Talaromyces sp. With multi-oxidase synergistic expression ability, fermentation method and application of talaromyces sp.

Abstract

The invention discloses talaromyces sp. With a polyoxidase synergistic expression capability as well as a fermentation method and an application of the talaromyces sp. The talaromyces sp. Ta-lac is preserved in the China General Microbiological Culture Collection Center on July 11, 2025, and the preservation number of the talaromyces sp. Ta-lac is CGMCC (China General Microbiological Culture Collection Center) No.42125. The talaromyces sp. Ta-lac is named Ta-lac. Compared with an engineering strain for biologically synthesizing laccase, the strain has relatively high environmental adaptability, laccase synthesis can be naturally carried out, a genome is relatively stable, potential risks of exogenous genes do not exist, and the safety is relatively high. The talaromyces sp. Is short in growth cycle, high in yield of laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase in the fermentation process, and high in lignin degradation activity. The talaromyces talarosus Ta-lac is applied to degradation of toxin dye wastewater, and the toxicity reduction and decolorization effects are remarkable.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, in particular to a Talaromyces with multi-oxidase synergistic expression ability and a fermentation method and application thereof. BACKGROUND

[0002] In the field of environmental pollutant degradation, the efficient decomposition of complex organic compounds such as lignin and polycyclic aromatic hydrocarbons has always been a major challenge. These substances have highly stable aromatic ring structures, and traditional physical and chemical methods are difficult to completely degrade and are prone to secondary pollution. In nature, microorganisms achieve efficient conversion of these recalcitrant substances by secreting an oxidase system, in which laccase, monooxygenase and dioxygenase constitute the key catalytic system.

[0003] As a multicopper oxidase, laccase catalyzes the oxidation of phenolic and non-phenolic substrates through a single electron transfer mechanism, while reducing molecular oxygen to water. This property makes it play a core role in lignin depolymerization. However, the catalytic efficiency of laccase alone on non-phenolic substrates is limited. At this time, monooxygenase and dioxygenase work synergistically by introducing hydroxyl groups and cleaving aromatic rings, which can significantly expand the substrate degradation range and improve the mineralization efficiency. This multi-enzyme synergistic cascade reaction is the key to the efficient degradation of complex pollutants by microorganisms.

[0004] The main bottleneck of current industrial applications is that natural strains have low enzyme production and poor stability, and genetically engineered bacteria have unstable expression systems. Therefore, screening wild strains with multi-oxidase synergistic expression ability from natural environment and optimizing their fermentation characteristics become a feasible way to break through the limitations of existing technology. SUMMARY

[0005] To solve the above technical problems, the present application provides a Talaromyces with multi-oxidase synergistic expression ability and a fermentation method and application thereof. Based on enzyme mechanism and metabolic network analysis, the present application establishes an efficient screening system to screen a complex enzyme-producing strain with high enzyme activity and good environmental adaptability. This strain can produce laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase, providing high-quality microbial resources for industrial biodegradation.

[0006] In the first aspect, the present application provides a Talaromyces with multi-oxidase synergistic expression ability, which is realized by the following technical solution.

[0007] A Talaromyces with multi-oxidase synergistic expression ability (Talaromyces) Talaromyces sp. The Talaromyces has been deposited with the China General Microbiological Culture Collection Center on July 11, 2025, and the deposit number is CGMCC No.42125.

[0008] Further, the fermentation of the basket fungus Ta-lac can simultaneously produce laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase.

[0009] The basket fungus Ta-lac screened by the application is an obligate aerobic bacterium, and the optimum growth temperature is 30-37 DEG C, and the optimum growth pH is 2.5-3.5. The colony of the basket fungus presents a circular or irregular shape, and the surface has dense rope-shaped mycelium, and the whole is smooth with neat wet edges, and the texture is viscous, and the color of the colony is bean green, and has certain gloss and elasticity. The carbon sources that can be utilized include lignin, corn cob powder, fructose, maltose, sucrose, starch, arabinose, cellulose, etc., and the nitrogen sources that can be utilized include ammonium sulfate, ammonium chloride, potassium nitrate, urea, yeast powder, etc. Compared with the existing laccase production strains, the basket fungus Ta-lac has strong adaptability, high safety and good stability, and the yield of laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase is high when the carbon source (lignin or corn cob powder) is used as the substrate for fermentation, and the substrate degradation rate is high.

