A laccase fusion protein, a fermentation preparation method and application thereof in mycotoxin detoxification

Abstract

This invention belongs to the field of agricultural biotechnology, specifically relating to a laccase fusion protein and its fermentation preparation method, and its application in fungal toxin detoxification. The fusion protein is derived from laccase of *Auricularia ochracea*. So Lac, linker peptides, and xylanase from Aspergillus niger An XynB composition. This invention constructs xylanase. An XynB and Laccase So The Lac fusion protein expression vector was transformed into Pichia pastoris for heterologous expression of the fusion protein, which significantly improved laccase expression. So Lac expression level. The recombinant laccase obtained in this invention. So Lac and xylanase An XynB fusion protein can effectively detoxify mycotoxins such as aflatoxin B1 and zearalenone, and can be widely used in the field of mycotoxin detoxification in food and feed.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a laccase fusion protein and its fermentation preparation method, and its application in the detoxification of fungal toxins. Background Technology

[0002] Mycotoxins are secondary metabolites produced by fungi, seriously endangering food and feed safety. Aflatoxin B1 is the most common and most dangerous, exhibiting high stability and requiring temperatures above 260°C to decompose. Zearalenone has the structure of m-dihydroxybenzoic acid lactone. The molecular structure of zearalenone and its metabolites is similar to the endogenous estrogen 17-estradiol; therefore, it can bind to estrogen receptors, thereby inhibiting estrogen binding to receptors in tissues and affecting reproductive system function. Mycotoxin contamination in food, feed, and feed ingredients is mostly co-contamination of several toxins, while most discovered enzymes only have the ability to degrade specific types of mycotoxins. Therefore, broad-spectrum mycotoxin-degrading enzymes have greater application potential.

[0003] Lacase (EC 1.10.3.2) is a copper-containing polyphenol oxidase widely distributed in plants, fungi, a few insects, and bacteria, capable of oxidizing a variety of aromatic compounds. Among these, laccases from Basidiomycetes dominate industrial applications due to their broad substrate spectrum, high enzyme activity, and strong redox potential. However, the yield of laccase from natural strains is typically low, making it difficult to meet industrial demands; therefore, achieving low-cost and efficient production is crucial. Currently, heterologous expression systems are considered ideal for large-scale laccase production due to their cost-effectiveness and environmental friendliness. Among numerous hosts, Pichia pastoris (… Pichia pastoris With its high cell density culture characteristics and efficient protein secretion capacity, fungal laccase has become the preferred platform for the production of food-grade laccase. However, the active expression of fungal laccase in Pichia pastoris still faces challenges, which limits its industrial application.

[0004] Furthermore, the conversion ability of laccases to fungal toxins (such as aflatoxin B1 and zearalenone) is highly dependent on their subtype characteristics. For example, in king oyster mushrooms (… Pleurotus eryngii Laccases require mediators such as 2,2'-adiazono-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) to degrade toxins, which not only increases application costs but also poses food safety risks due to mediator residues. Therefore, identifying basidiomycete laccases that can efficiently degrade toxins without mediators and achieving their expression in Pichia pastoris is a necessary step to promote the application of fungal laccases in the field of food and feed detoxification.

[0005] Xylanase, a common feed additive, can break down the xylan components of plant cell walls in feed and regulate the gut microbiota, thereby improving feed conversion ratio. Due to its high expression rate in Pichia pastoris, the fusion expression of xylanase and laccase can potentially increase laccase production. Simultaneously, this fusion protein can perform the dual function of eliminating mycotoxins in feed and promoting feed digestion.

[0006] To obtain a fusion protein of xylanase and fungal laccase with excellent enzymatic properties, the selection of xylanase is crucial. For example, xylanase derived from *Aspergillus niger* (…) Aspergillus niger Xylanases (xylanases) are capable of adapting to a wide range of pH and temperature conditions, exhibiting good stability under different pH conditions, and possessing highly efficient catalytic activity. An (XynB). However, the direct fusion of the two catalytically functional domains of xylanase and fungal laccase may lead to consequences such as protein misfolding and impaired enzyme activity. Summary of the Invention

[0007] The purpose of this invention is to provide a laccase. So Lac and xylanase An XynB fusion protein.

[0008] Another object of the present invention is to provide the encoding gene of the above-mentioned fusion protein.

[0009] Another object of the present invention is to provide a recombinant expression vector containing the above-mentioned coding gene.

[0010] Another object of the present invention is to provide a recombinant strain containing the gene encoding the above-mentioned fusion protein.

[0011] Another object of the present invention is to provide a method for preparing laccase by fermentation.

[0012] Another object of the present invention is to provide the application of the above-mentioned fusion protein in the detoxification of fungal toxins.

[0013] According to the present invention, the laccase fusion protein is composed of xylanase. An XynB, linker peptides and laccase So Lac composition. The laccase. So Lac is Ochraea rubra ( Steccherinum ochraceum The xylanase is derived from [a source], and its amino acid sequence is shown in SEQ ID No:1. An XynB is Aspergillus niger ( Aspergillus niger The amino acid sequence of the linker peptide is shown in SEQ ID No:2, and the amino acid sequence of the linker peptide is shown in SEQ ID No:3.

[0014] SEQ ID No:1

[0015] AIGPIADLHINNSNISPDGFTRPAVLAGGTFPGPLIQGNKGDNFQINVIDELTDENQLKSTSIHWHGLFQHGTNWADGAAFVTQCPIATGHSFLYNFDVPDQAGTYWYHSHLSTQYCDGLRGPFVVYDPDDVHQQLYDIDNEDTVITLADWYHILARQEPPGPPVPDSTLINGFGRFPGQTTPSDLAVITVEQGKRYRLRLVNIACDPNYQFSIDNHNLTVIEVDGVSTQALTVTSLTIFAGQRYSVILHANQDNKGDQGNYWIRAKPNTGADTSFNGGLNSAILRYVGANPVDPETTMGMDNAPLNEVNLRPFISTPVPGQPHAGGADFVKNLAFTFAAGLFAVDGTPFVPPTVPVLLQILSGAQTAQDLLPTGSIIELPPNKVIEFSMPGGVVGGGHPIHLHGHTFWVVRSAGSSTYNYNDAILRDVVNIGVAGDNVTIRFVTDNPGPWFLHCHIDWHLDTGFAVVMAEDIPGTAAANPVPADWSQLCPLYDALPPEDL;

[0016] wherein the enzyme comprises 502 amino acids and has a theoretical molecular weight of 54.2 kDa.

