Laccase from coprinus cinereus as well as recombinant expression strain and efficient expression method thereof

Abstract

The invention discloses coprinus cinereus sourced laccase as well as a recombinant expression strain and a high-efficiency expression method thereof, and belongs to the technical field of microbiology and bioengineering. According to the invention, aspergillus niger MA70.15 is used as an experimental strain, and a random integration strategy is adopted, so that a pC3-PcitA-SPGlaA-lcc9-TtrpC-pyrG expression vector is integrated in an upstream region of a promoter of host bacteria citrate synthase in a form of nine continuous copies. In order to explore the influence of particles and osmotic regulators in a fermentation culture medium on laccase production through fermentation of recombinant aspergillus niger, the particles and osmotic regulators of different types and concentrations are added, so that the enzyme activity of the laccase fermented in a shake flask of the recombinant strain AnRcgL1 reaches 1566.7 U / L, the enzyme activity fermented in a 1L fermentation tank reaches 1961 U / L, and the recombinant high-efficiency expression of the laccase Lcc9 in the aspergillus niger is successfully realized.

Description

Technical Field

[0001] The invention relates to a recombinant Aspergillus niger bacterium and a method for efficiently expressing basidiomycete laccase thereof, belonging to the technical fields of microbiology and bioengineering. Background Art

[0002] Aspergillus niger ( Aspergillus niger ) is a filamentous fungus widely distributed in nature. Aspergillus niger has simple growth conditions and a wide range of temperatures and pH conditions, growing within a range of 6-47°C and a pH of 1.4-9.8. Certified as GRAS by the U.S. Food and Drug Administration, Aspergillus niger is suitable for use in the food industry and other industries. Due to its exceptional protein secretion and growth and reproduction capabilities, Aspergillus niger has been developed into a versatile cell factory over the past century and is widely used in industrial fermentation to produce various products.

[0003] At present, there are two ways to express exogenous genes in the Aspergillus niger expression system. The first way is to use an integration plasmid to integrate the target gene coding frame into the chromosome for genomic integration expression. The corresponding integration methods can be divided into site-specific integration and random integration. The integration site of the target gene also has an important influence on protein expression. Since the target gene is integrated into the host genome, it will specifically destroy the relevant sites and produce some potential unknown effects. Therefore, the exogenous protein is based on different gene sites in the chassis host genome for site-specific integration expression, and the extracellular expression level of the recombinant protein obtained may be significantly different. The gene sites encoding glucoamylase and α-amylase in the Aspergillus niger genome are commonly used integration sites for exogenous gene integration expression. Integrating exogenous genes into the above sites usually increases the expression level of the target protein.

[0004] Although integrative expression has high genetic stability, the copy number of integrative expression vectors is low, making it difficult to achieve efficient recombinant extracellular expression of proteins. Of the 14,165 open reading frames predicted in the Aspergillus niger genome, only 2% have been characterized, and there are still more than 6,000 protein-coding genes with unknown functions. Such a high unknown rate seriously limits the screening of potential integration sites for efficient protein expression in Aspergillus niger. Therefore, in previous studies, random integration strategies were often used to randomly integrate the target gene into the genome to increase the expression level of the target protein. In addition, based on the random integration strategy, the target gene will also be inserted into the chassis host genome in the form of multiple copies, thereby further improving the protein expression level.

[0005] Different culture methods can result in varying strain morphologies. In liquid culture, filamentous fungi produce a range of morphologies, including nearly spherical pellets, irregular clumps, scattered hyphal fragments, or a combination of these growth types. These morphological differences may affect the expression level of the target protein. Protein secretion in fungal hyphae is primarily located in the apical region. In Aspergillus niger pellets grown in liquid culture, protein production reaches its maximum within a thin layer on the pellet surface, with production significantly decreasing within the pellet interior. Numerous studies have investigated the use of microparticles in culture media to manipulate strain morphology and, in turn, target protein expression. Osmoregulators can indirectly influence protein stability by altering solvent properties to facilitate proper protein folding. Furthermore, laccase is a multi-copper enzyme. Copper ions bind to the enzyme's active site during laccase synthesis and can enhance laccase gene expression at the transcriptional level. Therefore, optimizing the type and concentration of microparticles, the type and concentration of osmoregulator, and the copper ion concentration during liquid fermentation plays a crucial role in enhancing the efficient expression of laccase in Aspergillus niger. Summary of the Invention

[0006] The present invention provides a laccase derived from Coprinus cinereus and its recombinant expression strain and efficient expression method. The present invention uses Aspergillus niger MA70.15 as the experimental strain and adopts a random integration strategy to make pC3-P citA -SP GlaA - lcc9- T trpC - pyrG The expression vector was integrated into the upstream region of the citrate synthase promoter of the host strain in the form of nine consecutive copies. To investigate the effects of microparticles and osmotic regulators in the fermentation medium on laccase production by recombinant Aspergillus niger, the present invention added different types and concentrations of microparticles and osmotic regulators, resulting in an enzyme activity of 1566.7 U / L in shake flask fermentation of the recombinant strain AnRcgL1 and 1961 U / L in 1 L fermentor tank fermentation, successfully achieving recombinant and efficient expression of laccase Lcc9 in Aspergillus niger.

