[0042] Depend on Figure 2 It can be seen that a high absorption peak appears at around 300 nm, but no high absorption peak appears at 610 nm, which confirms that PnLac1 is a unique fungal laccase.
[0043] Example 3
[0044] Determination of the optimal pH for in vitro enzyme reaction: The reaction system was constructed using 880 μL of 100 mM buffer, 100 μL of 10 mM reaction substrate, and 10 μL of laccase PnLac1 enzyme solution diluted 10 times.
[0045] The buffer solution is sodium acetate buffer, BR buffer solution, or phosphate-disodium hydrogenphosphate; the reaction substrate is 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) or 2,6-xylenol (2,6-DMP).
[0046] When the buffer solution is sodium acetate buffer, the reaction system is constructed according to the above ratio, and two pH gradient points of 4.0 and 5.0 are set. Using a UV spectrophotometer, the absorbance is measured at 420 nm when ABTS is used as the substrate and at 469 nm when 2,6-DMP is used as the substrate. After adding the enzyme solution, the change in absorbance is measured at 10-second intervals.
[0047] When the buffer solution is BR buffer or phosphate-citrate buffer, seven pH gradient points of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 are set. Using a UV spectrophotometer, the absorbance is measured at 420 nm when ABTS is used as the substrate and at 469 nm when 2,6-DMP is used as the substrate. After adding the enzyme solution, the change in absorbance is measured at 10-second intervals.
[0048] The optimal pH for the in vitro reaction of the enzyme was determined as follows: Figure 3 As shown.
[0049] Depend on Figure 3 It was found that the optimal pH for laccase PnLac1 was determined using two substrates in three buffer solutions. With ABTS as the substrate, laccase PnLac1 achieved its highest activity in phosphate-citrate buffer at pH 2.2; with 2,6-DMP as the substrate, laccase PnLac1 achieved its highest activity in sodium acetate buffer at pH 4.
[0050] Example 4
[0051] Crystal violet was dissolved in 20 mM phosphate-citrate buffer (pH 5) to prepare a dye system with a final concentration of 1 mg / ml. Then, 0.5 mM ABTS and 1 U / ml laccase PnLac1 enzyme solution were added to the dye system to prepare the laccase-mediator group (CV-PnLac1). Simultaneously, a dye system containing 0.5 mM ABTS and an equal volume of PBS buffer was used as the blank group (CV-LMS). Both the laccase-mediator group and the blank group were reacted at 30℃ and 200 rpm for 48 h. The dye decolorization rate was measured periodically during the reaction. The results are shown below. Figure 4 and Figure 5 As shown.
[0052] Decolorization rate calculation formula:
[0053] Where I is the decolorization rate; A0 is the initial dye absorbance value; and A1 is the detected dye absorbance value.
[0054] Depend on Figure 4 and Figure 5 It is known that the novel laccase PnLac1 provided by the present invention has the function of decolorizing crystal violet, and the decolorization ability is greatly improved after adding ABTS as a medium. The decolorization efficiency is very high, and the decolorization rate of crystal violet changes significantly with time, reaching 75% in 5 hours.
[0055] Example 5
[0056] 1. A three-dimensional structure of wild-type laccase PnLac1 was constructed using alphafold3. Several key amino acids were identified by analyzing the active structure, namely H348, M403, L467, and D166. The inventors of this application speculate that these four sites are located near the active center and highly conserved regions, and that site-directed mutagenesis on them is very likely to improve the enzyme activity of laccase PnLac1. Therefore, site-directed mutagenesis was performed on them.
[0057] Using the recombinant vector pPICZαA-PnLac1 containing the wild-type laccase PnLac1 encoding gene constructed in Example 1 as a template, H348F, D166A, L467M, and M403F were selected as mutation sites. PCR amplification was performed using mutation primers to introduce mutations at the mutation sites, resulting in single-point mutant plasmids H348F, D166A, L467M, and M403F.
[0058] The mutation primers used include:
[0059] L467M forward: 5'-CTGGTatgGCCATCGTATTTGCAGAGGACACT-3'.
