Laccase as well as mutant and application thereof

Abstract

The invention relates to laccase as well as a mutant and application thereof, and belongs to the technical field of biology. The amino acid sequence of the laccase PnLac1 is as shown in SEQ ID NO. 2, and the nucleotide sequence of the laccase PnLac1 is as shown in SEQ ID NO. 1. The mutant is characterized in that aspartic acid at the 166th position of laccase PnLac1 is subjected to site-specific mutagenesis, histidine at the 348th position of laccase PnLac1 is subjected to site-specific mutagenesis, methionine at the 403th position of laccase PnLac1 is subjected to site-specific mutagenesis, or leucine at the 467th position of laccase PnLac1 is subjected to site-specific mutagenesis, amino acid sequences are shown as SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO.10, and nucleotide sequences are shown as SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.9. The decoloring efficiency of the laccase PnLac1 on crystal violet reaches 5h, and the degradation rate reaches 75%. When the ABTS is used as a substrate, the enzyme activities of the laccase mutants H348F and L467M are respectively improved by 1.89 times and 1.65 times, and the ABTS has the potential of degrading stubborn substances; when guaiacol is used as a substrate, the enzyme activities of the laccase mutants D166A and M403F are respectively improved by 2.07 times and 3.72 times, and the guaiacol has a relatively high application prospect in the field of lignin.

Description

Technical Field

[0001] The present invention relates to a laccase and its mutants and applications, belonging to the field of biotechnology. Background Art

[0002] Laccase (EC 1.10.3.2), also known as polyphenol oxidase, belongs to the multi-copper oxidase superfamily. It can oxidize various aromatic compounds such as phenolic substances, polyphenolic substances, and anilines, using molecular oxygen as an electron acceptor to produce the only by-product water. Due to its broad substrate spectrum and green catalytic characteristics, laccase is widely used in organic synthesis, biosensor construction, fuel cell development, biomass valorization, and exogenous biodegradation.

[0003] Laccase is widely distributed in nature and can be classified into insect laccase, plant laccase, bacterial laccase, and fungal laccase according to its source. Currently, it is known that laccase can oxidize more than 250 substrates, and its huge application potential has led people to focus on research, development, and industrial production applications.

[0004] Most of the laccases reported currently are from bacteria. Fungal genomes also contain abundant laccase genes. Compared with bacteria, fungal laccase is an extracellular enzyme, the protein purification process is simpler, and it has higher enzyme activity and stability. Fungi secreting laccase mainly focus on higher fungi such as basidiomycetes, ascomycetes, and deuteromycetes. The enzymatic properties and laccase-producing abilities of laccases from different species and genera vary greatly. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a laccase and its mutants and applications.

[0006] The technical solution of the present invention is as follows:

[0007] A novel laccase PnLac1, whose amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0008] A mutant of laccase PnLac1, 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] Preferably according to the present invention, the mutant is a laccase mutant D166A, whose amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3; it is a mutation of aspartic acid at position 166 of the laccase amino acid sequence to alanine.

[0010] Preferably according to the present invention, the mutant is a laccase mutant H348F, whose amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5; it is the mutation of histidine at position 348 of the laccase amino acid sequence into phenylalanine.

[0011] Preferably according to the present invention, the mutant is a laccase mutant M403F, whose amino acid sequence is shown in SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7; it is the mutation of methionine at position 403 of the laccase amino acid sequence into phenylalanine.

[0012] Preferably according to the present invention, the mutant is a laccase mutant L467M, whose amino acid sequence is shown in SEQ ID NO.10, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.9; it is the mutation of leucine at position 467 of the laccase amino acid sequence into methionine.

[0013] A recombinant vector is obtained by inserting the encoding gene of the above-mentioned laccase PnLac1, laccase mutant D166A, laccase mutant H348F, laccase mutant M403F or laccase mutant L467M into a plasmid vector.

[0014] Preferably according to 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] Preferably according to the present invention, the host cell is Pichia pastoris.

[0017] The application of the above-mentioned laccase PnLac1, laccase mutant D166A, laccase mutant H348F, laccase mutant M403F or laccase mutant L467M in biodegradation.

[0018] Preferably according to the present invention, the biodegradation refers to the degradation of organic dyes such as crystal violet or organic polymers such as lignin using ABTS as a medium.

[0019] The experimental operations not described in detail in the present invention can be carried out according to the conventional experimental operations in this technical field.