[0017] SEQ ID No: 2:

[0018] VPHDSVAQRSDALHMLSERSTPSSTGENNGFYYSFWTDGGGDVTYTNGDAGAYTVEWSNVGNFVGGKGWNPGSAQDITYSGTFTPSGNGYLSVYGWTTDPLIEYYIVESYGDYNPGSGGTYKGTVTSDGSVYDIYTATRTNAASIQGTATFTQYWSVRQNKRVGGTVTTSNHFNAWAKLGMNLGTHNYQIVATEGYQSSGSSSITVQ,

[0019] wherein the enzyme comprises 207 amino acids and has a theoretical molecular weight of 22.2 kDa.

[0020] SEQ ID No: 3:

[0021] SASSGGTTPTTTHM,

[0022] The linker peptide consists of 14 amino acids and has a theoretical molecular weight of 1.3 kDa.

[0023] The present invention provides a coding sequence for the above-mentioned fusion protein.

[0024] The present invention also provides a xylanase comprising the above-mentioned xylanase. An XynB, linker peptides and laccase So Recombination vector of Lac-encoded sequences.

[0025] This invention also provides a method for preparing laccase by fermentation, comprising the following steps:

[0026] (1) Using laccase containing encoding So The recombinant expression vector of the Lac fusion protein gene was transformed into host cells to obtain recombinant strains;

[0027] (2) Recombinant strains were cultured in a fermenter and laccase was induced by methanol. So Lac fusion protein expression;

[0028] (3) Purification of laccase So Lac fusion protein.

[0029] The present invention also provides the above-mentioned laccase. So The application of Lac fusion protein can effectively degrade aflatoxin B1 and zearalenone, and can be applied in fields such as biomass energy, food industry and feed industry.

[0030] The beneficial effects of this invention are:

[0031] According to the technical solution of this application, a linker peptide is introduced between xylanase and fungal laccase. However, linker peptides with different sequences can significantly affect the activity of fusion proteins by influencing the steric hindrance and conformational freedom, domain orientation, substrate accessibility and protein stability. Therefore, this application screens suitable linker peptide sequences to obtain fusion proteins with high laccase activity.

[0032] This invention utilizes xylanase derived from Aspergillus niger. An XynB and laccase So Fusion expression of Lac in Pichia pastoris successfully achieved heterologous and efficient expression of fungal laccase with high redox potential in Pichia pastoris, significantly improving laccase efficiency. So Lac expression levels. Meanwhile, this fusion expression strategy does not affect laccase expression. So Lac activity in degrading fungal toxins. Attached Figure Description

[0033] Figure 1 Showing laccase SoMap of the expression plasmid pPICZα(A)-Solac for Lac protein;

[0034] Figure 2 Showing laccase So Map of the expression plasmid pPICZα(A)-AnxynB-linker-Solac for the Lac fusion protein;

[0035] Figure 3 This demonstrates the expression of laccase using different linker peptides. So Lac fusion protein laccase activity results;

[0036] Figure 4 This study demonstrated the synthesis of laccase using Pichia pastoris X33 / AnxynB-linker2-Solac strain in a fermentation tank. So Results of changes in laccase activity with methanol induction time for Lac fusion protein.

[0037] Figure 5 This study demonstrated the synthesis of laccase using Pichia pastoris X33 / AnxynB-linker2-Solac strain in a fermentation tank. So Results of changes in xylanase activity with methanol induction time in Lac fusion protein;

[0038] Figure 6 This study demonstrated the synthesis of laccase using Pichia pastoris X33 / AnxynB-linker2-Solac strain in a fermentation tank. So Results of cell wet weight changes with methanol induction time when Lac fusion protein was induced;

[0039] Figure 7 The linear relationship between laccase and xylanase in the fermentation broth during the fermentation process using Pichia pastoris X33 / AnxynB-linker2-Solac strain was shown.

[0040] Figure 8 This study demonstrated the synthesis of laccase using Pichia pastoris X33 / AnxynB-linker2-Solac strain in a fermentation tank. So SDS-PAGE electrophoresis image of the purified Lac fusion protein;

[0041] Figure 9 The mass spectrometry identification results of the purified fusion protein are shown;

[0042] Figure 10 Showing laccase So Optimal pH results for Lac fusion protein using ABTS, SGZ and 2,6-DMP as substrates;

[0043] Figure 11 Showing laccaseSo The optimal catalytic temperature for Lac fusion protein with ABTS as substrate was determined.

