A kind of gray cap of coprinus mushroom source of lacquer enzyme and its recombinant expression strain and high expression method

Abstract

This invention discloses a laccase derived from *Coprinus gracilis*, its recombinant expression strain, and a highly efficient expression method, belonging to the fields of microbiology and bioengineering technology. Using *Aspergillus niger* MA70.15 as the experimental strain, this invention employs a random integration strategy to achieve pC3-P... citA -SP GlaA - lcc9‑ T trpC ‑pyrG The expression vector was integrated into the upstream region of the promoter of the host bacterium citrate synthase 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, this invention added different types and concentrations of microparticles and osmotic regulators, resulting in a laccase activity of 1566.7 U / L in shake-flask fermentation of recombinant strain AnRcgL1 and 1961 U / L in a 1 L fermenter, successfully achieving efficient recombinant expression of laccase Lcc9 in Aspergillus niger.

Description

Technical Field

[0001] This invention relates to a recombinant Aspergillus niger strain and a method for efficiently expressing laccase from basidiomycetes, belonging to the fields of microbiology and bioengineering technology. Background Technology

[0002] Aspergillus niger ( Aspergillus niger Aspergillus niger is a filamentous fungus widely distributed in nature. Its growth conditions are simple, with a wide range of temperature and pH requirements, allowing it to grow within a range of 6–47°C and pH 1.4–9.8. Aspergillus niger is a GRAS-certified safe fungus by the U.S. Food and Drug Administration (FDA) and can be used in the food and other industries. Due to its excellent protein secretion and reproductive capabilities, Aspergillus niger has been gradually developed into a multi-purpose cell factory over the past century and is now widely used in industrial fermentation to produce various products.

[0003] Currently, there are two methods for expressing exogenous genes in the *Aspergillus niger* expression system. The first method involves using integration plasmids to integrate the target gene coding frame into the chromosome for genomic integration expression. This integration can be categorized into site-directed integration and random integration. The integration site of the target gene also significantly impacts protein expression. Because the integration of the target gene into the host genome specifically disrupts relevant sites, it can produce some potential unknown effects. Therefore, site-directed integration expression of exogenous proteins based on different gene loci in the host genome may result in significant differences in the extracellular expression levels of the recombinant proteins. The gene loci encoding glucoamylase and α-amylase in the *Aspergillus niger* genome are commonly used integration sites for exogenous gene integration expression; integrating exogenous genes into these sites typically increases the expression level of the target protein.

[0004] Although integrative expression exhibits high genetic stability, the low copy number of integrative expression vectors makes it difficult to achieve efficient recombinant extracellular protein expression. Of the 14,165 open reading frames predicted in the *Aspergillus niger* genome, only 2% have been characterized, leaving over 6,000 protein-coding genes with unknown functions. This high unknown rate severely limits the screening of potential integration sites for efficient protein expression in *Aspergillus niger*. Therefore, previous studies have often utilized random integration strategies to randomly integrate target genes into the genome to enhance target protein expression levels. Furthermore, random integration strategies can also insert target genes in multiple copies into the host genome, further increasing protein expression levels.

[0005] Different cultivation methods result in different strain morphologies. In liquid culture, filamentous fungi produce a range of morphologies, including near-spherical clusters, irregular clumps, scattered hyphal fragments, or combinations of these growth types. These morphological differences can affect the expression levels of target proteins. Protein secretion in fungal hyphae primarily occurs in the apical region. In Aspergillus niger liquid culture pellets, protein production reaches its maximum only within a thin layer on the surface of the pellet, significantly decreasing within the pellet. Numerous studies have explored the use of microparticles in the culture medium to regulate strain morphology and thus influence target protein expression levels. Osmotic regulators can indirectly affect protein stability by altering the properties of the solvent, aiding in proper protein folding. Furthermore, laccase is a copper-rich 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, osmotic regulators, and copper ion concentration during liquid fermentation plays a crucial role in improving the efficient expression of laccase in Aspergillus niger. Summary of the Invention

[0006] This invention provides a laccase derived from *Coprinus gracilis*, its recombinant expression strain, and a highly efficient expression method. Using *Aspergillus niger* MA70.15 as the experimental strain, this invention employs a random integration strategy to achieve pC3-P... citA -SP GlaA - lcc9- T trpC - pyrG The expression vector was integrated into the upstream region of the promoter of the host bacterium citrate synthase 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, this invention added different types and concentrations of microparticles and osmotic regulators, resulting in a laccase activity of 1566.7 U / L in shake-flask fermentation of recombinant strain AnRcgL1 and 1961 U / L in a 1 L fermenter, successfully achieving efficient recombinant expression of laccase Lcc9 in Aspergillus niger.

