Laccase fusion protein, fermentation preparation method thereof and application of laccase fusion protein in mycotoxin detoxification

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to laccase fusion protein, a fermentation preparation method and application of the laccase fusion protein to detoxification of fungaltoxin. The synthetic protein is composed of laccase SoLac from ochrachium ochraceum, a connecting peptide and xylanase AnXynB from aspergillus niger. According to the invention, a fusion protein expression vector of xylanase AnXynB and laccase SoLac is constructed and transferred into pichia pastoris for heterologous expression of the fusion protein, so that the expression quantity of laccase SoLac is greatly increased. The recombinant laccase SoLac and xylanase AnXynB fusion protein obtained by the invention can realize effective detoxification of mycotoxins such as aflatoxin B1, zearalenone and the like, and can be widely applied to the field of mycotoxin detoxification of foods and feeds.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and in particular to a laccase fusion protein and a fermentation preparation method thereof, and application thereof in the detoxification of fungal toxins. Background Art

[0002] Mycotoxins are secondary metabolites produced by fungi and pose a serious threat to food and feed safety. Aflatoxin B1 is the most common and dangerous type. It is highly stable and can only be decomposed at temperatures above 260°C. The structure of zearalenone is dihydroxybenzoic acid phenol lactone. The molecular structure of zearalenone and its metabolites is similar to that of the endogenous estrogen 17-estradiol. Therefore, they can bind to estrogen receptors, thereby inhibiting estrogen binding to receptors in tissues and affecting the function of the reproductive system. Mycotoxin contamination in food, feed, and feed ingredients is mostly a combination of several toxins, and most discovered enzymes are only capable of degrading specific types of mycotoxins. Therefore, broad-spectrum mycotoxin-degrading enzymes have greater application potential.

[0003] Laccase (EC 1.10.3.2) is a polyphenol oxidase containing copper ions. It is widely distributed in plants, fungi, a few insects and bacteria, and can oxidize a variety of aromatic compounds. Among them, basidiomycete fungal laccases have a dominant position in industrial applications due to their advantages such as a broad substrate spectrum, high enzyme activity, and strong redox potential. However, the laccase yield of natural strains is usually low, which is difficult to meet the needs of industrialization. Therefore, achieving low-cost and efficient production has become the key. At present, heterologous expression systems are considered to be ideal solutions for large-scale production of laccases due to their high cost-effectiveness and environmental friendliness. Among the many hosts, Pichia pastoris ( Pichia pastoris ) has become a preferred platform for food-grade laccase production due to its high cell density culture characteristics and efficient protein secretion capacity. However, the active expression of fungal laccases in Pichia pastoris remains challenging, limiting its industrial application.

[0004] In addition, the ability of laccase to transform mycotoxins such as aflatoxin B1 and zearalenone is highly dependent on its isoform characteristics. Pleurotus eryngii Laccases require mediators such as 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) to degrade toxins, which not only increases application costs but also poses food safety risks due to residual mediators. Therefore, identifying basidiomycete laccases that can efficiently degrade toxins without mediators and expressing them in Pichia pastoris are essential steps to promote the application of fungal laccases in food and feed detoxification.

[0005] Xylanase, a common feed additive, can break down xylan, a component of plant cell walls in feed, regulate intestinal microbial communities, and improve feed conversion efficiency. Due to its high expression in Pichia pastoris, the fusion of xylanase with laccase has the potential to increase laccase production. Furthermore, this fusion protein can perform the dual functions of eliminating mycotoxins in feed and promoting feed digestion.

[0006] In order to obtain a fusion protein of xylanase and fungal laccase with excellent enzymatic properties, the selection of xylanase is important. Aspergillus niger ) can adapt to a wide range of pH and temperature, has good stability under different pH conditions and high catalytic activity ( An However, the direct fusion of two catalytic domains of xylanase and fungal laccase may lead to protein misfolding and affect enzyme activity. Summary of the Invention

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

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

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

[0010] Another object of the present invention is to provide a recombinant strain containing the gene encoding the 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 use of the above fusion protein in the detoxification of fungal toxins.

[0013] According to the present invention, the laccase fusion protein is composed of xylanase An XynB, connecting peptide and laccase So Lac composition. So Lac is Ochroodon ochratoides ( Steccherinum ochraceum ) source, and its amino acid sequence is shown in SEQ ID No: 1. The xylanase An XynB is Aspergillus niger ( Aspergillus niger ) source, the amino acid sequence of which is shown in SEQ ID No: 2, and the amino acid sequence of the connecting peptide is shown in SEQ ID No: 3.