[0007] The laccase Lcc9 derived from Coprinus cinerea of ​​the present invention has an amino acid sequence as shown in SEQ ID NO: 1 or a sequence having a homology higher than 85% with the amino acid sequence shown in SEQ ID NO: 1.

[0008] The gene encoding the laccase Lcc9 from Coprinus cinerea has a nucleotide sequence as shown in SEQ ID NO: 2 or a sequence having a homology higher than 85% with the nucleotide sequence shown in SEQ ID NO: 2.

[0009] The present invention also provides a recombinant expression vector pC3-P containing the coding gene citA -SPGlaA - lcc9- T trpC - pyrG , which was constructed based on the vector pC3.

[0010] The present invention also provides a recombinant Aspergillus niger strain AnRcgL1 expressing the laccase from Coprinus cinerea, which uses Aspergillus niger MA70.15 as the expression host and pC3-P citA -SP GlaA - lcc9- T trpC- pyrG It is an expression vector constructed by random integration.

[0011] Furthermore, the pC3-P citA -SP GlaA - lcc9- T trpC -pyrG The expression vector was integrated into the P citA Upstream of the promoter.

[0012] In one embodiment of the present invention, the expression vector pC3-P citA -SP GlaA - lcc9- T trpC- pyrG Transformation into Aspergillus niger protoplasts is based on polyethylene glycol (PEG)-mediated protoplast transformation, including mixing the protoplast suspension, expression vector and PEG buffer solution, placing it in an ice bath, then adding PEG buffer solution, osmotic pressure stabilizer and soft agar medium, mixing and transferring to regeneration medium, regenerating transformants, and picking the transformants to screening plates for screening.

[0013] Furthermore, the transformation method is as follows: 150-200 μL of the protoplast suspension is aspirated into a centrifuge tube, an expression vector (4-10 μg) and a PEG buffer solution are added, the tube is placed on ice for 15-40 minutes, the PEG buffer solution is added, and the tube is placed at room temperature for 5-30 minutes.

[0014] Furthermore, the protoplast regeneration method is as follows: the transformation mixed solution, the osmotic pressure stabilizer and the soft agar medium are mixed and then transferred to the regeneration medium, and cultured upright at 28° C. for 4 to 7 days to obtain the transformant.

[0015] Transformants were screened by placing transformants onto screening plates. The plate composition (w / v) included: glucose 1.5-3%, NaNO₃ 0.2-0.4%, KCl 0.1-0.3%, MgSO₄·7H₂O 0.04-0.06%, KH₂PO₄ 0.05-0.15%, FeSO₄·7H₂O 0.001%, agar powder 1.5-2%, ABTS 0.4-0.6 mM, and CuSO₄ 0.05-0.15 mM.

[0016] The invention discloses an application of the Aspergillus niger recombinant strain AnRcgL1 in the efficient preparation of laccase Lcc9 derived from Coprinus cinerea.

[0017] The specific steps include: Step 1: Spread the recombinant Aspergillus niger strain AnRcgL1 on a PDA solid plate, culture for 3-5 days, add a physiological saline solution containing 0.05% Tween 80, scrape the spores, and prepare a spore suspension; Step 2: Inoculate the fresh spore suspension into the fermentation medium and culture at 30°C and 200 rpm for 5-7 days to obtain laccase Lcc9.

[0018] Furthermore, the fermentation medium includes 0.1%-2% (w / v) CaCO3.

[0019] Furthermore, the fermentation medium also includes 0.05-0.2 M glycine.

[0020] The solid plate used for activation culture is a PDA solid plate. 1 L of the culture medium contains the following components: 150-250 g of potatoes, 15-25 g of glucose, and 15-20 g of agar, and 10 mM uracil needs to be additionally added.

[0021] The liquid culture medium is YPD medium. 1 L of the culture medium contains the following components: 15-25 g of peptone, 5-15 g of yeast extract, and 15-25 g of glucose, and 10 mM uracil needs to be additionally added.