[0007] The present invention relates to laccase Lcc9 derived from Coprinus gracilis, the amino acid sequence of which is shown in SEQ ID NO:1 or a sequence having a homology of more than 85% with the amino acid sequence shown in SEQ ID NO:1.

[0008] The gene encoding laccase Lcc9 from *Coprinus gracilis* has a nucleotide sequence as shown in SEQ ID NO:2 or a sequence with more than 85% homology to 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 It was obtained by constructing based on the pC3 carrier.

[0010] This invention also provides a recombinant Aspergillus niger strain AnRcgL1 expressing laccase derived from the aforementioned *Coprinus pygmae*, using *Aspergillus niger* MA70.15 as the expression host and pC3-P... citA -SP GlaA - lcc9- T trpC- pyrG It is obtained through random integration as an expression carrier.

[0011] Furthermore, the pC3-P in the recombinant Aspergillus niger strain AnRcgL1 citA -SP GlaA - lcc9- T trpC -pyrG The expression vector is integrated into P in the form of nine consecutive copies. 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 was performed using a polyethylene glycol (PEG)-mediated protoplast transformation method. This involved mixing protoplast suspension, expression vector, and PEG buffer solution in an ice bath, then adding PEG buffer solution, osmotic stabilizer, and soft agar medium. After mixing, the mixture was transferred to a regeneration medium to regenerate the transformed strains. The transformed strains were then selected and screened onto screening plates.

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

[0014] Furthermore, the protoplast regeneration method is as follows: after mixing the transformation mixture, osmotic pressure stabilizer and soft agar medium, the mixture is transferred to the regeneration medium and cultured upright at 28°C for 4 to 7 days to obtain the transformed strain.

[0015] Furthermore, the screening method for transformants is as follows: transformants are picked and placed onto screening plates for screening. The screening plate components (w / v) involved include: 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%, and ABTS 0.4-0.6 mM, CuSO4 0.05-0.15 mM.

[0016] The present invention relates to the application of the recombinant Aspergillus niger strain AnRcgL1 in the efficient preparation of laccase Lcc9 from Coprinus gracilis.

[0017] Specifically, the steps include the following:

[0018] Step 1: Spread the recombinant strain AnRcgL1 of Aspergillus niger onto a PDA solid plate, and after culturing for 3-5 days, add a physiological saline solution containing 0.05% Tween 80, scrape off the spores, and prepare a spore suspension;

[0019] Step 2: Inoculate the fresh spore suspension into the fermentation medium and culture at 30℃ and 200 rpm for 5-7 days to obtain laccase Lcc9.

[0020] Furthermore, the fermentation medium contains 0.1%-2% (w / v) CaCO3.

[0021] Furthermore, the fermentation medium also includes 0.05-0.2 M glycine.

[0022] The solid plates used for activation culture are PDA solid plates. 1 L of culture medium contains the following components: 150-250 g potato, 15-25 g glucose, 15-20 g agar, and requires the addition of 10 mM uracil.

[0023] The liquid culture medium is YPD medium. 1 L of the medium contains the following components: 15-25 g peptone, 5-15 g yeast extract, 15-25 g glucose, and requires the addition of 10 mM uracil.

[0024] The osmotic pressure stabilizer component comprises: CaCl2 40-60 mM, sorbitol 1.0-1.4 mM, Tris-HCl 8-12 mM, pH 7.5.

[0025] The PEG buffer solution comprises: PEG 4000 40-80% (w / v), CaCl2 40-60 mM, Tris-HCl 5-15 mM, pH 7.5.

[0026] The hypertonic soft agar medium composition (w / v) is as follows: sucrose 30-35%, NaNO3 0.2-0.4%, KCl 0.1-0.3%, MgSO4·7H2O 0.02-0.07%, KH2PO4 0.1-0.2%, FeSO4·7H2O 0.001-0.003%, and agar powder 1%.