[0044] Figure 12 Showing laccase So pH stability of Lac fusion protein when ABTS is used as substrate;

[0045] Figure 13 Showing laccase So Temperature stability of the Lac fusion protein when ABTS was used as a substrate;

[0046] Figure 14 Showing laccase So Results of the optimal temperature for Lac fusion protein to degrade aflatoxin B1;

[0047] Figure 15 Showing laccase So The optimal pH result for the degradation of aflatoxin B1 by the Lac fusion protein;

[0048] Figure 16 Showing laccase So Effects of Lac fusion protein dosage and reaction time on the degradation of aflatoxin B1;

[0049] Figure 17 Experimental results showing the cytotoxic effects of different concentrations of aflatoxin B1 on HepG2 cells;

[0050] Figure 18 Showing laccase So Experimental results on the detoxification effect of Lac fusion protein on aflatoxin B1;

[0051] Figure 19 Showing laccase So Results of the optimal temperature for Lac fusion protein to degrade zearalenone in zearalenone;

[0052] Figure 20 Showing laccase So The optimal pH result for the degradation of zearalenone by the Lac fusion protein;

[0053] Figure 21 Showing laccase So The effects of Lac fusion protein dosage and reaction time on the degradation of zearalenone;

[0054] Figure 22 Experimental results showing the cytotoxicity of different concentrations of zearalenone to HepG2 cells;

[0055] Figure 23 Showing laccase So Experimental results on the detoxification effect of Lac fusion protein on zearalenone in zearalenone. Detailed Implementation

[0056] Experimental materials and reagents

[0057] 1. Vectors and genes: expression vector pPICZα(A) and Pichia pastoris strain X33;

[0058] 2. Enzymes and other biochemical reagents: restriction endonucleases, recombinases, aflatoxin B1, and zearalenone;

[0059] 3. Escherichia coli culture medium: LLB medium (1% peptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0), with bleomycin added to a final concentration of 25 mg / L if necessary. Solid plates are prepared with 2% agar.

[0060] 4. Pichia pastoris culture media: YPD medium (2% glucose, 2% peptone, 1% yeast extract), with 100 mg / L bleomycin added if necessary; solid plates with 2% agar added; BMGY medium (1% glycerol, 2% peptone, 1% yeast extract, 10% YNB, 1‰ biotin); BMMY medium (0.5% methanol, 2% peptone, 1% yeast extract, 10% YNB, 1‰ biotin); fermentation medium (glycerol 40 g / L, KH2PO4 5 g / L, CaSO4 0.93 g / L, K2SO4 10 g / L, MgSO4 10 g / L, NH4H2PO4 40 g / L, KOH 1.5 g / L); PTM1 solution (copper sulfate 6.0 g / L, potassium iodide 0.09 g / L, manganese sulfate 3.0 g / L, sodium molybdate 0.2 g / L, boric acid 0.02 g / L). (g / L, cobalt chloride 0.5 g / L, zinc chloride 20 g / L, ferrous sulfate 65 g / L, sulfuric acid 5.0 ml / L).

[0061] Example 1: Construction of expression vectors for laccase SoLac fusion proteins containing different linking peptides

[0062] Ochratus erythrorhizon ( Steccherinum ochraceum Laccase from Solac Genes and Aspergillus niger ( Aspergillus niger xylanase from ) Anxynb The gene was optimized and synthesized based on the codon bias of Pichia pastoris, including the optimized laccase. Solac The nucleotide sequence of the gene is shown in SEQ ID No: 4, and the optimized xylanase... Anxynb The nucleotide sequence of the gene is shown in SEQ ID No: 5.

[0063] SEQ ID No: 4:

[0064]

[0065] SEQ ID No: 5:

[0066] Gttccacacgactctgttgctcaaagatccgatgccttgcacatgttgtctgagagatccactccatcttccaccggtgaaaacaacggtttctactactccttctggactgatggtggtggtgacgttacttacactaacggtgatgctggtgcttacactgttgagtggtctaacgttggtaacttcgtcggtggtaaaggttggaacccaggttctgctcaggacattacttactccggtactttcactccatccggtaacggttacttgtccgtttacggttggactactgacccactgatcgagtactacatcgttgaatcctacggtgactacaaccctggttctggtggtacttacaagggtactgttacttccgacggttccgtctacgatatctacactgctactagaactaacgccgcttccattcaaggtactgctactttcacccaatactggtccgtcagacagaacaagagagttggaggtactgtcaccacttccaaccactttaacgcttgggctaagctgggtatgaacttgggtactcacaactaccagatcgttgctaccgaaggttaccaatcttctggttcctcctccattactgttcaa。

[0067] Using the synthesized gene sequence as a template, the laccase gene fragment or the laccase and xylanase linked by different linkers was amplified by PCR Solac gene fragments or laccase linked with different linkers (linker) [[ID=*11]] Solac and xylanase AnxynB Note: There seems to be an issue with line break preservation in the provided translation rules as the original text has 13 lines and the translated text has 14 lines. The adjustment in the translation of line 8 was made to try to fit the content better while still following the rules as closely as possible. If strict line break preservation is required, further adjustments might be needed in the translation process.Gene fragment. PCR primers used: Solac-F (SEQ ID No: 6), Solac-R (SEQ ID No: 7); AnxynB-F (SEQ ID No: 8), AnxynB-R (SEQ ID No: 9); L0-Solac-F (SEQ ID No: 10); L1-Solac-F (SEQ ID No: 11); L2-Solac-F (SEQ ID No: 12); L3-Solac-F (SEQ ID No: 13); L4-Solac-F (SEQ ID No: 14); L5-Solac-F (SEQ ID No: 15); L6-Solac-F (SEQ ID No: 16); L7-Solac-F (SEQ ID No: 17); L8-Solac-F (SEQ ID No: 18); L9-Solac-F (SEQ ID No: 19).

[0068] Linking peptide sequence used:

[0069] Linker peptide 1: GGGGS (SEQ ID No: 20), nucleotide sequence (SEQ ID No: 21);

[0070] Linker peptide 2: SASSGGTTPTTTHM (SEQ ID No: 3) nucleotide sequence (SEQ ID No: 22);

[0071] Linker peptide 3: RSGLEKREAEA (SEQ ID No: 23), nucleotide sequence (SEQ ID No: 24);

[0072] Linker peptide 4: GGGGSGGGGSGGGGS (SEQ ID No: 25), nucleotide sequence (SEQ ID No: 26);

[0073] Linker peptide 5: GGGGGGGG (SEQ ID No: 27), nucleotide sequence (SEQ ID No: 28);

[0074] Linker peptide 6: GGGGSKEEEKGGGGS (SEQ ID No: 29), nucleotide sequence (SEQ ID No: 30);

[0075] Linker peptide 7: EPKSCDKTHTCPPCP (SEQ ID No: 31), nucleotide sequence (SEQ ID No: 32).