[0022] The osmotic pressure stabilizer component comprises: CaCl2 40-60 mM, sorbitol 1.0-1.4 mM, Tris-HCl 8-12 mM, pH 7.5.

[0023] The PEG buffer component comprises: PEG 4000 40-80% (w / v), CaCl2 40-60 mM, Tris-HCl 5-15 mM, pH 7.5.

[0024] The components (w / v) of the hypertonic soft agar medium are: 30-35% sucrose, 0.2-0.4% NaNO3, 0.1-0.3% KCl, 0.02-0.07% MgSO4·7H2O, 0.1-0.2% KH2PO4, 0.001-0.003% FeSO4·7H2O, and 1% agar powder.

[0025] The components of the regeneration culture medium are the same as those of the hypertonic soft agar culture medium, except that the amount of agar powder added (w / v) is 1.5-2%.

[0026] The screening medium components (w / v) include: glucose 1.5-3%, NaNO3 0.2-0.4%, KCl 0.1-0.3% (w / v), MgSO4·7H2O 0.04-0.06%, KH2PO4 0.05-0.15%, FeSO4·7H2O 0.001%, agar powder 1.5-2%, ABTS 0.4-0.6 mM, CuSO4 0.05-0.15 mM.

[0027] The fermentation medium components (w / v) include: 2-5 mM CuSO4, 50-80 mM NaNO3, 5-10 mM KCl, 100-300 mM K2HPO4, 1-5 mM MgSO4, 1%-2% glucose (w / v), and trace elements (1000× mother liquor: 70-80 mM ZnSO4, 20-30 mM MnCl2, 15-20 mM FeSO4, 6-8 mM CoCl2, 6-8 mM CuSO4, 6-8 mM Na2MoO4, and 150-200 mM EDTA). The fermentation medium was adjusted to pH 5.5 using citric acid.

[0028] In one embodiment of the present invention, the composition (w / v) of a 1 L jar fermentation medium comprises: 0.1%-2% CaCO3, 0.05-0.2 M glycine, 2-5 mM CuSO4, 50-80 mM NaNO3, 5-10 mM KCl, 100-300 mM K2HPO4, 1-5 mM MgSO4, 1%-2% (w / v) glucose, and trace elements (1000× mother liquor: 70-80 mM ZnSO4, 20-30 mM MnCl2, 15-20 mM FeSO4, 6-8 mM CoCl2, 6-8 mM CuSO4, 6-8 mM Na2MoO4, and 150-200 mM EDTA). The fermentation medium is adjusted to pH 5.5 using citric acid.

[0029] The beneficial effects of the present invention are embodied in: (1) The present invention successfully achieved the efficient expression of the basidiomycete Coprinus cinereus laccase in Aspergillus niger lcc9 , using the technical solution of the present invention, using Aspergillus niger MA70.15 as the expression host, and using pC3-P citA -SP GlaA -T trpC- lcc9 As an expression vector, a random integration strategy was used to successfully construct the recombinant Aspergillus niger strain AnRcgL1 expressing the basidiomycete Coprinus cinereus laccase Lcc9, with an enzyme activity of 86 U / L in shake flask fermentation.

[0030] (2) Using the recombinant Aspergillus niger strain AnRcgL1 constructed by the present invention, and exploring the effects of microparticles and osmotic regulators on its fermentation and laccase production, the enzyme activity of laccase Lcc9 in the shake flask fermentation supernatant was increased to 1566.7 U / L. When fermented in a 1 L fermenter, the extracellular laccase Lcc9 activity of the recombinant Aspergillus niger strain reached 1961.1 U / L. High-efficiency expression of Coprinus cinereus laccase Lcc9 in Aspergillus niger was successfully achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The laccase expression vector pC3-P constructed by the present invention citA -SP GlaA - lcc9- T trpC -pyrG .

[0032] Figure 2 The laccase expression vector pC3-P constructed by the present invention citA -SP GlaA - lcc9 -T trpC -pyrG Verification results; A is: colony PCR identification, 1~2 are: transformant 1, transformant 2; B is: plasmid enzyme digestion verification, 1~2 are: transformant 1, transformant 2.

[0033] Figure 3 These are the plate color development results and active protein gel results of the recombinant Aspergillus niger AnRcgL1 of the present invention; wherein A is: the color development of the recombinant Aspergillus niger AnRcgL1 on a plate containing ABTS, and B is: the active protein gel result of the supernatant of the shake flask fermentation of the recombinant Aspergillus niger AnRcgL1.

[0034] Figure 4 Sequencing confirmation of recombinant Aspergillus niger AnRcgL1 lcc9 Schematic diagram of the copy number and integration sites.