[0027] The regeneration culture medium components are the same as those of hypertonic soft agar culture medium, except that the amount of agar powder added (w / v) is 1.5-2%.

[0028] The screening medium components (w / v) comprise: 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, and CuSO4 0.05-0.15 mM.

[0029] The fermentation medium components (w / v) comprise: 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× 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). The pH of the fermentation medium is adjusted to 5.5 using citric acid.

[0030] In one embodiment of the invention, the 1 L fermentation medium composition (w / v) 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% glucose (w / v), and 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). The fermentation medium is adjusted to pH 5.5 using citric acid.

[0031] The beneficial effects of this invention are reflected in:

[0032] (1) This invention successfully achieved efficient expression of laccase from the basidiomycete Coprinus gracilistylus in Aspergillus niger. lcc9 Using the technical solution of this invention, Aspergillus niger MA70.15 is used as the expression host, and pC3-P... citA -SP GlaA -T trpC- lcc9 Using a random integration strategy, a recombinant Aspergillus niger strain AnRcgL1 expressing laccase Lcc9 from the basidiomycete Coprinus gracilistylus was successfully constructed as an expression vector, with an enzyme activity of 86 U / L during shake-flask fermentation.

[0033] (2) Using the recombinant Aspergillus niger strain AnRcgL1 constructed in this invention, and to investigate the effects of microparticles and osmotic regulators on its fermentation production of laccase, the enzyme activity of laccase Lcc9 in the shake-flask fermentation supernatant was increased to 1566.7 U / L. 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 laccase Lcc9 in Aspergillus niger was successfully achieved. Attached Figure Description

[0034] Figure 1 The laccase expression vector pC3-P constructed in this invention citA -SP GlaA - lcc9- T trpC -pyrG .

[0035] Figure 2 The laccase expression vector pC3-P constructed in this invention citA -SP GlaA - lcc9 -T trpC -pyrG The verification results are as follows: A is colony PCR identification, and 1-2 are transformant 1 and transformant 2, respectively; B is plasmid enzyme digestion verification, and 1-2 are transformant 1 and transformant 2, respectively.

[0036] Figure 3 The results of plate color development and active protein gel examination of recombinant Aspergillus niger AnRcgL1 of the present invention are shown in Figure A, where A represents the color development of recombinant Aspergillus niger AnRcgL1 on a plate containing ABTS, and B represents the active protein gel examination of the supernatant from shake-flask fermentation of recombinant Aspergillus niger AnRcgL1.

[0037] Figure 4 Sequencing confirmed the recombinant Aspergillus niger AnRcgL1. lcc9 A schematic diagram of copy number and integration site.

[0038] Figure 5 The effect of adding microparticles to the liquid fermentation medium of recombinant Aspergillus niger AnRcgL1 on laccase expression was shown in this invention. A, B, C, D, and E are different concentrations of MgSiO3, Al2O3, CaCO3, quartz sand, and ZnCO3, respectively.

[0039] Figure 6 The effect of adding osmotic regulators to the liquid fermentation medium of recombinant Aspergillus niger AnRcgL1 on laccase expression was shown in this invention. A, B, C, D, and E are different concentrations of glycine, proline, TMAO, mannitol, and calcium chloride, respectively.

[0040] Figure 7 The images show the laccase Lcc9 enzyme activity (A) and (B) bands in the supernatant of the 1 L fermenter fermentation of recombinant Aspergillus niger AnRcgL1 of this invention. Detailed Implementation

[0041] Unless otherwise specified, the implementation methods in the following embodiments are all conventional methods.

[0042] The Aspergillus niger MA70.15 used in this invention is disclosed in patent CN2023108680572, and in Guo SX, Yao GF, Ye HR, et al. Functional Characterization of a Cystathionine β-Synthase Gene in Sulfur Metabolism and Pathogenicity of AspergillusnigerIn Pear Fruit. Journal of Agricultural and Food Chemistry. 2019, 67 (16): 4435-4443, Vera Meyer, Mark Arentshorst, et al. Highly efficient gene targeting in the Aspergillus niger kusA mutant. Journal of Biotechnology. 128 (2007):770-775, etc. Our research group initially purchased the technology 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.

[0043] The synthetic gene PUC57 involved in the following examples lcc9 PUC57-P citA PUC57-T trpC Both are based on commercially available vector skeletons and corresponding genomes, and both were sent to the company for synthesis.