[0076] Linker peptide 8: EPKSSDKTHTSPPSP (SEQ ID No: 33), nucleotide sequence (SEQ ID No: 34);

[0077] Linker peptide 9: GGGSLEKREAEA (SEQ ID No: 35), nucleotide sequence (SEQ ID No: 36).

[0078] Specifically, Solac-F and Solac-R are used to amplify the Solac fragment, AnxynB-F and AnxynB-R are used to amplify the AnxynB fragment, L0-Solac-F and Solac-R are used to amplify the linker0-Solac fragment, L1-Solac-F and Solac-R are used to amplify the linker1-Solac fragment, L2-Solac-F and Solac-R are used to amplify the linker2-Solac fragment, L3-Solac-F and Solac-R are used to amplify the linker3-Solac fragment, and L4-Solac... L-F and Solac-R are used to amplify the linker4-Solac fragment, L5-Solac-F and Solac-R are used to amplify the linker5-Solac fragment, L6-Solac-F and Solac-R are used to amplify the linker6-Solac fragment, L7-Solac-F and Solac-R are used to amplify the linker7-Solac fragment, L8-Solac-F and Solac-R are used to amplify the linker8-Solac fragment, and L9-Solac-F and Solac-R are used to amplify the linker9-Solac fragment.

[0079] After amplification, the PCR products were detected by nucleic acid electrophoresis. The band sizes of the Solac fragment, Anxynb fragment, linker0-Solac fragment, linker1-Solac fragment, linker2-Solac fragment, linker3-Solac fragment, linker4-Solac fragment, linker5-Solac fragment, linker6-Solac fragment, linker7-Solac fragment, linker8-Solac fragment, and linker9-Solac fragment were 1556 bp, 648 bp, 1555 bp, 1566 bp, 1596 bp, 1587 bp, 1599 bp, 1578 bp, 1599 bp, 1599 bp, 1599 bp, and 1590 bp, respectively. After purifying the linker-Solac fragments and the xylanase Anxynb fragment, overlap PCR was performed to amplify the fusion protein AnxynB and the coding sequences of each linker-Solac fragment, using primers AnxynB-F and Solac-R. The amplified laccase... So Lac protein-coding sequence fragments and fusion protein-coding sequence fragments (AnxynB-Solac, AnxynB-linker1-Solac, AnxynB-linker2-Solac, AnxynB-linker3-Solac, AnxynB-linker4-Solac, AnxynB-linker5-Solac, AnxynB-linker6-Solac, AnxynB-linker7-Solac, AnxynB-linker8-Solac, and AnxynB-linker9-Solac) were respectively combined with a recombinant kit and EcoR I / Not The pPICZα(A) vector, which was double-digested with enzyme I, was ligated. The recombinant product was transformed into *E. coli* TransI cloning host and plated on LLB agar plates containing 25 mg / L bleomycin. Single clones were picked and sequenced to verify the correct laccase construction. So The recombinant plasmid pPICZα(A)-Solac, directly expressed by Lac, and various laccases SoLac fusion expression recombinant plasmids pPICZα(A)-AnxynB-Solac, pPICZα(A)-AnxynB-linker1-Solac, pPICZα(A)-AnxynB-linker2-Solac, pPICZα(A)-AnxynB-linker3-Solac, pPICZα(A)-AnxynB-linker4-Sola c, pPICZα(A)-AnxynB-linker5-Solac, pPICZα(A)-AnxynB-linker6-Solac, pPICZα(A)-AnxynB-linker7-Solac, pPICZα(A)-AnxynB-linker8-Solac and pPICZα(A)-AnxynB-linker9-Solac.

[0080] Laccase So Lac protein expression alone and laccase So The vector maps for Lac fusion protein expression are as follows: Figure 1 and Figure 2 As shown.

[0081] Example 2: Construction and screening of Pichia pastoris engineered strains expressing laccase SoLac alone and in fusion.

[0082] 1. Expression of laccase So Construction of engineered Pichia pastoris strains of Lac

[0083] Laccase So The vector pPICZα(A)-Solac for expressing Lac protein alone and laccases containing different linker peptides So Lac fusion expression vector utilization Dra Linearization was performed using enzyme digestion. The digested products were recovered using a PCR product purification kit. The recovered linearized plasmid was electroporated into Pichia pastoris X33 competent cells and plated on bleomycin-resistant (100 mg / L) YPD medium plates to obtain laccase. So Lac-directly expressed transformants of Pichia pastoris X33 / Solac and various laccases SoThe Lac fusion expression transformants are Pichia pastoris X33 / AnxynB-Solac, Pichia pastoris X33 / AnxynB-linker1-Solac, Pichia pastoris X33 / AnxynB-linker2-Solac, Pichia pastoris X33 / AnxynB-linker3-Solac, Pichia pastoris X33 / AnxynB-linker4-Solac, Pichia pastoris X33 / AnxynB-linker5-Solac, Pichia pastoris X33 / AnxynB-linker6-Solac, Pichia pastoris X33 / AnxynB-linker7-Solac, X33 / AnxynB-linker8-Solac, and X33 / AnxynB-linker9-Solac.