[0035] Figure 5The present invention shows the effect of adding microparticles to the liquid fermentation medium of the recombinant Aspergillus niger AnRcgL1 on the expression of laccase, wherein A, B, C, D, and E are MgSiO3, Al2O3, CaCO3, quartz sand, and ZnCO3 with different concentrations, respectively.

[0036] Figure 6 The present invention shows the effect of adding osmotic regulators to the liquid fermentation medium of the recombinant Aspergillus niger AnRcgL1 on the expression of laccase, wherein A, B, C, D, and E are glycine, proline, TMAO, mannitol, and calcium chloride at different concentrations, respectively.

[0037] Figure 7 Figure 1 shows the laccase Lcc9 activity in the supernatant of 1 L fermentation tank fermentation of the recombinant Aspergillus niger AnRcgL1 of the present invention (A). Figure 1 shows the activity gel color bands (B). DETAILED DESCRIPTION

[0038] The implementation methods in the following examples are all conventional methods unless otherwise specified.

[0039] The Aspergillus niger MA70.15 used in the present invention is disclosed in patent CN2023108680572, and Guo SX, Yao GF, Ye HR, et al. Functional Characterization of a Cystathionine β-Synthase Gene in Sulfur Metabolism and Pathogenicity of Aspergillus nigerinPear Fruit. Journal of Agricultural and Food Chemistry. 2019, 67 (16): 4435-4443,Vera Meyer, Mark Arentshorst, et al. Highly efficient gene targeting inthe Aspergillus niger kusA mutant. Journal of Biotechnology. 128 (2007):770-775 and other existing technologies. This research group initially purchased the product from Leiden Univ, Inst Biol, Wassenaarseweg 64, NL-2333 AL Leiden, Netherlands;E-mail Addresses:AFJRam@biology.leidenuniv.nl;Addresses:Leiden Univ, Inst Biol, NL-2333 AL Leiden, Netherlands。

[0040] The synthetic gene PUC57 involved in the following examples lcc9 、PUC57-P citA 、PUC57-T trpC , are all composed of commercially available vector skeletons and corresponding genes, and are all sent to the company for synthesis.

[0041] The culture medium and solution involved in the following examples are as follows: PDA solid medium (1 L): 200 g potatoes, 20 g glucose, 15-20 g agar, and 10 mM uracil as needed.

[0042] YPD medium (w / v): 2% peptone, 1% yeast extract, 2% glucose, supplemented with 10 mM uracil as needed.

[0043] Hypertonic solid CD medium (1 L): sucrose 34% (w / v), NaNO3 0.3% (w / v), KCl 0.2% (w / v), MgSO4·7H2O 0.05% (w / v), KH2PO4 0.1% (w / v), FeSO4·7H2O 0.001% (w / v), agar powder 1.5-2% (w / v).

[0044] Hypertonic CD soft agar medium (w / v): sucrose 34%, NaNO3 0.3%, KCl 0.2%, MgSO4·7H2O 0.05%, KH2PO4 0.1%, FeSO4·7H2O 0.001%, agar powder 1%.

[0045] Screening medium (w / v): glucose 2%, NaNO3 0.3%, KCl 0.2%, MgSO4·7H2O 0.05%, KH2PO4 0.1%, FeSO4·7H2O 0.001%, agar powder 1.5-2%, ABTS 0.5 mM, CuSO4 0.1 mM.

[0046] STC buffer: anhydrous CaCl2 50 mM, sorbitol 1.2 M, Tris-HCl 10 mM, pH 7.5.

[0047] PEG buffer: PEG 4000 60% (w / v), CaCl2 50 mM, Tris-HCl 10 mM, pH 7.5.

[0048] Fermentation medium A: 2-5mM CuSO4, 50-80 mM NaNO3, 5-10 mM KCl, 100-300 mM K2HPO4, 1-5 mM MgSO4, glucose 1%-2% (w / v), trace elements (1000× stock solution: 70-80 mM ZnSO4, 20-30 mM MnCl2, 15-20 mM FeSO4, 6-8 mM CoCl2, 6-8 mM CuSO4, 6-8 mM Na2MoO4, and 150-200 mM EDTA).

[0049] Fermentation medium B: 10.1%-2% CaCO3, 0.05-0.2 M glycine, 2-5 mM CuSO4, 50-80 mM NaNO3, 5-10 mM KCl, 100-300 mM K2HPO4, 1-5 mM MgSO4, glucose 1%-2% (w / v), trace elements (1000× stock solution: 70-80 mM ZnSO4, 20-30 mM MnCl2, 15-20 mM FeSO4, 6-8 mM CoCl2, 6-8 mM CuSO4, 6-8 mM Na2MoO4, and 150-200 mM EDTA).