[0014] SEQ ID No: 1 AIGPIADLHINNSNISPDGFTRPAVLAGGTFPGPLIQGNKGDNFQINVIDELTDENQLKSTSIHWHGLFQHGTNWADGAAFVTQCPIATGHSFLYNFDVPDQAGTYWYHSHLSTQYCDGLRGPFV VYDPDDVHQQLYDIDNEDTVITLADWYHILARQEPPGPPVPDSTLINGFGRFPGQTTPSDLAVITVEQGKRYRLRLVNIACDPNYQFSIDNHNLTVIEVDGVSTQALTVTSLTIFAGQRYSVILHA NQDNKGDQGNYWIRAKPNTGADTSFNGGLNSAILRYVGANPVDPETTMGMDNAPLNEVNLRPFISTPVPGQPHAGGADFVKNLAFTFAAGLFAVDGTPFVPPTVPVLLQILSGAQTAQDLLPTGS IIELPPNKVIEFSMPGGVVGGGHPIHLHGHTFWVVRSAGSSTYNYNDAILRDVVNIGVAGDNVTIRFVTDNPGPWFLHCHIDWHLDTGFAVVMAEDIPGTAAANPVPADWSQLCPLYDALPPEDL; The enzyme consists of 502 amino acids and has a theoretical molecular weight of 54.2 kDa.

[0015] SEQ ID No: 2: VPHDSVAQRSDALHMLSERSTPSSTGENNGFYYSFWTDGGGDVTYTNGDAGAYTVEWSNVGNFVGGKGWNPGSAQDITYSGTFTPSGNGYLSVYGWTTDPLIEY YIVESYGDYNPGSGGTYKGTVTSDGSVYDIYTATRTNAASIQGTATFTQYWSVRQNKRVGGTVTTSNHFNAWAKLGMNLGTHNYQIVATEGYQSSGSSSITVQ, The enzyme consists of 207 amino acids and has a theoretical molecular weight of 22.2 kDa.

[0016] SEQ ID No: 3: SASSGGTTPTTTHM, Among them, the connecting peptide includes 14 amino acids and has a theoretical molecular weight of 1.3 kDa.

[0017] The present invention provides a coding sequence encoding the above fusion protein.

[0018] The present invention also provides a method comprising the above-mentioned xylanase An XynB, connecting peptide and laccase So Recombinant vector containing Lac coding sequence.

[0019] The present invention also provides a method for preparing laccase by fermentation, comprising the following steps: (1) using a gene encoding laccase So The recombinant expression vector of the Lac fusion protein gene is transformed into a host cell to obtain a recombinant strain; (2) Cultivate the recombinant strain in a fermenter and use methanol to induce laccase So Lac fusion protein expression; (3) Purification of laccase So Lac fusion protein.

[0020] 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 biomass energy, food industry, feed industry and other fields.

[0021] Beneficial effects of the present invention: According to the technical solution of the present application, a connecting peptide is introduced between xylanase and fungal laccase. However, connecting peptides of different sequences can significantly affect the activity of the fusion protein by affecting the steric hindrance and conformational freedom, domain orientation, substrate accessibility and stability of the protein. Therefore, the present application screened for a suitable connecting peptide sequence to obtain a fusion protein with high laccase activity.