[0084] 2. Laccase So Screening of recombinant Pichia pastoris strains that successfully expressed Lac

[0085] Single colonies were picked from YPD agar plates containing bleomycin and inoculated into 10 mL tubes containing 3 mL BMGY medium. The tubes were incubated at 30°C and 220 rpm for 48 h. After centrifugation at 4500 rpm, the supernatant was discarded, and 1.5 mL of BMMY medium containing 1% methanol and 1 mM CuSO4 was added. The mixture was then induced at 30°C and 220 rpm for 48 h. Methanol was added every 24 h to reach a final concentration of 1%. After fermentation, the supernatant was collected by centrifugation and then inoculated with ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid) (ε420=36,000 M). -1 ·cm -1 Laccase activity was determined using 50 mM acetate-sodium acetate buffer (pH 4.8) with 1 mM ABTS as the substrate. The assay was incubated at 30 °C for 3 min, and absorbance was measured at 420 nm. Under standard assay conditions, the amount of enzyme producing 1 μM of product per minute was defined as 1 unit (1 U) of laccase activity.

[0086] Expressing laccase in different forms So The highest laccase activity in the supernatant of the microtube fermentation broth of the Lac clone was as follows: Figure 3 As shown. Directly perform laccase processing. So No laccase activity was detected in the supernatant of the fermentation broth of the host strain expressing Lac, indicating that laccase... So Lac could not be directly heterologously expressed in Pichia pastoris X33. Lacase activity was detected in the supernatant of fermentation broth from Pichia pastoris strains expressing the complete xylanase-laccase fusion protein, and xylanase activity was also detected.An XynB and laccase So The linker peptide sequence between Lac proteins significantly affects laccase activity. When no linker peptide exists between laccase and xylanase, the enzyme activity in the supernatant of the tube fermentation broth is 7.2 U / L. When linker peptides with different sequences are added between the fusion proteins, the linker peptide in the fusion protein expressed by the *Pichia pastoris* strain with the highest supernatant enzyme activity in the tube fermentation broth is linker peptide 2 (SASSGGTTPTTTHM). This linker peptide is the natural sequence between the catalytic domain and the carbohydrate-binding module (CBM) of cellulase derived from *Bacillus subtilis* 41M-1. The highest enzyme activity in the supernatant of the tube fermentation broth of this strain (*Pichia pastoris* X33 / AnxynB-linker2-Solac) reached 94 U / L.

[0087] Example 3: Horizontal fermentation and protein purification of laccase SoLac fusion protein in a 15 L fermenter

[0088] 1. Pichia pastoris X33 / AnxynB-linker2-Solac fermentation in a top-tank.

[0089] Laccase processing was carried out using a 15 L fermenter on Pichia pastoris X33 / AnxynB-linker2-Solac. So Fermentation production of Lac fusion protein. Pichia pastoris X33 strain, frozen at -80℃ for recombinant protein production, was inoculated into 50 mL of YPD medium and cultured at 30℃ and 220 rpm for 48 h. The primary seed culture was then transferred to three 200 mL bottles of BMGY medium and cultured overnight at 30℃ and 220 rpm. 7.5 L of fermentation medium was prepared and sterilized by steam at 121℃ for 30 min. After the temperature dropped to 30℃, the secondary seed culture was inoculated into the fermenter. The strain inoculated into the fermenter was cultured for 18-22 h at 800 rpm, dissolved oxygen controlled at 20-40%, and temperature 30℃. The pH was stabilized at approximately 4.5 using ammonia. After the glycerol used as a carbon source in the previous steps was depleted, 50% glycerol containing 12 mL / L PTM1 solution was added at a rate of 18 mL / h / L, and the culture was continued for 3-6 h. When the cell wet weight reached 160-180 g / L, methanol containing 12 mL / L PTM1 solution was added at a rate of 3.2 mL / h / L, maintaining the pH at approximately 5.5. 10 mL of 0.5 M CuSO4 was added every 24 h. Laccase and xylanase activities and yeast cell wet weight were measured every 24 h. During the induction of recombinant protein expression, laccase, xylanase activities, and cell wet weight generally showed an increasing trend.

[0090] like Figure 4 , 5As shown in Figure 6, after 6 days of induction, the laccase and xylanase activities in the fermentation broth were 5947.8 U / L and 92700.0 U / L, respectively. Subsequently, on day 9, they reached their peak values ​​of 9102.0 U / L and 150300.0 U / L, respectively. After 8 days of induction, the cell wet weight reached its maximum value (442.7 g / L).

[0091] Furthermore, the experimental results also showed a significant linear relationship (R0) between the enzyme activities of xylanase and laccase in the fermentation broth. 2 = 0.9792)( Figure 7 This indicates that xylanase An XynB and laccase So Lac was expressed heterogeneously in a fusion manner.

[0092] 2. Laccase So Purification of Lac fusion protein

[0093] After fermentation is complete, the fermentation broth is collected and sterilized and concentrated using hollow fiber optics. Laccase is then added. So The Lac fusion protein was displaced into 20 mM acetate-sodium acetate buffer (pH 5.5). Purification of the fusion protein was performed using a GE Healthcare HiTrap Q HP anion exchange column. Solution A was 20 mM acetate-sodium acetate buffer (pH 5.5), and solution B was solution A with the addition of 1 M NaCl. A linear gradient elution was performed using NaCl solutions with concentrations of 0–1 M, and each eluted fraction was collected for SDS-PAGE analysis. The purified fractions were combined and displaced into protein storage buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5). The purification results are shown below. Figure 8 As shown, the obtained protein was detected by SDS-PAGE, and the band was approximately 80.0 kDa, which is close to the theoretical molecular weight of the fusion protein (77.7 kDa). The target band was excised from the SDS-PAGE gel and identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The equipment used was an EASY-nLC 1000 micro-liquid chromatography system coupled with a Q-Exactive mass spectrometer. MS / MS data were compared with the expected protein sequence using PEAKS Studio software. The results showed that 18 peptide sequences matched the target protein sequence, and these sequences covered the fusion protein. An XynB- So 51.5% of Lac ( Figure 9 ).