[0044] The culture media and solutions involved in the following examples are as follows:

[0045] PDA solid medium (1 L): 200 g potato, 20 g glucose, 15-20 g agar, with an additional 10 mM uracil as needed.

[0046] YPD medium (w / v): 2% peptone, 1% yeast extract, 2% glucose, with an additional 10mM uracil as needed.

[0047] 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).

[0048] Hyperosmolar 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%.

[0049] 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.

[0050] STC buffer: Anhydrous CaCl2 50 mM, Sorbitol 1.2 M, Tris-HCl 10 mM, pH 7.5.

[0051] PEG buffer: PEG 4000 60% (w / v), CaCl2 50 mM, Tris-HCl 10 mM, pH 7.5.

[0052] Fermentation medium A: 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), 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).

[0053] 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, 1%-2% glucose (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).

[0054] The detection methods involved in the following embodiments are as follows:

[0055] Laccase activity: Laccase activity was determined using the ABTS method. Specifically, 950 μL of sodium tartrate buffer (100 mmol / L, pH 4.0) was mixed with 33 μL of 15 mmol / L ABTS and 17 μL of appropriately diluted enzyme solution. The mixture was thoroughly mixed and then incubated in a water bath at 30°C for 3 min. The reaction was terminated by incubating on ice for 30 s. The absorbance of the reaction solution was measured at a wavelength of λ = 420 nm.

[0056] Formula for calculating laccase activity: E (U / L) = OD 420 ×Dilution factor×555.56.

[0057] Laccase expression: Laccase expression in the fermentation broth was detected by native-pAGE. Specific procedures: The supernatant of the fermentation broth, diluted to a certain extent, was mixed with 2× loading buffer, and 10 μL was loaded into the wells of the active gel. Electrophoresis was performed in a VE-180A miniature vertical electrophoresis tank at 80 V for 30 min, followed by a further electrophoresis at 120 V for 3 h. The active gel was then removed and incubated at room temperature in 100 mmol / L pH 4.0 sodium tartrate buffer (0.5 mmol / L ABTS).

[0058] Example 1: Construction of a laccase expression vector containing Coprinus grayis.

[0059] expression vector pC3-P citA -SP GlaA - lcc9- - pyrG It was constructed based on the vector pC3, which is 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 pC3 vector, with laccase lcc9 Expression box, filter tags pyrGRecombination. Expression vector pC3-P citA -SP GlaA - lcc9- T trpC -pyrG See the schematic diagram of the carrier structure. Figure 1 .

[0060] The specific steps are as follows:

[0061] The promoter, signal peptide, and other components were obtained respectively. lcc9 Termination of sub-sub ... pyrG Five elements were obtained, and the five fragments were combined to form a long fragment by fusion PCR. Homologous recombination ligation was then performed between the long fragment and the linearized pC3 fragment using the CloneExpress II Onestep Cloning Kit from Novizan.

[0062] The amino acid sequence of Lcc9 is shown in SEQ ID NO:1. The gene PUC57- was synthesized using primers F1 / R1. lcc9 The template was obtained by PCR. lcc9 The sequence is shown in SEQ ID NO:2. The sequences of primers F1 / R1 are shown in Table 1. The promoter P mediates Lcc9 expression. citA The sequence is shown in SEQ ID NO:3, and was obtained by PCR using primer F2 / R2 with the synthesized gene as a template. The sequences of primer F2 / R2 are shown in Table 1. The signal peptide SP mediates Lcc9 expression. GlaA The sequence is shown in SEQ ID NO:4. The gene PUC57-SP was synthesized using primers F3 / R3. GlaA The template was obtained by PCR. The lower sequences of primers F3 / R3 are shown in Table 1. Terminator T... trpC The sequence is shown in SEQ ID NO:5. The gene PUC57-T was synthesized using primers F4 / R4. trpC The primers were obtained by PCR. The sequences of primers F4 / R4 are shown in Table 1. pyrG The sequences already present on the pC3 vector were obtained by PCR using primers F5 / R5 with plasmid pC3 as a template. The sequences of primers F5 / R5 are shown in Table 1.