[0020] Beneficial effects

[0021] 1. The present invention discovers a novel laccase PnLac1, which has a very high decolorization efficiency for crystal violet, and the degradation rate reaches 75% in 5 h, and can be used for degrading organic dyes.

[0022] 2. Based on the novel laccase PnLac1, site-directed mutagenesis was performed at its 166th, 348th, 403rd or 467th position. The aspartic acid at the 166th position was mutated to alanine (D166A), the histidine at the 348th position was mutated to phenylalanine (H348F), the methionine at the 403rd position was mutated to phenylalanine (M403F), or the leucine at the 467th position was mutated to methionine (L467M), resulting in laccase mutants H348F, D166A, L467M and M403F. Compared with the wild-type laccase PnLac1, the laccase mutants have higher enzyme activity. When using ABTS as a substrate, the enzyme activities of laccase mutants H348F and L467M were increased by 1.89-fold and 1.65-fold respectively compared with the wild-type PnLac1, showing the potential to degrade recalcitrant substances using ABTS as a mediator; when using guaiacol as a substrate, the enzyme activities of laccase mutants D166A and M403F were increased by 2.07-fold and 3.72-fold respectively compared with the wild-type PnLac1, and they can be used to degrade organic polymers such as lignin, showing high application prospects in the field of lignin. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the result diagram of sodium dodecyl sulfate 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 It is the spectral scan diagram of wild-type laccase PnLac1.

[0026] Figure 3 It is the determination result of the optimal reaction pH of wild-type laccase PnLac1 in vitro reaction.

[0027] Figure 4 It is the process diagram of wild-type laccase PnLac1 degrading crystal violet using ABTS as a mediator.

[0028] Figure 5 It is the diagram showing the change of decolorization rate of wild-type laccase PnLac1 degrading crystal violet and bromophenol blue over time with or without a mediator.

[0029] Figure 6 It is the relative enzyme activity of wild-type laccase PnLac1 and its mutants when using ABTS as a substrate.

[0030] Figure 7 It is the relative enzyme activity of wild-type laccase PnLac1 and its mutants when using guaiacol as a substrate. DETAILED DESCRIPTION OF THE INVENTION

[0031] The technical solution of the present invention will be further described below in conjunction with the embodiments and the accompanying drawings of the specification, 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 well-known to those skilled in the art.

[0032] Example 1: Heterologous expression of novel laccase PnLac1

[0033] During the bioinformatics database search using the BLASTp algorithm, the inventors found that the amino acid sequence formed by a gene in Phellopilus nigrolimitatus had a considerable degree of homology with the laccase amino acid sequence. Therefore, the inventors of the present application speculated that the protein product expressed by this gene might have laccase activity, and named this gene PnLac1. Then, the PnLac1 gene was heterologously expressed in Pichia pastoris, and its expressed protein product was named PnLac1. Its amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0034] The heterologous expression and purification of laccase PnLac1 are as follows:

[0035] The PnLac1 gene was artificially synthesized by Tsingke Biological Company 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 the competent cells of Pichia pastoris X-33 by electroporation and cultured on LB solid medium containing bleomycin (50 μg / mL) for 12 h to screen for transformants and obtain positive transformants.

[0036] Single colonies of the positive transformants of laccase PnLac1 were picked and inoculated into a long glass tube containing 2 mL of YPG liquid medium, and cultured at 200 rpm and 30 °C with an inclined placement for 30 h to obtain a seed solution. Then, the seed solution was inoculated into 125 ml of BMGY liquid medium at a volume ratio of 1% and cultured at 130 rpm and 30 °C for 20 h until OD600 = 3. After centrifugation at 4 °C and 3500 rpm for 20 min, the cells were collected.

[0037] The 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 keep the final concentration of methanol at 1%, and the total culture time was 72 h. After the culture, the culture broth was collected by centrifugation at 8000 rpm for 20 min, and the culture broth was concentrated about 10-fold using a 30 KDa ultrafiltration module; finally, the recombinant protein was purified using a nickel ion affinity chromatography column, and the purified protein was ultrafiltered and desalted using a retention tube (50 kDa) to obtain the target protein laccase PnLac1.

[0038] The purified laccase PnLac1 was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the results were as Figure 1 shown.