[0050] The detection methods involved in the following embodiments are as follows: Laccase activity: Laccase activity was determined using the ABTS method. Specifically, 950 μL of sodium tartrate buffer (100 mmol / L, pH 4.0) was added with 33 μL of 15 mmol / L ABTS and 17 μL of appropriately diluted enzyme solution. After thorough mixing, the mixture was incubated in a 30°C water bath for 3 min and then incubated on ice for 30 s to terminate the reaction. The absorbance of the reaction solution was measured at a wavelength of λ = 420 nm.

[0051] Laccase activity calculation formula: E (U / L) = OD 420 × dilution factor × 555.56.

[0052] Laccase expression: Laccase expression in the fermentation broth was assessed by native-PAGE. The fermentation broth supernatant, diluted to a certain degree, was mixed with 2× loading buffer and 10 μL was applied to the wells of the native-PAGE gel. Electrophoresis was performed in a VE-180A micro-vertical electrophoresis tank at 80 V for 30 minutes and then at 120 V for 3 hours. The gel was removed and incubated in 100 mmol / L sodium tartrate buffer (pH 4.0) (0.5 mmol / L ABTS) at room temperature. Example 1: Construction of an expression vector containing laccase from Coprinus cinerea

[0053] Expression vector pC3-P citA -SP GlaA - lcc9- - pyrG It was constructed based on the vector pC3, which was disclosed in Guo SX, Yao GF, Ye HR, et al. Functional Characterization of aCystathionine β-Synthase Gene in Sulfur Metabolism and Pathogenicity of Aspergillus niger in Pear Fruit. Journal of Agricultural and Food Chemistry.2019, 67 (16): 4435-4443. Expression vector pC3-P citA -SP GlaA - lcc9- T trpC -pyrG Based on the vector pC3, laccase lcc9 Expression cassette, screening marker pyrG Recombinant expression vector pC3-PcitA -SP GlaA - lcc9- T trpC -pyrG The vector structure diagram is shown in Figure 1 .

[0054] The specific steps are as follows: Promoter, signal peptide, lcc9 , terminator, pyrG The five fragments were fused into a long fragment by PCR and then connected with the linearized pC3 fragment by homologous recombination using the CloneExpress II Onestep Cloning Kit from Novozymes.

[0055] The amino acid sequence of Lcc9 is shown in SEQ ID NO: 1. The gene PUC57- lcc9 The template was obtained by PCR, lcc9 The sequence is shown in SEQ ID NO: 2. The sequence of primer F1 / R1 is shown in Table 1. The promoter P that mediates Lcc9 expression citA The sequence is shown in SEQ ID NO: 3 and was obtained by PCR using primers F2 / R2 with the synthetic gene as a template. The sequence of primers F2 / R2 is shown in Table 1. GlaA The sequence is shown in SEQ ID NO: 4, and the gene PUC57-SP was synthesized using primers F3 / R3. GlaA The primers F3 / R3 were used as templates for PCR. The sequence of primers F3 / R3 is shown in Table 1. trpC The sequence is shown in SEQ ID NO: 5, and the gene PUC57-T was synthesized using primers F4 / R4. trpC The sequence of primers F4 / R4 is shown in Table 1. pyrG This is a sequence already present in the pC3 vector and was obtained by PCR using primers F5 / R5 and plasmid pC3 as a template. The sequence of primers F5 / R5 is shown in Table 1.

[0056]

[0057] The fusion PCR employed was a two-step process. The first step involved recombination and fusion using the homologous sequences between the components without primers. The fusion PCR system is shown in Table 2. The second step involved PCR amplification of the recombinant product from the first step using primers F1 / R4. Linearization of the vector pC3 was achieved using inverse PCR. The sequences of primers F5 / R5 are shown in Table 1.

[0058]

[0059] PCR conditions: 94°C pre-denaturation for 5 min; 15 cycles of 98°C denaturation for 30 s, 55°C annealing for 15 s, and 72°C extension for 1 min 49 s; and 72°C for 10 min. PCR products were recovered by gel extraction.

[0060] The ligation system between the fusion fragment and the linearized vector pC3 is shown in Table 3.

[0061]

[0062] Ligation conditions: 37°C for 30 min.