[0022] The present invention uses xylanase derived from Aspergillus niger An XynB and laccase So Lac was fused and expressed in Pichia pastoris, and the heterologous expression of fungal laccase with high redox potential in Pichia pastoris was successfully achieved, which greatly improved the laccase So At the same time, this fusion expression strategy does not affect the expression of laccase So Lac mycotoxin-degrading activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Picture 1 Laccase So Map of the Lac protein expression plasmid pPICZα(A)-Solac; Picture 2 Laccase So Map of the expression plasmid pPICZα(A)-AnxynB-linker-Solac for Lac fusion protein; Picture 3 Demonstrating the expression of laccase using different linker peptides So Lacase activity results when Lac fusion protein was used; Picture 4 The synthesis of laccase by Pichia pastoris X33 / AnxynB-linker2-Solac strain was demonstrated So The results of the changes of laccase activity with methanol induction time when Lac fusion protein was used; Picture 5 The synthesis of laccase by Pichia pastoris X33 / AnxynB-linker2-Solac strain was demonstrated So The results of the changes of xylanase activity with methanol induction time when Lac fusion protein was used; Picture 6 The synthesis of laccase by Pichia pastoris X33 / AnxynB-linker2-Solac strain was demonstrated So The results of the changes of cell wet weight with methanol induction time when Lac fusion protein was used; Picture 7 The linear relationship between laccase and xylanase in the fermentation broth during the tank fermentation using the Pichia pastoris X33 / AnxynB-linker2-Solac strain was shown. Picture 8 The synthesis of laccase by Pichia pastoris X33 / AnxynB-linker2-Solac strain was demonstrated So SDS-PAGE electrophoresis of Lac fusion protein after purification; Picture 9 The mass spectrometry identification results of the purified fusion protein are shown; Picture 10 Laccase So The optimal pH results of Lac fusion protein using ABTS, SGZ and 2,6-DMP as substrates; Picture 11 Laccase So The optimal catalytic temperature of Lac fusion protein with ABTS as substrate; Picture 12 Laccase So The pH stability results of Lac fusion protein were measured using ABTS as substrate; Picture 13 Laccase So The temperature stability results of Lac fusion protein were measured using ABTS as substrate; Picture 14 Laccase So The optimal temperature results of Lac fusion protein for degradation of aflatoxin B1; Picture 15 Laccase So The optimal pH results of Lac fusion protein for degradation of aflatoxin B1; Picture 16 Laccase So Effects of Lac fusion protein dosage and reaction time on the degradation of aflatoxin B1; Picture 17 The experimental results show the toxicity of different concentrations of aflatoxin B1 on HepG2 cells; Picture 18 Laccase So Experimental results on the detoxification effect of Lac fusion protein on aflatoxin B1; Picture 19 Laccase So The optimal temperature results of Lac fusion protein for degradation of zearalenone; Picture 20 Laccase So The optimal pH results of Lac fusion protein for degradation of zearalenone; Picture 21 Laccase So Effects of Lac fusion protein dosage and reaction time on the degradation of zearalenone; Picture 22 The experimental results show the toxicity of different concentrations of zearalenone on HepG2 cells; Picture 23 Laccase So Experimental results of the detoxification effect of Lac fusion protein on zearalenone. DETAILED DESCRIPTION

[0024] Experimental materials and reagents 1. Vector and gene: expression vector pPICZα(A) and Pichia pastoris X33 strain; 2. Enzymes and other biochemical reagents: restriction endonucleases, recombinant enzymes, aflatoxin B1 and zearalenone; 3. E. coli culture medium: LLB medium (1% peptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0). If necessary, add bleomycin to a final concentration of 25 mg / L. Add 2% agar to the solid plate.

[0025] 4. Pichia pastoris culture media: YPD medium (2% glucose, 2% peptone, 1% yeast extract), with 100 mg / L bleomycin added if necessary, and 2% agar for solid plates; 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, cobalt chloride 0.5 g / L, zinc chloride 20 g / L, ferrous sulfate 65 g / L, sulfuric acid 5.0 ml / L). Example 1 Construction of Laccase SoLac Fusion Protein Expression Vectors Containing Different Connector Peptides

[0026] The ochre yellow tooth fungus ( Steccherinum ochraceum ) from laccase Solac genes and Aspergillus niger ( Aspergillus niger ) derived xylanase Anxynb The gene was optimized according to the codon preference of Pichia pastoris and synthesized, and 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.

[0027] SEQ ID No: 4: SEQ ID No: 5: Gttccacacgactctgttgctcaaagatccgatgccttgcacatgttgtctgagagatccactccatcttccaccggtgaaaacaacggtttctactactccttctggactgatggtggtggtgacgttacttacactaacggtgatgctggtgcttacactgttgagtggtctaacgttggtaacttcgtcggtggtaaaggttggaacccaggttctgctcaggacattacttactccggtactttcactccatccggtaacggttacttgtccgtttacggttggactactgacccactgatcgagtactacatcgttgaatcctacggtgactacaaccctggttctggtggtacttacaagggtactgttacttccgacggttccgtctacgatatctacactgctactagaactaacgccgcttccattcaaggtactgctactttcacccaatactggtccgtcagacagaacaagagagttggaggtactgtcaccacttccaaccactttaacgcttgggctaagctgggtatgaacttgggtactcacaactaccagatcgttgctaccgaaggttaccaatcttctggttcctcctccattactgttcaa。

[0028] Using the synthesized gene sequence as a template, the laccase gene fragment or the laccase and xylanase linked with different linkers were amplified by PCR method Solac [[ID=८]]Gene fragment or laccase linked with different linkers Solac [[ID=१०]]And xylanase[[ID=११]] AnxynB It should be noted that there seems to be some incorrect numbering in the original Chinese text (e.g., "漆酶 " should probably be a continuous text without such sudden ID insertions). Also, the translation of the last part might need some adjustment depending on the exact context and intended meaning. The above translation is a literal one based on the provided text.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).