[0063]

[0064] The fusion PCR used consisted of two steps. The first step involved recombination and fusion of homologous sequences between the elements without primers; the fusion PCR system is shown in Table 2. The second step involved large-scale PCR amplification of the recombination product from the first step using primers F1 / R4. Linearization of the vector pC3 was achieved by inverse PCR; the sequences of primers F5 / R5 are shown in Table 1.

[0065]

[0066] PCR conditions: 94℃ pre-denaturation, 5 min; 98℃ denaturation, 30 s; 55℃ annealing, 15 s; 72℃ extension, 1 min 49 s, 15 cycles; 72℃, 10 min. PCR products were recovered by gel electrophoresis.

[0067] The connection system between the fusion fragment and the linearized vector pC3 is shown in Table 3.

[0068]

[0069] Connection conditions: React at 37℃ for 30 min.

[0070] 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 .

[0071] Example 2: Construction of recombinant Aspergillus niger and expression of laccase

[0072] (1) Preparation of protoplasts

[0073] A. Activation culture of the strain

[0074] After appropriately diluting the Aspergillus niger MA70.15 glycerol tube preservation material stored at -80℃, spread it onto PDA solid medium (add 10 mM uracil) and incubate at 28℃ for 3-5 days.

[0075] B. Culture of mycelium (liquid culture method)

[0076] Take an activated plate covered with spores, cut a piece of mycelium and inoculate it into YPD liquid medium (add 10 mM uracil), and incubate at 28-32℃ and 180-220 rpm for 48 h.

[0077] C. Collection of mycelium

[0078] Filter the bacterial cells using a Buchner funnel lined with double-layered filter paper until the cells are dry. Then wash the cells twice with sterile deionized water and twice with NaCl solution, filtering again until the cells are dry. Scrape off the cells with a clean spatula and collect them in an Erlenmeyer flask or EP tube, then weigh them.

[0079] D. Enzymatic hydrolysis of mycelium

[0080] Add the enzymatic hydrolysate at a ratio of mycelium to enzymatic hydrolysate of 1:10 (w / v), and place it in a water bath shaker at 37℃ and 100 rpm for 3 h for enzymatic hydrolysis.

[0081] The enzymatic hydrolysate consisted of: 2% (w / v) cellulase, 1% (w / v) lysozyme, 0.5% (w / v) lysozyme, 1% (w / v) snailase, 0.8 M NaCl, and 10% (v / v) 100 mM pH 6.0 sodium phosphate buffer.

[0082] E. Collection of protoplasts

[0083] Filter the enzyme digest using lens paper (smooth side) to collect the protoplasts. Then rinse the test tubes containing the enzyme digest and the lens paper with NaCl. Centrifuge at 3000-4000 rpm for 10 min, discard the supernatant, and collect the protoplasts obtained using different enzyme digests.

[0084] F. Purification of protoplasts

[0085] Add STC buffer (osmotic stabilizer), gently aspirate with a de-pointed pipette tip to resuspend the protoplasts, and centrifuge at 3000-4000 rpm for 10 min at 4°C to collect the protoplasts. Repeat this step once. Add an appropriate amount of STC buffer (osmotic stabilizer), gently aspirate with a de-pointed pipette tip to resuspend the protoplasts, and obtain a protoplast suspension.

[0086] (2) Transformation of protoplasts

[0087] Pipe 150–200 μL of protoplast suspension into 2 mL centrifuge tubes, then add 20 μL of expression vector pC3-P. citA -SP GlaA - lcc9- T trpC -pyrG Add 4~10 μg of PEG buffer solution and 50 μL of PEG buffer solution, mix gently by inverting the container once every 10 min, incubate on ice for 30 min, then add 1.5 mL of PEG buffer solution and let stand at room temperature for 25 min.

[0088] (3) Regeneration of protoplasts

[0089] Add 2.5 mL of STC buffer and 5 mL of hypertonic CD soft agar medium to a 10 mL centrifuge tube, mix well, transfer the protoplast transformation mixture to a 10 mL centrifuge tube, mix with a pre-cooled 5 mL de-pointed pipette tip, and then 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.

[0090] (4) Screening and identification of transformants

[0091] Twenty transformants from the regeneration plate were selected for qualitative analysis on ABTS plates. One of them was viable, the recombinant Aspergillus niger strain MA70.15 / pC3-P. citA -SP GlaA - lcc9- T trpC -pyrG The strain was named AnRcgL1. The transformed strain was inoculated onto the selection medium and cultured at 28℃ for 2-3 days. The growth and color development of the strain were then observed.