[0060] L467M Reverse: 5'-TACGATGGCcatACCAGCCTCCAAATGCCAGT-3';
[0061] H348F forward: 5'-GATGGTAGAttcTTGGTTAATGGAGTCACCTTCATC-3'.
[0062] H348F Reverse: 5'-ACCAAgaaTCTACCATCACCAACATTACCGGT-3';
[0063] D166A forward: 5'-TAACGTTgctCCAGTTCCAAACACTACAGTTATCAA-3',
[0064] D166A reverse: 5'-GAACTGGagcAACGTTACCCAGATTTGGAAACA-3';
[0065] M403F forward orientation: 5'-CCCTCATCCAtttCACTTGCATGGACATAACTTCGA-3'.
[0066] M403 Reverse: 5'-AGTGaaaTGGATGAGGGAATGCACCTGGCATA-3'.
[0067] PCR amplification system: Phanta Max Super-Fidelity DNA Ploymerase 1 μL, 2×MaxBuffer 25 μL, dNTP Mix 1 μL, forward primer 2 μL, reverse primer 2 μL, template plasmid (20 ng / μL) 0.2 μL, ddH2O 19.8 μL, total 51 μL.
[0068] PCR amplification program: pre-denaturation, 95℃ for 30 seconds; denaturation, 95℃ for 15 seconds; annealing, 60℃ for 30 seconds; extension, 72℃ for 55 seconds, 32 cycles; termination extension, 72℃ for 2 minutes; final incubation at 4℃.
[0069] Following the expression method of wild-type laccase PnLac1 described in Example 1, X-33 was used as the expression host, and single-point mutant plasmids H348F, D166A, L467M and M403F were used as exogenous genes to express four laccase mutants: H348F, D166A, L467M and M403F.
[0070] 2. Following the enzyme activity system and enzyme activity assay method described in Example 3, the relative enzyme activities of laccase mutants H348F, D166A, L467M, and M403F were determined using ABTS and guaiacol as substrates, respectively. The results are as follows: Figure 6 and Figure 7 As shown.
[0071] Depend on Figure 6 It can be seen that when using ABTS as a substrate, the enzyme activities of laccase mutants H348F and L467M are increased by 1.89 times and 1.65 times respectively compared with wild-type PnLac1. The higher enzyme activity of the laccase mutants shows that they have the potential to use ABTS as a medium to degrade stubborn substances. These two mutants can be used to degrade organic dyes such as crystal violet.
[0072] Depend on Figure 7 It is known that when guaiacol is used as a substrate, the enzyme activities of the laccase mutants D166A and M403F are increased by 2.07 times and 3.72 times, respectively, compared with the wild-type PnLac1. Guaiacol is one of the lignin monomers, and one of the important roles of laccase in nature is to participate in the synthesis and degradation of lignin. Higher enzyme activity corresponds to higher lignin degradation ability. These two mutants can be used to degrade lignin and other organic polymers, and have high application prospects in the field of lignin.
Claims
1. A laccase mutant, characterized in that, The laccase mutants are: D166A, which is obtained by mutating aspartic acid at position 166 of the PnLac1 amino acid sequence to alanine; H348F, which is obtained by mutating histidine at position 348 of the PnLac1 amino acid sequence to phenylalanine; M403F, which is obtained by mutating methionine at position 403 of the PnLac1 amino acid sequence; and L467M, which is obtained by mutating leucine at position 467 of the PnLac1 amino acid sequence. The amino acid sequence of the laccase mutant D166A is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.
3. The amino acid sequence of the laccase mutant H348F is shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5; The amino acid sequence of the laccase mutant M403F is shown in SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7; The amino acid sequence of the laccase mutant L467M is shown in SEQ ID NO.10, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.
9.
2. A recombinant vector, characterized in that, The encoding gene of the laccase mutant described in claim 1 is inserted into a plasmid vector.
3. A recombinant bacterial strain, characterized in that, It is obtained by converting the recombinant vector described in claim 2 into host cells.
4. The application of the laccase mutant according to claim 1 in biodegradation, characterized in that, The biodegradation refers to the degradation of crystal violet or lignin.