[0039] As Figure 1 can be seen, due to the influence of over-glycosylation of the Pichia pastoris expression system, the molecular weight of laccase PnLac1 in the SDS-PAGE result was about 100 KDa, indicating the successful heterologous expression of laccase PnLac1.

[0040] Example 2

[0041] The laccase PnLac1 prepared in Example 1 was diluted to a laccase solution with a protein concentration of 1 mg / ml using 20 mM, pH = 7 citrate-phosphate buffer. At the same time, PBS buffer was used as a blank solution, and the laccase solution and the blank solution were scanned at full wavelength from 200 to 800 nm using an ultraviolet spectrophotometer. The results were as Figure 2 shown.

[0042] As Figure 2 can be seen, a high absorption peak appeared at about 300 nm, but no high absorption peak appeared at 610 nm. It can be determined that PnLac1 is a unique fungal laccase.

[0043] Example 3

[0044] Determination of the optimal reaction pH for the in vitro reaction of the enzyme: A reaction system was constructed according to 880 μL of 100 mM buffer, 100 μL of 10 mM reaction substrate, and 10 μL of laccase PnLac1 enzyme solution diluted 10-fold;

[0045] Among them, the buffer solution is acetate buffer (Ace acid-sodium acetate buffer), BR buffer solution (BR buffer solution), or phosphate-citrate buffer (Citric acid-disodium hydrogenphosphate); the reaction substrate is 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) or 2,6-dimethylphenol (2,6-DMP);

[0046] When the buffer solution is acetate buffer, construct the reaction system according to the above ratio, set two pH gradient points of 4.0 and 5.0, use an ultraviolet spectrophotometer, measure the absorbance at 420 nm when using ABTS as the substrate, and measure the absorbance at 469 nm when using 2,6-DMP as the substrate. After adding the enzyme solution, measure the change in absorbance at intervals of 10 seconds.

[0047] When the buffer solution is BR buffer solution or phosphate-citrate buffer, set seven pH gradient points of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0. Use an ultraviolet spectrophotometer, measure the absorbance at 420 nm when using ABTS as the substrate, and measure the absorbance at 469 nm when using 2,6-DMP as the substrate. After adding the enzyme solution, measure the change in absorbance at intervals of 10 seconds.

[0048] The measurement results of the optimal reaction pH for the in vitro reaction of the enzyme are as Figure 3 shown.

[0049] From Figure 3 it can be seen that the optimal pH of laccase PnLac1 was determined using two substrates in three buffer solutions. When using ABTS as the substrate, laccase PnLac1 reached the highest enzyme activity in phosphate-citrate buffer at pH 2.2; when using 2,6-DMP as the substrate, laccase PnLac1 reached the highest enzyme activity in acetate buffer at pH = 4.

[0050] Example 4

[0051] Dissolve crystal violet in 20 mM phosphate-citrate buffer at pH = 5 to prepare a dye system with a final concentration of 1 mg / ml. Then, continue to add ABTS with a final concentration of 0.5 mM and laccase PnLac1 enzyme solution with a concentration of 1 U / ml to the dye system as the laccase-medium group (CV-PnLac1); at the same time, use the dye system with 0.5 mM ABTS and an equal amount of PBS buffer as the blank group (CV-LMS); react the laccase-medium group and the blank group at 30 °C and 200 rpm for 48 h, and regularly measure the dye decolorization rate during this period. The results are as Figure 4 and Figure 5 shown.

[0052] Calculation formula for decolorization rate:

[0053] Wherein, I is the decolorization rate; A0 is the initial dye absorbance value; A1 is the detected dye absorbance value.

[0054] From Figure 4 and Figure 5 it can be seen that the novel laccase PnLac1 provided by the present invention has the function of decolorizing crystal violet, and after adding ABTS as a medium, its decolorization ability is greatly improved, the decolorization efficiency is very high, the decolorization rate of crystal violet is obvious with time change, and the degradation rate reaches 75% at 5 h.

[0055] Example 5

[0056] 1. The three-dimensional structure diagram of wild-type laccase PnLac1 was constructed using alphafold3. By analyzing the active architecture, several key amino acids were found, namely H348, M403, L467, and D166. The inventors of the present application speculated that these 4 sites are located near the active center and highly conserved region, and site-directed mutagenesis of them is very likely to improve the enzyme activity of laccase PnLac1. Therefore, site-directed mutagenesis was carried out on them.