[0029] Connector peptide sequence used: Connector peptide 1: GGGGS (SEQ ID No: 20), nucleotide sequence (SEQ ID No: 21); Connector peptide 2: SASSGGTTPTTTHM (SEQ ID No: 3) nucleotide sequence (SEQ ID No: 22); Connector peptide 3: RSGLEKREAEA (SEQ ID No: 23), nucleotide sequence (SEQ ID No: 24); Connector peptide 4: GGGGSGGGGSGGGGS (SEQ ID No: 25), nucleotide sequence (SEQ ID No: 26); Connector peptide 5: GGGGGGGG (SEQ ID No: 27), nucleotide sequence (SEQ ID No: 28); Connector peptide 6: GGGGSKEEEKGGGGS (SEQ ID No: 29), nucleotide sequence (SEQ ID No: 30); Connector peptide 7: EPKSCDKTHTCPPCP (SEQ ID No: 31), nucleotide sequence (SEQ ID No: 32), Connector peptide 8: EPKSSDKTHTSPPSP (SEQ ID No: 33), nucleotide sequence (SEQ ID No: 34); Connecting peptide 9: GGGSLEKREAEA (SEQ ID No: 35), nucleotide sequence (SEQ ID No: 36).

[0030] Among them, 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, L4-Solac -F and Solac-R were used to amplify the linker4-Solac fragment, L5-Solac-F and Solac-R were used to amplify the linker5-Solac fragment, L6-Solac-F and Solac-R were used to amplify the linker6-Solac fragment, L7-Solac-F and Solac-R were used to amplify the linker7-Solac fragment, L8-Solac-F and Solac-R were used to amplify the linker8-Solac fragment, and L9-Solac-F and Solac-R were used to amplify the linker9-Solac fragment.

[0031] 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, and 1590 bp, respectively. Each linker-Solac fragment and xylanase Anxynb fragment were recovered and purified, and then overlap PCR was performed to amplify the fusion protein AnxynB and each linker-Solac coding sequence fragment. The primers used were AnxynB-F and Solac-R. SoThe 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 recombined with EcoR I / Not The recombinant product was transformed into E. coli TransI cloning host and plated on LLB medium containing 25 mg / L bleomycin. Single clones were picked and sequenced to obtain the correctly constructed laccase. So Lac direct expression recombinant plasmid pPICZα(A)-Solac and various laccases So Lac 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.

[0032] Laccase So Lac protein expression alone and laccase So The vector maps for Lac fusion protein expression are as follows: Picture 1 and Picture 2 shown. Example 2 Construction and Screening of Pichia pastoris Engineered Strains Expressing Laccase SoLac Alone or in Fusion

[0033] 1. Expression of Laccase So Construction of Pichia pastoris engineered strains expressing Lac Laccase SoLac protein expression vector pPICZα(A)-Solac and laccases containing different linker peptides So Lac fusion expression vector Dra I was used for enzyme linearization. The enzyme digestion product was recovered using a PCR product purification kit. The recovered linearized plasmid was transformed into Pichia pastoris X33 competent cells by electroporation and spread on YPD medium plates containing bleomycin resistance (100 mg / L) to obtain laccase. So Lac direct expression transformant Pichia pastoris X33 / Solac and various laccases So Lac fusion expression transformants 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.

[0034] 2. Laccase So Screening of recombinant Pichia pastoris strains expressing Lac successfully A single colony was picked from a YPD plate containing bleomycin and inoculated into a 10 mL tube containing 3 mL of BMGY medium. The culture was incubated at 30°C, 220 rpm, and incubated for 48 h. Centrifugation was performed at 4500 rpm, the supernatant discarded, and 1.5 mL of BMMY medium containing 1% methanol and 1 mM CuSO4 was added. The culture was induced at 30°C, 220 rpm for 48 h. Methanol was added every 24 h to a final concentration of 1%. After fermentation, the supernatant was collected by centrifugation and the tube was eluted with ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)) (ε420 = 36,000 M). -1 cm -1 Laccase activity was determined using 1 mM ABTS as the substrate in 50 mM acetic acid-sodium acetate buffer (pH 4.8). The assay was incubated at 30°C for 3 minutes, and absorbance was measured at 420 nm. Under standard assay conditions, 1 unit (1 U) of laccase activity was defined as the amount of enzyme that produces 1 μM product per minute.