[0094] Example 4 Determination of the enzymatic properties of the fusion-expressed recombinant laccase SoLac

[0095] 1. Fusion-expressed recombinant laccase So Determination of optimal pH and optimal temperature for Lac

[0096] At 30℃, ABTS and syringaldehyde-2-azine (SGZ, ε530=65,000 M) were used in the system with a final concentration of 1 mM. -1 ·cm -1 ) and 2,6-dimethoxyphenol (2,6-DMP, ε470=49,600 M) -1 ·cm -1 Using laccase as a substrate, the recombinant laccase expressed by fusion was measured. So The optimal pH for Lac was determined. The pH range was 2.0–12.0, using 100 mM glycine-HCl buffer (pH 2.0), 100 mM Na₂HPO₄-citric acid buffer (pH 3.0–7.0), 100 mM Tris-HCl buffer (pH 7.0–9.0), and 100 mM glycine-NaOH buffer (pH 9.0–12.0). Results are as follows: Figure 10 As shown, when ABTS is used as a substrate, the recombinant laccase expressed by fusion... So The optimal pH for Lac is 4.0. When SGZ and 2,6-DMP are used as substrates, the recombinant laccase expressed via fusion... So The optimal pH for all Lac was 5.0. Using ABTS at a final concentration of 1 mM as a substrate, the recombinant laccase expressed by fusion was measured. So The optimal temperature for Lac. The measurement temperature range was 30-90℃. Results are as follows: Figure 11 As shown, when ABTS is used as a substrate, laccase So The optimal temperature for Lac is 70℃, and it exhibits high activity in the temperature range of 50℃ to 80℃.

[0097] 2. Fusion-expressed recombinant laccase So Determination of pH and temperature stability of Lac

[0098] At 30°C, appropriately diluted laccase So After treating the Lac fusion protein in a buffer solution with pH 2.0 to 12.0 for 1 h, the remaining laccase activity was determined using ABTS as a substrate. Figure 12 As shown, laccase So Lac was most stable at pH 9.0. After 1 h of treatment, its residual enzyme activity was 95.6%. Within the pH range of 5.0 to 10.0, its residual enzyme activity remained above 69.1%, while at pH 4.0 and 11.0, it decreased to 44.0% and 36.6% of the untreated protein, respectively. This indicates that laccase... SoLac exhibits good stability under both weakly acidic and alkaline conditions.

[0099] Laccase So The Lac fusion protein was appropriately diluted with 100 mM Tris-HCl buffer (pH 9.0) and then incubated within the range of 40 to 70 °C. Samples were taken at 5 min, 10 min, 20 min, and 30 min after treatment, and the residual enzyme activity was determined using ABTS as a substrate. The results showed that the fusion-expressed recombinant laccase exhibited good thermostability at 40 °C and 50 °C. Figure 13 As shown, after incubation at 40℃ and 50℃ for 5 min, the enzyme activities were 97.0% and 94.5% of the initial enzyme activities, respectively. After incubation for 30 min, the laccase... So Lac retained 89.2% and 88.6% of its enzyme activity. However, when the temperature exceeded 50℃, the enzyme activity decreased significantly. After incubation at 60℃ and 70℃ for 5 min, the relative enzyme activities were 83.6% and 38.1% of the initial enzyme activity, respectively. After incubation for 20 min, the relative enzyme activities decreased to 70.5% and 2.6% of the initial enzyme activity, respectively.

[0100] Example 5: Laccase SoLac fusion protein's degradation of aflatoxin B1 independent of mediators.

[0101] 1. Laccase So The optimal temperature for the direct degradation of aflatoxin B1 by the Lac fusion protein

[0102] Aflatoxin B1 was dissolved using dimethyl sulfoxide (DMSO) to prepare a 100 mg / L stock solution of aflatoxin B1. The following reaction system was prepared: 20 μL of the aflatoxin B1 stock solution and 20 μL of enzyme solution (laccase concentration 6000 U / L). The reaction was carried out in 100 mM Tris-HCl buffer (pH 7.0) at 20℃–50℃ for 10 h. Each reaction system was repeated in triplicate, with a reaction system without laccase fusion protein used as a negative control. The reaction was terminated by adding three volumes of methanol after completion. The concentration of aflatoxin B1 and its degradation rate were analyzed using a Shimadzu Nexera UHPLC system. The chromatographic column was a Zorbax SB-C18 (4.6 × 250 mm, 5 μm), and the mobile phases were A (0.06% TFA in water) and B (0.05% TFA in acetonitrile). Gradient elution conditions: 0% B elution for 4 min, 0%-100% B elution for 15 min, and 100% B elution for 10 min. The samples were detected using a UV detector at a wavelength of 365 nm.

[0103] like Figure 14As shown, laccase at 50℃ So Lac exhibited the highest degradation rate for aflatoxin B1, at 38.9%. However, when the temperature exceeded 60°C, the degradation rate of aflatoxin B1 decreased significantly to 5.6%.

[0104] 2. Laccase So The optimal pH for Lac fusion protein to degrade aflatoxin B1

[0105] The following reaction system was prepared: 20 μL of aflatoxin B1 stock solution, 20 μL of enzyme solution (laccase concentration in the system was 6000 U / L), and 160 μL of buffer solutions at different pH values. The buffer solutions used were 100 mM glycine-HCl buffer solution (pH 2.0), 100 mM Na2HPO4-citric acid buffer solution (pH 3.0-6.0), and 100 mM Tris-HCl buffer solution (pH 7.0). Each reaction system was repeated in triplicate, with a reaction system without laccase as a negative control. The reaction was carried out at 50 °C, and after 10 h, three volumes of methanol were added to terminate the reaction. The degradation rate of aflatoxin B1 was analyzed by high-performance liquid chromatography (HPLC).

[0106] The results are as follows Figure 15 As shown, the degradation rate of aflatoxin B1 increased in the pH range of 2.0-7.0, reaching a peak at pH 7.0 with a conversion rate of 32.0%.