[0092] Conclusion: Obvious color development was observed on the ABTS plate (see...) Figure 3 A) indicates the presence of laccase activity.

[0093] (5) Shake-flask fermentation of recombinant Aspergillus niger

[0094] The obtained recombinant strain AnRcgL1 was spread on a PDA solid plate and cultured for 3-5 days until it had fully sporulated. Then, a physiological saline solution containing 0.05% Tween 80 was added, and spores were scraped off to prepare a spore suspension. The fresh spore suspension was inoculated into fermentation medium A (1×10⁻⁶). 5 (spores / mL), cultured at 30℃ and 200 rpm for 7 days. 1 mL of fermentation broth sample was taken daily, centrifuged at 12000×g for 5 min, and the supernatant was stored at 4℃. The fermentation supernatant was used to determine laccase activity and expression.

[0095] Laccase expression: After mixing the fermentation broth supernatant with 2× loading buffer, 10 μL was loaded into the wells of the active gel and electrophoresed at 80 V for 30 min in a VE-180A miniature vertical electrophoresis tank, followed by electrophoresis at 120 V for 3 h. The active gel was then removed and incubated at room temperature in 100 mmol / L pH 4.0 sodium tartrate buffer (0.5 mmol / L ABTS).

[0096] The results showed that laccase activity reached its peak on day 5, at 86 U / L, and the active gel showed a distinct colored band. Figure 3 B) indicates that laccase expression was detected.

[0097] (6) Sequencing analysis

[0098] The recombinant strain AnRcgL1 was subjected to whole-genome sequencing to analyze its integration site and copy number.

[0099] The results show that ( Figure 4 ), lcc9The expression frame was integrated upstream of the citrate synthase promoter in a 9-copy configuration.

[0100] Example 3: Effect of microparticles in fermentation medium on laccase production by recombinant Aspergillus niger.

[0101] The obtained recombinant strain AnRcgL1 was spread on a PDA solid plate and cultured for 3-5 days until it had fully sporulated. Then, a physiological saline solution containing 0.05% Tween 80 was added, and spores were scraped off to prepare a spore suspension. The fresh spore suspension was inoculated into a fermentation medium (1×10⁻⁶). 5 Different types and concentrations of microparticles (CaCO3, MgSiO3, Al2O3, quartz sand, ZnCO3) were added to the fermentation medium. The microparticles were suspended in 50 mM sodium acetate buffer (pH 6.5) and autoclaved at 121°C for 20 minutes. Subsequently, the microparticles were added to the fermentation medium. To further investigate the effect of microparticle concentration on laccase activity, different concentrations of 0.5%, 1%, and 2% were selected. The medium was incubated at 30°C and 200 rpm for 7 days. 1 mL of fermentation broth was collected daily, centrifuged at 12000×g for 5 min, and the supernatant was stored at 4°C. The fermentation supernatant was used to determine laccase activity.

[0102] Laccase activity assay: The ABTS method was used. Specifically, 950 μL of sodium tartrate buffer (100 mmol / L, pH 4.0) was mixed with 33 μL of 15 mmol / L ABTS and 17 μL of appropriately diluted enzyme solution. The mixture was thoroughly mixed, incubated at 30°C for 3 min, and then stopped by incubating on ice for 30 s. 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.

[0103] The results show that ( Figure 5 C) When 1% calcium carbonate was added to the fermentation medium, the laccase activity reached its highest level of 925.6 U / L on day 5, which was 10.8 times higher than the control.

[0104] Example 4: Effect of osmotic regulators in fermentation medium on laccase production by recombinant Aspergillus niger.

[0105] The obtained recombinant strain AnRcgL1 was spread on a PDA solid plate and cultured for 3-5 days until it had fully sporulated. Then, a physiological saline solution containing 0.05% Tween 80 was added, and spores were scraped off to prepare a spore suspension. The fresh spore suspension was inoculated into a fermentation medium (1×10⁻⁶). 5The fermentation broth was incubated at 30°C and 200 rpm for 7 days. 1% CaCO3 was added, along with various osmotic regulators (glycine, proline, TMAO, mannitol, and calcium chloride). To further investigate the effect of osmotic regulator concentrations on laccase activity, different concentrations were selected: glycine (0.05-0.4 M), proline (0.1-0.5 M), TMAO (0.05-0.4 M), mannitol (0.1-0.5 M), and calcium chloride (0.05-0.5 M). The broth was centrifuged daily at 12000×g for 5 min, and the supernatant was stored at 4°C. The fermentation supernatant was used to determine laccase activity.