[0035] Expressing laccase in different forms So The highest laccase activity in the supernatant of the fermentation broth of the Lac clone is Picture 3 Direct laccase So No laccase activity was detected in the supernatant of the fermentation broth of the host strain expressing Lac, indicating that laccase So Lac cannot be directly expressed heterologously in Pichia pastoris X33. Lacase activity was detected in the supernatant of the fermentation broth of Pichia pastoris strains expressing the complete fusion protein of xylanase and laccase, and xylanase An XynB and laccase So The linker sequence between Lac proteins significantly affects laccase activity. When no linker peptide was present between the laccase and xylanase, the enzyme activity in the fermentation supernatant was 7.2 U / L. When different linker peptide sequences were added between the fusion proteins, the Pichia pastoris strain expressing the fusion protein with the highest enzyme activity in the fermentation supernatant was the linker peptide 2 (SASSGGTTPTTTHM). This linker peptide represents the native sequence between the catalytic domain and the carbohydrate binding module (CBM) of the cellulase from Bacillus subtilis 41M-1. The highest enzyme activity in the fermentation supernatant of this strain (Pichia pastoris X33 / AnxynB-linker2-Solac) reached 94 U / L. Example 3 Horizontal fermentation of laccase SoLac fusion protein in a 15 L fermenter and protein purification

[0036] 1. Pichia pastoris X33 / AnxynB-linker2-Solac fermentation Pichia pastoris X33 / AnxynB-linker2-Solac was fermented in a 15 L fermenter SoFermentation production of Lac fusion proteins. Pichia pastoris X33 strains (prepared for recombinant protein production) stored at -80°C were inoculated into 50 mL of YPD medium and cultured in a shaker at 30°C at 220 rpm for 48 hours. The primary seed solution was then transferred to three 200 mL bottles of BMGY medium and cultured overnight in a shaker at 30°C at 220 rpm. 7.5 L of fermentation medium was prepared and sterilized by steam sterilization at 121°C for 30 minutes. After the temperature dropped to 30°C, the secondary seed solution was inoculated into a fermentor. The inoculated strains were cultured in the fermentor for an additional 18-22 hours at 800 rpm, with dissolved oxygen maintained at 20-40%, and at 30°C. Ammonia was used to stabilize the pH at approximately 4.5. After the glycerol used as the carbon source in the previous step was depleted, 12 mL / L of PTM1 solution in 50% glycerol was added at a rate of 18 mL / h / L and the culture continued for 3-6 hours. When the yeast wet weight reached 160-180 g / L, methanol containing 12 mL / L of LPTM1 solution was added at a rate of 3.2 mL / h / L, while maintaining a pH of approximately 5.5. 10 mL of 0.5 M CuSO₄ was added every 24 hours. Laccase and xylanase activities and yeast cell wet weight were measured every 24 hours. During the induction of recombinant protein expression, laccase and xylanase activities and cell wet weight generally increased.

[0037] like Picture 4 、 5 As shown in Figures 6 and 7, 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 peaks 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).

[0038] In addition, the experimental results also showed that there was a significant linear relationship between the enzyme activities of xylanase and laccase in the fermentation broth (R 2 = 0.9792)( Picture 7 ), which indicates that xylanase An XynB and laccase So Lac was heterologously expressed as a fusion.

[0039] 2. Laccase So Purification of Lac fusion protein After the fermentation is completed, the fermentation liquid is collected and sterilized and concentrated using hollow fibers. SoThe Lac fusion protein was replaced with a 20 mM acetic acid-sodium acetate buffer solution (pH 5.5). The fusion protein was purified using a HiTrap Q HP anion exchange column from GE healthcare. Solution A is a 20 mM acetic acid-sodium acetate buffer solution (pH 5.5), and solution B is 1 M NaCl added to solution A. Linear gradient elution was performed using a solution with a NaCl concentration of 0-1 M, and the eluted fractions were collected for SDS-PAGE detection. The qualified purified fractions were combined and replaced with a protein storage buffer solution (50 mM Tris-HCl, 150 mM NaCl, pH 7.5). The purification results are shown in Figure 2. Picture 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 cut from the SDS-PAGE gel and identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The equipment used was a micro-liquid chromatography system EASY-nLC 1000 coupled with a mass spectrometer Q-Exactive. The MS / MS data were aligned with the expected protein sequence using PEAKS Studio software. The results showed that 18 peptide sequences could match the target protein sequence, and these sequences covered the fusion protein. An XynB- So 51.5% of Lac ( Picture 9 ). Example 4 Determination of Enzymatic Properties of Fusion-Expressed Recombinant Laccase SoLac

[0040] 1. Fusion expression of recombinant laccase So Determination of the Optimal pH and Temperature of Lac At 30°C, ABTS and syringaldazine (SGZ, ε530 = 65,000 M) were added to the system at a final concentration of 1 mM. -1 cm -1 ) and 2,6-dimethoxyphenol (2,6-DMP, ε470 = 49,600 M -1 cm -1 ) as substrate, and the expression of recombinant laccase was determined So The optimal pH of Lac was determined in the pH range of 2.0-12.0. The buffers used were 100 mM glycine-HCl buffer solution (pH 2.0), 100 mM Na2HPO4-citric acid buffer solution (pH 3.0-7.0), 100 mM Tris-HCl buffer solution (pH 7.0-9.0), and 100 mM glycine-NaOH buffer solution (pH 9.0-12.0). The results are shown in Figure 2. Picture 10As shown, when ABTS was used as substrate, the recombinant laccase expressed by fusion So The optimal pH of Lac is 4.0. When SGZ and 2,6-DMP are used as substrates, the recombinant laccase expressed by the fusion So The optimal pH of Lac is 5.0. The final concentration of 1 mM ABTS was used as substrate to measure the expression of recombinant laccase. So The optimum temperature of Lac. The test temperature range is 30-90℃. Picture 11 As shown, when ABTS is used as substrate, laccase So The optimum temperature of Lac was 70℃, and it showed high activity in the temperature range of 50℃ to 80℃.