[0063] Depend on Figure 2 It can be seen that the expression vector pC3-P has been successfully constructed through colony PCR, enzyme digestion verification and sequencing. citA -SP GlaA - lcc9- T trpC -pyrG . Example 2: Construction of recombinant Aspergillus niger and expression of laccase

[0064] (1) Preparation of protoplasts A. Activation culture of strains The glycerol tube stock of Aspergillus niger MA70.15 stored at -80°C was appropriately diluted and spread onto PDA solid medium (supplemented with 10 mM uracil) and cultured at 28°C for 3-5 days.

[0065] B. Mycelial culture (liquid culture method) Take the activated spore-filled plate, cut the mycelial block and inoculate it into YPD liquid medium (supplemented with 10 mM uracil), and culture it at 28-32℃ and 180-220 rpm for 48 h.

[0066] C. Collection of mycelium Filter through a Büchner funnel lined with double-layer filter paper until the cells are dry. Wash the cells twice with sterile deionized water and twice with NaCl solution, then filter until dry. Scrape the cells with a clean spatula, collect them in a conical flask or EP tube, and weigh them.

[0067] D. Enzymatic hydrolysis of mycelium Enzyme hydrolysis solution was added at a ratio of mycelium to enzyme hydrolysis solution of 1:10 (w / v), and the cells were placed in a water bath shaker at 37°C and 100 rpm for enzymatic hydrolysis for 3 h.

[0068] The enzymatic hydrolysis solution is as follows: the components of the enzymatic hydrolysis solution are: cellulase 2% (w / v), lytic enzyme 1% (w / v), lysozyme 0.5% (w / v), snail enzyme 1% (w / v), NaCl 0.8 M, 100 mM sodium phosphate buffer pH 6.0 10% (v / v).

[0069] E. Protoplast Collection Filter the enzymatic hydrolysate through a smooth lens paper to collect the protoplasts. Rinse the test tube containing the enzymatic hydrolysate and the lens paper with NaCl. Centrifuge at 3000-4000 rpm for 10 minutes, discard the supernatant, and collect the protoplasts obtained using the different enzymatic hydrolysates.

[0070] F. Purification of protoplasts Add STC buffer (osmotic pressure stabilizer) and gently pipette with a cut pipette tip to resuspend the protoplasts. Centrifuge at 3000-4000 rpm for 10 minutes at 4°C to collect the protoplasts. Repeat this step once. Add an appropriate amount of STC buffer (osmotic pressure stabilizer) and gently pipette with a cut pipette tip to resuspend the protoplasts to obtain a protoplast suspension.

[0071] (2) Protoplast transformation Pipette 150-200 μL of protoplast suspension into 2 mL centrifuge tubes, and then add 20 μL of expression vector pC3-P citA -SP GlaA - lcc9- T trpC -pyrG (4-10 μg) and 50 μL PEG buffer solution, gently mix by inverting, mix once every 10 minutes, ice bath for 30 minutes, add 1.5 mL PEG buffer solution, and let it stand at room temperature for 25 minutes.

[0072] (3) Protoplast regeneration Add 2.5 mL of STC buffer and 5 mL of hypertonic CD soft agar medium to a 10 mL centrifuge tube and mix thoroughly. Transfer the protoplast transformation mixture to a 10 mL centrifuge tube. Mix thoroughly with a pre-cooled 5 mL pipette tip and pipette 5 mL onto two hypertonic solid CD medium plates. Gently rotate the plate to spread the soft agar evenly on the plate. Incubate at 28°C, upright, for 4-7 days.

[0073] (4) Screening and identification of transformants 20 transformants on the regeneration plate were picked and qualitatively tested on ABTS plates. One of them was active, the recombinant Aspergillus niger strain MA70.15 / pC3-P citA -SP GlaA - lcc9- TtrpC -pyrG The strain was named AnRcgL1, and the transformant was inoculated onto the screening medium. After culturing at 28°C for 2-3 days, the growth and color development of the strain were observed.

[0074] Conclusion: There is obvious color development on the ABTS plate (see Figure 3 A), indicating laccase activity.

[0075] (5) Shake flask fermentation of recombinant Aspergillus niger The obtained recombinant bacteria AnRcgL1 was spread on a PDA solid plate and cultured for 3-5 days. After sufficient spore production, a physiological saline solution containing 0.05% Tween 80 was added and the spores were scraped to prepare a spore suspension. The fresh spore suspension was inoculated into fermentation medium A (1×10 5 spores / mL) and cultured at 30°C and 200 rpm for 7 days. Daily, 1 mL of fermentation broth was collected and centrifuged at 12,000 × g for 5 min. The supernatant was stored in a refrigerator at 4°C. The fermentation supernatant was used to determine laccase activity and expression.