[0106] Laccase activity assay: The ABTS method was used. Specifically, 950 μL of sodium tartrate buffer (100 mmol / L, pH 4.0) was mixed with 33 μL of 15 mmol / L ABTS and 17 μL of appropriately diluted enzyme solution. After thorough mixing, the mixture was incubated at 30°C for 3 min, followed by an ice bath 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.

[0107] The results show that ( Figure 6 A): When 0.1 M glycine osmotic regulator was added to the fermentation medium, the laccase activity reached its peak on the fifth day, at 1566.7 U / L, which was 1.7 times higher than that of adding 1% CaCO3.

[0108] Example 5: Fermentation of recombinant Aspergillus niger in a 1 L tank

[0109] The obtained recombinant strain AnRcgL1 was spread on PDA solid plates and cultured for 3-5 days until it had fully sporulated. Then, a physiological saline solution containing 0.05% Tween 80 was added, and spores were scraped off to prepare a spore suspension. The fresh spore suspension was inoculated into fermentation medium (1×10⁻⁶). 5 The fermentation medium was supplemented with 1% CaCO3, 0.1 M glycine, and 3 mM CuSO4. The fermentation tank volume was 1 L, and the initial culture conditions were 30℃, pH 5.5, 200 rpm, and an air flow rate of 50 L / h. From day 2 onwards, the oxygen flow rate was maintained at 20 L / h. Every 24 h, 5 mL of sample was taken, centrifuged at 12000×g for 5 min, and the supernatant was collected and stored at 4℃. The fermentation supernatant was used to determine laccase activity and laccase expression.

[0110] Laccase expression: After mixing the fermentation broth supernatant with 2× loading buffer, 10 μL was loaded into the wells of the active gel and electrophoresed at 80 V for 30 min in a VE-180A miniature vertical electrophoresis tank, followed by electrophoresis at 120 V for 3 h. The active gel was then removed and incubated at room temperature in 100 mmol / L pH 4.0 sodium tartrate buffer (0.5 mmol / L ABTS).

[0111] The results show that ( Figure 7 A) Laccase activity reached its peak on day 5, at 1961 U / L, which is 1.3 times the level observed during shake-flask fermentation. The active gel showed distinct colored bands. Figure 7 B).

[0112] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of recombinant Aspergillus niger strain AnRcgL1 in the efficient preparation of laccase Lcc9 from Coprinus macrocephala, characterized in that... Includes the following steps: Step 1: Spread the recombinant strain AnRcgL1 of Aspergillus niger onto a PDA solid plate, and after culturing for 3-5 days, add a physiological saline solution containing 0.05% Tween 80, scrape off the spores, and prepare a spore suspension; Step 2: Inoculate the fresh spore suspension into the fermentation medium and culture at 30℃ and 200 rpm for 5-7 days to obtain laccase Lcc9; the fermentation medium contains 1%-2% CaCO3; The recombinant Aspergillus niger strain AnRcgL1 uses Aspergillus niger MA70.15 as the expression host and pC3-P as the expression medium. citA -SP GlaA -lcc9-T trpC- pyrG is the expression vector, obtained through random integration; the promoter P... citA The sequence is shown in SEQ ID NO.3, signal peptide SP GlaA The sequence is shown in SEQ ID NO.4; The expression vector pC3-P citA -SP GlaA -lcc9-T trpC- pyrG was transformed into Aspergillus niger protoplasts using a polyethylene glycol-mediated transformation method. The transformation involved mixing protoplast suspension, expression vector, and PEG buffer solution in an ice bath, then adding PEG buffer solution, osmotic stabilizer, and soft agar medium. After mixing, the mixture was transferred to regeneration medium to regenerate and obtain transformants. Transformants were then selected and screened on screening plates to obtain the final product.

2. The application according to claim 1, characterized in that: The fermentation medium also contains 0.05-0.2 M glycine.

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