[0041] 2. Fusion expression of recombinant laccase So Determination of pH and temperature stability of Lac At 30℃, the appropriately diluted laccase So After the Lac fusion protein was treated in a buffer solution with a pH range of 2.0 to 12.0 for 1 h, the remaining laccase activity was determined using ABTS as a substrate. Picture 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%. In the pH range of 5.0 to 10.0, its residual enzyme activity was higher than 69.1%, while at pH 4.0 and 11.0, it dropped to 44.0% and 36.6% of the untreated protein, respectively, indicating that laccase So Lac has good stability under both weakly acidic and alkaline conditions.

[0042] Laccase So The Lac fusion protein was diluted appropriately with 100 mM Tris-HCl buffer (pH 9.0) and then incubated at 40 to 70°C. Samples were taken at 5, 10, 20, and 30 minutes after treatment, and the residual enzyme activity was determined using ABTS as a substrate. The results showed that the recombinant laccase expressed by the fusion had good thermal stability at 40 and 50°C. Picture 13 As shown in Figure 2, after incubation at 40℃ and 50℃ for 5 min, the enzyme activities were 97.0% and 94.5% of the initial enzyme activities, respectively. So Lac still retained 89.2% and 88.6% of its enzyme activity. However, when the temperature exceeded 50°C, the enzyme activity decreased significantly. After incubation at 60°C and 70°C for 5 minutes, the relative enzyme activities were 83.6% and 38.1% of the initial activity, respectively. After incubation for 20 minutes, the relative enzyme activities dropped to 70.5% and 2.6% of the initial activity, respectively. Example 5 Laccase SoLac fusion protein degrades aflatoxin B1 independently of mediator

[0043] 1. Laccase So Optimal temperature for direct degradation of aflatoxin B1 by Lac fusion protein Aflatoxin B1 was dissolved in dimethyl sulfoxide to prepare a 100 mg / L aflatoxin B1 stock solution. The following reaction system was prepared: 20 μL of aflatoxin B1 stock solution and 20 μL of enzyme solution (laccase concentration was 6000 U / L). The reaction was carried out in 100 mM Tris-HCl buffer (pH 7.0) at 20°C-50°C for 10 h. Each reaction was replicated three times, and a reaction without laccase fusion protein served as a negative control. The reaction was terminated by adding three volumes of methanol. Aflatoxin B1 concentration and degradation rate were determined using a Shimadzu Nexera UHPLC system. The chromatographic separation was performed on a Zorbax SB-C18 column (4.6 × 250 mm, 5 μm), with mobile phases A (0.06% TFA in water) and B (0.05% TFA in acetonitrile). The gradient elution conditions were 0% B for 4 min, 0%-100% B for 15 min, and 100% B for 10 min. The samples were detected using a UV detector at a wavelength of 365 nm.

[0044] like Picture 14 As shown, laccase at 50℃ So Lac had the highest degradation rate of aflatoxin B1, at 38.9%. When the temperature exceeded 60°C, the degradation rate of aflatoxin B1 dropped significantly to 5.6%.

[0045] 2. Laccase So Optimal pH for degradation of aflatoxin B1 by Lac fusion protein 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 of different pH values. The buffers used were 100 mM glycine-HCl buffer (pH 2.0), 100 mM Na₂HPO₄-citrate buffer (pH 3.0-6.0), and 100 mM Tris-HCl buffer (pH 7.0). Each reaction system was replicated three times, and a reaction without laccase served as a negative control. The reaction was carried out at 50°C and terminated after 10 h by adding three volumes of methanol. The degradation rate of aflatoxin B1 was analyzed by high-performance liquid chromatography.

[0046] The results are as follows Picture 15As shown in the figure, in the pH range of 2.0-7.0, the degradation rate of aflatoxin B1 showed an upward trend and reached a peak at pH 7.0 with a conversion rate of 32.0%.