[0076] Laccase expression: After mixing the fermentation broth supernatant with 2× loading buffer, 10 μL was spotted onto the wells of the reactivation gel. Electrophoresis was performed in a VE-180A micro-vertical electrophoresis tank at 80 V for 30 minutes and then at 120 V for 3 hours. The reactivation gel was removed and incubated in 100 mmol / L sodium tartrate buffer (pH 4.0) (0.5 mmol / L ABTS) at room temperature.

[0077] The results showed that the laccase activity reached its highest level on the 5th day, with an activity of 86 U / L, and the active gel had obvious color bands ( Figure 3 B), showing that laccase expression was detected.

[0078] (6) Sequencing analysis The obtained recombinant bacterium AnRcgL1 was subjected to whole genome sequencing to analyze its integration site and copy number.

[0079] The results show that ( Figure 4 ), lcc9 The expression cassette was integrated upstream of the citrate synthase promoter in the form of 9 copies. Example 3: Effect of microparticles in fermentation medium on laccase production by recombinant Aspergillus niger

[0080] The obtained recombinant bacteria AnRcgL1 was spread on a PDA solid plate and cultured for 3-5 days. After sufficient spore production, a physiological saline solution containing 0.05% Tween 80 was added and the spores were scraped to prepare a spore suspension. The fresh spore suspension was inoculated into the fermentation medium (1×105 spores / mL), and microparticles of varying types and concentrations were added to the fermentation medium. Microparticles (CaCO₃, MgSiO₃, Al₂O₃, quartz sand, and ZnCO₃) were suspended in 50 mM sodium acetate buffer (pH 6.5) and sterilized by autoclaving at 121°C for 20 minutes. Subsequently, the microparticles were added to the fermentation medium. To further investigate the effect of microparticle addition concentration on laccase activity, concentrations of 0.5%, 1%, and 2% were selected. Cultures were maintained at 30°C and 200 rpm for 7 days. Daily, 1 mL of fermentation broth was sampled and centrifuged at 12,000 × g for 5 minutes. The supernatant was stored at 4°C and used to measure laccase activity.

[0081] Laccase activity was determined using the ABTS method. To 950 μL of sodium tartrate buffer (100 mmol / L, pH 4.0), 33 μL of 15 mmol / L ABTS and 17 μL of appropriately diluted enzyme solution were added. After thorough mixing, the mixture was incubated in a 30°C water bath for 3 min and then incubated on ice for 30 s to terminate the reaction. The absorbance of the reaction solution was measured at a wavelength of λ = 420 nm. Laccase activity was calculated using the formula: E (U / L) = OD 420 × dilution factor × 555.56.

[0082] The results show that ( Figure 5 C). When 1% calcium carbonate was added to the fermentation medium, the laccase activity reached its highest level on the 5th day, which was 925.6 U / L, a 10.8-fold increase compared with the control. Example 4: Effect of osmotic regulators in fermentation medium on laccase production by recombinant Aspergillus niger

[0083] The obtained recombinant bacteria AnRcgL1 was spread on a PDA solid plate and cultured for 3-5 days. After sufficient spore production, a physiological saline solution containing 0.05% Tween 80 was added and the spores were scraped to prepare a spore suspension. The fresh spore suspension was inoculated into the fermentation medium (1×10 5spores / mL), 1% CaCO3 was added, and different types of osmotic regulators (glycine, proline, TMAO, mannitol, and calcium chloride) were additionally added. To further explore the effect of osmotic regulator addition concentration on laccase activity, different addition concentrations were selected: glycine at concentrations of 0.05-0.4 M, proline at concentrations of 0.1-0.5 M, TMAO at concentrations of 0.05-0.4 M, mannitol at concentrations of 0.1-0.5 M, and calcium chloride at concentrations of 0.05-0.5 M. Cultures were carried out at 30°C and 200 rpm for 7 days. 1 mL of fermentation broth was sampled daily and centrifuged at 12,000 × g for 5 min. The supernatant was stored in a refrigerator at 4°C. The fermentation supernatant was used to determine laccase activity.

[0084] Laccase activity was determined using the ABTS method. To 950 μL of sodium tartrate buffer (100 mmol / L, pH 4.0), 33 μL of 15 mmol / L ABTS and 17 μL of appropriately diluted enzyme solution were added. After thorough mixing, the mixture was incubated in a 30°C water bath for 3 min and then ice-chilled for 30 s to terminate the reaction. The absorbance of the reaction solution was measured at a wavelength of λ = 420 nm. Laccase activity was calculated using the formula: E (U / L) = OD 420 × dilution factor × 555.56.