[0107] 3. Laccase So Effects of Lac fusion protein dosage and reaction time on the degradation of aflatoxin B1

[0108] The following reaction systems were prepared: 20 μL of aflatoxin B1 stock solution, 20 μL of enzyme solution (laccase concentrations of 2000 U / L, 4000 U / L, 6000 U / L, 8000 U / L, and 10000 U / L, respectively), and 160 μL of 100 mM Tris-HCl buffer solution (pH 7.0). Each reaction system was repeated in triplicate, with a reaction system without laccase serving as a negative control. The reaction was carried out at 50 °C. The reaction was terminated by adding three volumes of methanol at 0 h, 3 h, 6 h, 9 h, 12 h, and 20 h, respectively. The degradation rate of aflatoxin B1 was analyzed by high-performance liquid chromatography (HPLC). The results are shown below. Figure 16 As shown, the conversion rate of aflatoxin B1 increased with increasing reaction time. After 20 hours of incubation, when laccase... So When the concentration of Lac increased from 2000 U / L to 10000 U / L, the conversion rate of aflatoxin B1 gradually increased from 20.3% to 71.0%.

[0109] 4. Laccase So The detoxification effect of Lac fusion protein on aflatoxin B1

[0110] The cytotoxicity of the human hepatocellular carcinoma line HepG2 against aflatoxin B1 and its degradation products was evaluated. HepG2 cells were cultured at 37°C in a 5% CO2 incubator. The culture medium was DMEM containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / L streptomycin. Cells were sputtered at 5 × 10⁶ cells / mL. 4 Cells were seeded at a concentration of [number] cells / well in 96-well plates. After 24 h of culture, different final concentrations (0, 1, 2, 5, 10, 20, 30, 40, 50, 60 mg / L) of aflatoxin B1 or a system of aflatoxin B1 degraded by laccase were added to the culture medium, and the plates were cultured for another 48 h. The degradation system consisted of 100 mM Tris-HCl buffer (pH 7.0) containing 100 mg / L aflatoxin B1 and 8000 U / L laccase, and the reaction was carried out at 50 °C for 24 h. The culture medium was then discarded, and the cells were washed with phosphate-buffered saline (PBSA). Cell Counting Kit-8 (CCK-8) reagent was diluted with DMEM medium and added to each well of the 96-well plate at a concentration of 100 μL, and incubated at 37 °C for 2 h. After incubation, the absorbance of each sample in each well was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0111] like Figure 17 As shown, the viability of HepG2 cells decreased with increasing aflatoxin B1 concentration (0-60 mg / L). Treatment with 10 mg / L aflatoxin B1 resulted in a 40.2% decrease in cell viability compared to the untreated control group. Meanwhile, the fusion protein had almost no effect on cell viability. In contrast, treatment with laccase... So In the reaction system treated with aflatoxin B1 using the Lac fusion protein, the survival rate of HepG2 cells increased to 92.9%. These findings indicate that the use of laccase... So Treatment with Lac fusion protein significantly reduced the toxicity of aflatoxin B1. Figure 18 ).

[0112] Example 6 Degradation of zearalenone by laccase SoLac fusion protein

[0113] 1. Laccase So The optimal temperature for direct degradation of zearalenone by Lac fusion protein

[0114] Zearalenone was dissolved using dimethyl sulfoxide (DMSO) to prepare a 100 mg / L zearalenone stock solution. The following reaction system was prepared: 20 μL zearalenone stock solution and 20 μL enzyme solution (laccase concentration 6000 U / L). The reaction was carried out in 100 mM Tris-HCl buffer (pH 7.0) at 20℃–50℃ for 10 h. Each reaction system was repeated in triplicate, with a reaction system without laccase fusion protein used as a negative control. The reaction was terminated by adding three volumes of methanol. The concentration of zearalenone and the substrate degradation rate were analyzed using a Shimadzu Nexera UHPLC system. The chromatographic column was a Zorbax SB-C18 (4.6 × 250 mm, 5 μm). The mobile phase used was acetonitrile:H₂O:methanol = 46:46:8, and the flow rate was 0.8 mL / min. Zearalenone was detected using an RF-20A fluorescence detector with excitation and emission wavelengths of 274 nm and 440 nm, respectively.

[0115] like Figure 19 As shown, the degradation rate of zearalenone was highest at 50°C, reaching 43.0%. When the temperature rose to 60°C, the degradation rate of zearalenone decreased to 4.7%.

[0116] 2. Laccase So The optimal pH for Lac fusion protein to degrade zearalenone

[0117] The following reaction system was prepared: 20 μL of zearalenone stock solution, 20 μL of enzyme solution (laccase concentration in the system was 6000 U / L), and 160 μL of buffer solutions at different pH values. The buffer solutions used were 100 mM glycine-HCl buffer solution (pH 2.0), 100 mM Na₂HPO₄-citric acid buffer solution (pH 3.0-6.0), and 100 mM Tris-HCl buffer solution (pH 7.0-8.0). Each reaction system was repeated in triplicate, with the reaction system without laccase serving as a negative control. The reaction was carried out at 50 °C, and after 10 h, three volumes of methanol were added to terminate the reaction. The degradation rate of zearalenone was analyzed by high-performance liquid chromatography (HPLC).

[0118] The results are as follows Figure 20 As shown, the degradation rate of zearalenone showed an increasing trend in the pH range of 2.0-7.0 and reached a peak at pH 7.0, at which the degradation rate of zearalenone reached 61.1%.