[0047] 3. Laccase So Effects of Lac fusion protein dosage and reaction time on the degradation of aflatoxin B1 The following reaction system was prepared: 20 μL aflatoxin B1 stock solution, 20 μL enzyme solution (the concentrations of laccase in the system were 2000 U / L, 4000 U / L, 6000 U / L, 8000 U / L and 10000 U / L, respectively), and 160 μL 100 mM Tris-HCl buffer solution (pH 7.0). Each reaction system was repeated three times, and the reaction system without laccase was used as a negative control. The reaction was carried out at 50°C. Three volumes of methanol were added after 0 h, 3 h, 6 h, 9 h, 12 h and 20 h of reaction to terminate the reaction. The degradation rate of aflatoxin B1 was analyzed by high performance liquid chromatography. The results are shown in Figure 2. Picture 16 As shown in Figure 2, the conversion rate of aflatoxin B1 showed an upward trend with the extension of reaction time. 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%.

[0048] 4. Laccase So Detoxification effect of Lac fusion protein on aflatoxin B1 The cytotoxicity of aflatoxin B1 and its degradation products was evaluated in HepG2 cells, a human hepatocellular carcinoma cell line. HepG2 cells were cultured at 37°C in a 5% CO2 incubator using DMEM supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / L streptomycin. Cells were plated at a density of 5 × 10 4Cells were seeded in 96-well plates at a concentration of 100 μL / well. After 24 hours of culture, aflatoxin B1 or a laccase-degraded aflatoxin B1 system was added to the culture medium at various final concentrations (0, 1, 2, 5, 10, 20, 30, 40, 50, and 60 mg / L) and cultured for a further 48 hours. The degradation system consisted of 100 mg / L aflatoxin B1 and 8000 U / L laccase in 100 mM Tris-HCl buffer (pH 7.0). The reaction was incubated at 50°C for 24 hours. The culture medium was then discarded, and the cells were washed with phosphate-buffered saline. Cell Counting Kit-8 (CCK-8) reagent was diluted in DMEM and added to each well of the 96-well plate. 100 μL was added to each well and incubated at 37°C for 2 hours. After incubation, the absorbance of each well was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0049] like Picture 17 As shown in the figure, with the increase of aflatoxin B1 concentration (0-60 mg / L), the viability of HepG2 cells decreased with the increase of dose. After treatment with 10 mg / L aflatoxin B1, the cell viability decreased by 40.2% compared with the untreated control group. At the same time, the fusion protein had almost no effect on cell viability. In contrast, the laccase So The survival rate of HepG2 cells in the reaction system treated with aflatoxin B1 by Lac fusion protein increased to 92.9%. So Lac fusion protein treatment of aflatoxin B1 can significantly reduce its toxicity ( Picture 18 ). Example 6 Degradation of Zearalenone by Laccase SoLac Fusion Protein

[0050] 1. Laccase So Optimal temperature for direct degradation of zearalenone by Lac fusion protein Zearalenone was dissolved in dimethyl sulfoxide to prepare a 100 mg / L zearalenone stock solution. The following reaction system was prepared: 20 μL of the zearalenone stock solution and 20 μL of the enzyme solution (laccase concentration was 6000 U / L). The reaction was carried out in 100 mM Tris-HCl buffer (pH 7.0) at 20°C-50°C for 10 h. Each reaction was replicated three times, and a reaction without laccase fusion protein served as a negative control. The reaction was terminated by adding three volumes of methanol. Zearalenone concentration and substrate degradation rate were determined using a Shimadzu Nexera UHPLC system. The chromatographic separation was performed on a Zorbax SB-C18 column (4.6 × 250 mm, 5 μm). The mobile phase used was acetonitrile: H2O:methanol (46:46:8) at a flow rate of 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.

[0051] like Picture 19 As shown in the figure, the degradation rate of zearalenone was the highest at 43.0% when the reaction was carried out at 50°C. When the temperature was increased to 60°C, the degradation rate of zearalenone decreased to 4.7%.

[0052] 2. Laccase So Optimal pH for degradation of zearalenone by Lac fusion protein 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 of different pH values. The buffers used were 100 mM glycine-HCl buffer (pH 2.0), 100 mM Na₂HPO₄-citrate buffer (pH 3.0-6.0), and 100 mM Tris-HCl buffer (pH 7.0-8.0). Each reaction was repeated three times, and a reaction without laccase served as a negative control. The reaction was carried out at 50°C and terminated after 10 h by adding three volumes of methanol. The degradation rate of zearalenone was analyzed by high-performance liquid chromatography.

[0053] The results are as follows Picture 20 As shown in the results, 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%.