[0085] The results show that ( Figure 6 A): When 0.1 M glycine osmotic regulator was further added to the fermentation medium, the laccase activity reached its highest level on the fifth day, reaching 1566.7 U / L, which was 1.7 times higher than that of adding 1% CaCO3. Example 5: Fermentation of recombinant Aspergillus niger in 1 L tank

[0086] The obtained recombinant bacteria AnRcgL1 was spread on a PDA solid plate and cultured for 3-5 days. After sufficient spore production, a physiological saline solution containing 0.05% Tween 80 was added and the spores were scraped to prepare a spore suspension. The fresh spore suspension was inoculated into the fermentation medium (1×10 5 spores / mL), and the fermentation medium was supplemented with 1% CaCO₃, 0.1 M glycine, and 3 mM CuSO₄. The fermentation tank was filled with 1 L of liquid, and initial culture conditions were 30°C, pH 5.5, 200 rpm, and an air flow of 50 L / h. Starting on day 2, an oxygen flow of 20 L / h was maintained. Every 24 hours, 5 mL of samples were collected and centrifuged at 12,000 × g for 5 minutes. The supernatant was stored in a refrigerator at 4°C. The fermentation supernatant was used to determine laccase activity and expression.

[0087] Laccase expression: After mixing the fermentation broth supernatant with 2× loading buffer, 10 μL was spotted onto the wells of the reactivation gel. Electrophoresis was performed in a VE-180A micro-vertical electrophoresis tank at 80 V for 30 minutes and then at 120 V for 3 hours. The reactivation gel was removed and incubated in 100 mmol / L sodium tartrate buffer (pH 4.0) (0.5 mmol / L ABTS) at room temperature.

[0088] The results show that ( Figure 7 A), laccase activity reached its highest on the 5th day, with an activity of 1961 U / L, which was 1.3 times that of shake flask fermentation. Figure 7 B).

[0089] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A laccase from Coprinus cinerea, abbreviated as Lcc9, whose amino acid sequence is shown in SEQ ID NO: 1 or a sequence having a homology of higher than 85% with the amino acid sequence shown in SEQ ID NO:

1.

2. A gene encoding the laccase Lcc9 from Coprinus cinerea according to claim 1, wherein the nucleotide sequence thereof is as shown in SEQ ID NO: 2 or a sequence having a homology with the nucleotide sequence shown in SEQ ID NO: 2 being higher than 85%.

3. The recombinant expression vector pC3-P containing the coding gene of claim 2 citA -SP GlaA - lcc9- T trpC -pyrG , which is constructed based on the vector pC3.

4. The recombinant Aspergillus niger strain AnRcgL1 expressing the laccase derived from Coprinus cinerea according to claim 1, characterized in that: Aspergillus niger MA70.15 was used as the expression host and pC3-P citA -SP GlaA - lcc9- T trpC- pyr It is an expression vector constructed by random integration.

5. The Aspergillus niger recombinant strain AnRcgL1 according to claim 4, characterized in that: The pC3-P in the recombinant Aspergillus niger strain AnRcgL1 citA -SP GlaA - lcc9- T trpC -pyrG The expression vector was integrated into P citA Upstream of the promoter.

6. The recombinant Aspergillus niger strain AnRcgL1 according to claim 4, characterized in that: The expression vector pC3-P citA -SP GlaA - lcc9- T trpC- pyr The transformation is carried out into Aspergillus niger protoplasts. The transformation method is based on polyethylene glycol-mediated protoplast transformation, including mixing a protoplast suspension, an expression vector and a PEG buffer solution, placing the mixture in an ice bath, then adding a PEG buffer solution, an osmotic pressure stabilizer and a soft agar medium, mixing and transferring the mixture to a regeneration medium, regenerating a transformant, and picking the transformant to a screening plate for screening.

7. Use of the recombinant Aspergillus niger strain AnRcgL1 according to claim 4, 5 or 6 in the efficient preparation of laccase Lcc9 from Coprinus cinerea.

8. The use according to claim 7, characterized in that The steps include: Step 1: Spread the recombinant strain of Aspergillus niger AnRcgL1 on a PDA solid plate, culture for 3 to 5 days, add a physiological saline solution containing 0.05% Tween 80, scrape the spores, and prepare a spore suspension; Step 2: Inoculate the fresh spore suspension into the fermentation medium, and culture at 30°C and 200 rpm for 5-7 days to obtain laccase Lcc9.

9. The use according to claim 8, characterized in that The fermentation medium includes 0.1%-2% CaCO3.

10. The use according to claim 9, characterized in that The fermentation medium also includes 0.05-0.2 M glycine.

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