[0119] 3. Laccase So Effects of Lac fusion protein dosage and reaction time on zearalenone

[0120] The following reaction system was prepared: 20 μL of zearalenone stock solution, 20 μL of enzyme solution (laccase concentrations of 500 U / L, 1500 U / L, 2000 U / L, 4000 U / L, and 6000 U / L, respectively), and 160 μL of 100 mM Tris-HCl buffer solution (pH 7.0). Each reaction system was repeated in triplicate, with a reaction system without laccase serving as a negative control. The reaction was carried out at 50 °C. The reaction was terminated by adding three volumes of methanol at 0 h, 3 h, 6 h, 9 h, 12 h, and 20 h, respectively. The degradation rate of zearalenone was analyzed by high-performance liquid chromatography (HPLC). The results are shown below. Figure 21 As shown, the degradation rate of zearalenone in zearalenone increased with increasing reaction time. After 20 hours of incubation, when laccase... So When the concentration of Lac increased from 500 U / L to 4000 U / L, the degradation rate of zearalenone gradually increased from 20.3% to 71.0%. When the enzyme concentration was further increased to 6000 U / L, the conversion rate of zearalenone did not show a significant increase (it was 72.9%).

[0121] 4. Laccase So Detoxification effect of Lac fusion protein on zearalenone

[0122] The cytotoxicity of the human hepatocellular carcinoma line HepG2 against zearalenone and its degradation products was evaluated. HepG2 cells were cultured at 37°C in a 5% CO2 incubator. The culture medium was DMEM containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / L streptomycin. Cells were sputtered at 5 × 10⁶ cells / mL. 4 Cells were seeded at a concentration of [number] cells / well in 96-well plates. After 24 h of culture, different final concentrations (0, 1, 2, 5, 10, 20, 30, 40, 50, 60 mg / L) of zearalenone or a system of zearalenone degraded by laccase were added to the culture medium, and the plates were cultured for another 48 h. The degradation system consisted of 100 mM Tris-HCl buffer solution containing 100 mg / L zearalenone and 8000 U / L laccase, and the reaction was carried out at 50 °C for 24 h. The culture medium was then discarded, and the cells were washed with phosphate-buffered saline (PBRS). Cell Counting Kit-8 (CCK-8) reagent was diluted with DMEM medium and added to each well of the 96-well plate at a concentration of 100 μL, and incubated at 37 °C for 2 h. After incubation, the absorbance of each sample in each well was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0123] like Figure 22As shown, the viability of HepG2 cells decreased with increasing zearalenone concentration (0-60 mg / L). Treatment with 10 mg / L zearalenone resulted in a 25.0% decrease in cell viability compared to the untreated control group. Meanwhile, the fusion protein had almost no effect on cell viability. In contrast, treatment with laccase... So After treating HepG2 cells with the Lac fusion protein-treated zearalenone reaction system, the cell viability increased to 93.9%. These findings indicate that using laccase... So Treatment with Lac fusion protein significantly reduced the toxicity of zearalenone. Figure 23 ).

[0124] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. A laccase fusion protein, characterized in that, The laccase fusion protein consists of xylanase, a linker peptide, and laccase from the N-terminus to the C-terminus, wherein the amino acid sequence of the laccase is shown in SEQ ID No:1, the amino acid sequence of the xylanase is shown in SEQ ID No:2, and the amino acid sequence of the linker peptide is shown in SEQ ID No:

3.

2. The coding sequence of the laccase fusion protein of claim 1.

3. A recombinant expression vector comprising the coding sequence of claim 2.

4. A recombinant strain comprising the coding sequence of claim 2.

5. The recombinant strain according to claim 4, characterized in that, The recombinant strain is a recombinant yeast strain.

6. A method for preparing laccase by fermentation, characterized in that, The method includes the following steps: A recombinant expression vector containing the coding sequence of a laccase fusion protein was constructed, wherein the laccase fusion protein consists of xylanase, a linker peptide, and laccase from the N-terminus to the C-terminus, wherein the amino acid sequence of the laccase is shown in SEQ ID No:1, the amino acid sequence of the xylanase is shown in SEQ ID No:2, and the amino acid sequence of the linker peptide is shown in SEQ ID No:

3. The recombinant expression vector was transformed into host cells to obtain recombinant bacterial strains; The recombinant strain was fermented to obtain laccase.

7. The method for preparing laccase by fermentation according to claim 6, characterized in that, The recombinant strain is a recombinant yeast strain.

8. The method for preparing laccase by fermentation according to claim 6, characterized in that, The fermentation culture of the recombinant strain includes the following steps: Cultivating primary seed culture: The recombinant strain used for recombinant protein production is inoculated into YPD medium and cultured. Secondary seed culture: The primary seed culture is transferred to BMGY medium for cultivation; The secondary seed culture was inoculated into the fermentation medium in the fermenter; Cell culture: Continue to culture the strain inoculated in the fermenter for 18-22 h, with dissolved oxygen controlled at 20-40%, rotation speed at 800 rpm, temperature at 30℃, and pH value stabilized at 4.5; Glycerol feeding: After the carbon source in the solid culture step is exhausted, add 50% glycerol containing 12 mL / L PTM1 solution at a rate of 18 mL / h / L and continue culturing for 3-6 h; Methanol induction: When the cell wet weight reaches 160-180 g / L, methanol containing 12 mL / L PTM1 solution is added at a rate of 3.2 mL / h / L while controlling the pH value at around 5.

5. 10 mL of 0.5 M CuSO4 is added every 24 h. Enzyme activity detection.

9. The method for preparing laccase by fermentation according to claim 6, characterized in that, The fermentation medium is formulated as follows: glycerol 40 g / L, KH2PO4 5 g / L, CaSO4 0.93 g / L, K2SO4 10 g / L, MgSO4 10 g / L, NH4H2PO4 40 g / L, KOH 1.5 g / L; The PTM1 solution has the following composition: copper sulfate 6.0 g / L, potassium iodide 0.09 g / L, manganese sulfate 3.0 g / L, sodium molybdate 0.2 g / L, boric acid 0.02 g / L, cobalt chloride 0.5 g / L, zinc chloride 20 g / L, ferrous sulfate 65 g / L, and sulfuric acid 5.0 ml / L.

10. The use of the laccase fusion protein of claim 1 in the detoxification of fungal toxins.

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