[0054] 3. Laccase So Effects of Lac fusion protein dosage and reaction time on zearalenone The following reaction system was prepared: 20 μL of zearalenone mother solution, 20 μL of enzyme solution (the concentrations of laccase in the system were 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 three times, and the reaction system without laccase was used as a negative control. The reaction was carried out at 50°C. Three volumes of methanol were added after 0 h, 3 h, 6 h, 9 h, 12 h and 20 h of reaction to terminate the reaction. The degradation rate of zearalenone was analyzed by high performance liquid chromatography. The results are shown in Figure 2. Picture 21 As shown in Figure 2, the degradation rate of zearalenone showed an upward trend with the extension of reaction time. So As the Lac concentration 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 dosage was further increased to 6000 U / L, the conversion rate of zearalenone did not increase significantly (to 72.9%).

[0055] 4. Laccase So Detoxification effect of Lac fusion protein on zearalenone The cytotoxicity of zearalenone and its degradation products was evaluated in HepG2 cells, a human hepatocellular carcinoma cell line. HepG2 cells were cultured at 37°C in a 5% CO2 incubator using DMEM supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / L streptomycin. Cells were plated at a density of 5 × 10 4 Cells were seeded in 96-well plates at a concentration of 100 μL / well. After 24 hours of culture, zearalenone or zearalenone-degraded by laccase at various final concentrations (0, 1, 2, 5, 10, 20, 30, 40, 50, and 60 mg / L) was added to the culture medium, and the cells were cultured for another 48 hours. The degradation system consisted of 100 mg / L zearalenone and 8000 U / L laccase in 100 mM Tris-HCl buffer. The reaction was incubated at 50°C for 24 hours. The culture medium was then discarded, and the cells were washed with phosphate-buffered saline. Cell Counting Kit-8 (CCK-8) reagent was diluted in DMEM and added to each well of the 96-well plate. 100 μL was added to each well and incubated at 37°C for 2 hours. After incubation, the absorbance of each well was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0056] like Picture 22As shown in the figure, with the increase of zearalenone concentration (0-60 mg / L), the viability of HepG2 cells decreased with the increase of dose. After treatment with 10 mg / L zearalenone, the cell viability decreased by 25.0% compared with the untreated control group. At the same time, the fusion protein had almost no effect on cell viability. In contrast, the laccase So After the Lac fusion protein was treated with the reaction system of zearalenone, the cell survival rate increased to 93.9%. These findings indicate that the Lac fusion protein can be used to treat HepG2 cells. So Treatment of zearalenone with Lac fusion protein can significantly reduce its toxicity ( Picture 23 ).

[0057] The above embodiments are only used to understand the technical solutions of the present application and do not limit the scope of protection of the present application.

Claims

1. A laccase fusion protein, characterized in that The laccase fusion protein comprises xylanase, a connecting peptide and laccase, 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 connecting peptide is shown in SEQ ID No:

3.

2. A coding sequence encoding the laccase fusion protein according to 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 comprises the following steps: Constructing a recombinant expression vector comprising a coding sequence for a laccase fusion protein, wherein the laccase fusion protein comprises a xylanase, a connecting peptide, and a laccase, 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 connecting peptide is shown in SEQ ID No: 3; Transforming host cells with the recombinant expression vector to obtain a recombinant strain; The recombinant strain is fermented and cultured 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 step of fermenting and culturing the recombinant strain comprises the following steps: Cultivate the primary seed solution: inoculate the recombinant strain for recombinant protein production into YPD medium for cultivation; Secondary seed solution: transfer the primary seed solution to BMGY medium for culture; inoculating the secondary seed liquid into the fermentation medium of the fermentation tank; Bacterial culture: continue to culture the strain inoculated in the fermentation tank for 18-22 hours, control the dissolved oxygen at 20-40%, the rotation speed at 800 rpm, the temperature at 30°C, and stabilize the pH value at 4.5; Glycerol feeding: After the carbon source in the solid culture step is exhausted, 50% glycerol containing 12 mL / L PTM1 solution is fed at a rate of 18 mL / h / L and the culture is continued for 3-6 h; Methanol induction: When the wet weight of the cells reaches 160-180 g / L, add methanol containing 12 mL / L PTM1 solution at a rate of 3.2 mL / h / L while controlling the pH value at around 5.

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

9. The method for preparing laccase by fermentation according to claim 6, characterized in that: The formula of the fermentation medium is: 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 formula of the PTM1 solution is: 6.0 g / L copper sulfate, 0.09 g / L potassium iodide, 3.0 g / L manganese sulfate, 0.2 g / L sodium molybdate, 0.02 g / L boric acid, 0.5 g / L cobalt chloride, 20 g / L zinc chloride, 65 g / L ferrous sulfate, and 5.0 ml / L sulfuric acid.

10. Use of the laccase fusion protein according to claim 1 in the detoxification of fungal toxins.

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