[0057] Using the recombinant vector pPICZαA-PnLac1 containing the coding gene of wild-type laccase PnLac1 constructed in Example 1 as a template, H348F, D166A, L467M, and M403F were selected as mutation sites, and PCR amplification was carried out through mutant primers to introduce mutations at the mutation sites, obtaining single-site mutant plasmids H348F, D166A, L467M, and M403F.

[0058] Among them, the mutant primers used are:

[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] Forward of D166A: 5’-TAACGTTgctCCAGTTCCAAACACTACAGTTATCAA-3’,

[0064] Reverse of D166A: 5’-GAACTGGagcAACGTTACCCAGATTTGGAAACA-3’;

[0065] Forward of M403F: 5’-CCCTCATCCAtttCACTTGCATGGACATAACTTCGA-3’,

[0066] Reverse of M403F: 5’-AGTGaaaTGGATGAGGGAATGCACCTGGCATA-3’.

[0067] PCR amplification system: 1 μL of Phanta Max Super-Fidelity DNA Ploymerase, 25 μL of 2× MaxBuffer, 1 μL of dNTP Mix, 2 μL of forward primer, 2 μL of reverse primer, 0.2 μL of template plasmid (20 ng / μL), 19.8 μL of ddH2O, a total of 51 μL.

[0068] PCR amplification program: Pre-denaturation, 95 °C for 30 sec; Denaturation, 95 °C for 15 sec; Annealing, 60 °C for 30 sec; Extension, 72 °C for 55 sec, 32 cycles; Termination of extension, 72 °C for 2 min; Finally, keep at 4 °C.

[0069] According to the expression method of wild-type laccase PnLac1 described in Example 1, using X-33 as the expression host and single-point mutant plasmids H348F, D166A, L467M and M403F as foreign genes, four laccase mutants H348F, D166A, L467M and M403F were expressed.

[0070] 2. According to the enzyme activity system and enzyme activity measurement method described in Example 3, ABTS and guaiacol were used as substrates respectively to measure the relative enzyme activities of laccase mutants H348F, D166A, L467M and M403F. The results are as Figure 6 and Figure 7 shown.

[0071] It can be seen from Figure 6 that when using ABTS as the substrate, the enzyme activities of laccase mutants H348F and L467M are 1.89 times and 1.65 times higher than that of the wild-type PnLac1 respectively. The enzyme activities of laccase mutants are higher and have the potential to degrade stubborn substances using ABTS as a medium. These two mutants can be used to degrade organic dyes such as crystal violet.

[0072] It can be seen fromFigure 7 It can be seen 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. 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 PnLac1, characterized in that, The amino acid sequence is as shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.

1.

2. A laccase PnLac1 mutant, characterized in that, The mutant is a site-directed mutation of aspartic acid at position 166, a site-directed mutation of histidine at position 348, a site-directed mutation of methionine at position 403, or a site-directed mutation of leucine at position 467 of the laccase PnLac1 described in claim 1.

3. The laccase PnLac1 mutant according to claim 2, wherein The mutant is laccase mutant D166A, whose amino acid sequence is as shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.3; it is a mutation of aspartic acid at position 166 of the laccase amino acid sequence to alanine.

4. The laccase PnLac1 mutant according to claim 2, wherein The mutant is laccase mutant H348F, whose amino acid sequence is as shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.5; it is a mutation of histidine at position 348 of the laccase amino acid sequence to phenylalanine.

5. The laccase PnLac1 mutant according to claim 2, characterized in that, The mutant is laccase mutant M403F, whose amino acid sequence is as shown in SEQ ID NO.8, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.7; it is a mutation of methionine at position 403 of the laccase amino acid sequence to phenylalanine.

6. The laccase PnLac1 mutant according to claim 2, wherein, The mutant is laccase mutant L467M, whose amino acid sequence is as shown in SEQ ID NO.10, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.9; it is a mutation of leucine at position 467 of the laccase amino acid sequence to methionine.

7. A recombinant vector, characterized in that, It is to insert the encoding gene of the laccase PnLac1 described in claim 1 or the laccase mutant described in claim 2 into a plasmid vector.

8. A recombinant strain, characterized in that, It is obtained by transforming the recombinant vector described in claim 7 into a host cell.

9. Use of the laccase PnLac1 described in claim 1 or the laccase mutant described in claim 2 in biodegradation.

10. The application according to claim 9, characterized in that The biodegradation refers to the degradation of crystal violet or the degradation of lignin.

Images