[0010] In a second aspect, the application provides a first use of the basket fungus with the ability of synergistic expression of multiple oxidases, which is realized by the following technical scheme.

[0011] In a second aspect, the application provides a first use of the basket fungus with the ability of synergistic expression of multiple oxidases, which is realized by the following technical scheme.

[0012] In a third aspect, the application provides a second use of the basket fungus with the ability of synergistic expression of multiple oxidases, which is realized by the following technical scheme.

[0013] In a third aspect, the application provides a second use of the basket fungus with the ability of synergistic expression of multiple oxidases, which is realized by the following technical scheme.

[0014] Further, the environmental pollutants include dyes and lignin.

[0015] In a fourth aspect, the application provides a method for fermentatively producing laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase, which is realized by the following technical scheme.

[0016] A method for fermentatively producing laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase by using the basket fungus, which comprises the following steps: a. the basket fungus Ta-lac liquid stored at -80 DEG C is streak-inoculated on a CM culture medium, and cultured at 35-37 DEG C for 70-72 h; b. sterile distilled water is added to the cultured plate, and spores are scraped to collect and prepare a spore suspension; c. the spore suspension is diluted to 5×10 5 -5×10 6The fermentation medium is inoculated with an inoculation amount of 1 mL / mL at 35-37 DEG C, 180-200 rpm / min for 94-96 h.

[0017] Further, the formula of the CM medium is 1 wt% of glucose, 0.2 wt% of 1 mol / L MgSO4 mother liquor, 0.1 wt% of casein hydrolysate, 0.5 wt% of yeast extract and 0.08 wt% of ABTS.

[0018] Further, the formula of the fermentation medium is 1.7 wt% of lignin, 0.7 wt% of potassium nitrate, 0.1 wt% of casein hydrolysate and 0.08 wt% of ABTS, and the K + The final concentration is 0.3 wt%.

[0019] The application has the following beneficial effects.

[0020] 1. The application is based on the natural characteristics of the Talaromyces sp. in high-yield oxidation enzyme production, and the monooxygenase and dioxygenase produced in the fermentation process jointly play a role with the laccase, so that the efficiency of the Talaromyces sp. in producing laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase is significantly improved, and the total oxidation enzyme activity is increased from 21.7 U / L to 126.9 U / L after 96 h of shake flask fermentation.

[0021] 2. The application solves the problem of genetic instability caused by the introduction of plasmids to modify the metabolic pathway of the strain in the process of producing laccase by microbial fermentation. The Talaromyces sp. is a natural strain with strong environmental adaptability, can grow stably in the natural environment, and can form a biofilm on various surfaces, which not only enhances its adaptability to the environment, but also improves its tolerance to antibiotics.

[0022] 3. The Talaromyces sp. of the application naturally produces laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase, and has an effect on the degradation of different dyes, providing a new direction for dye decolorization and toxin degradation. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a gel map of the 18S rDNA PCR electrophoretic product of the Talaromyces sp. of the application (lanes 1-5 are all 18S rDNA PCR products); Figure 2 is an evolutionary tree map constructed after sequence comparison of the 18S rDNA of the Talaromyces sp. of the application; Figure 3 is a result map of the Talaromyces sp. Ta-lac under different temperature optimization; Figure 4is the optimization result diagram of different pH of the Talaromyces of the application Ta-lac; Figure 5 is the optimization result diagram of different carbon source of the Talaromyces of the application Ta-lac; Figure 6 is the optimization result diagram of different nitrogen source of the Talaromyces of the application Ta-lac; Figure 7 is the optimization result diagram of different metal ions of the Talaromyces of the application Ta-lac; Figure 8 is the SDS-PAGE verification result diagram of the fermentation liquid of the Talaromyces of the application Ta-lac; Figure 9 is the degradation result diagram of crystal violet by the Talaromyces of the application Ta-lac; Figure 10 is the degradation result diagram of malachite green by the Talaromyces of the application Ta-lac; Figure 11 is the degradation rate diagram of crystal violet by the Talaromyces of the application Ta-lac; Figure 12 is the degradation rate diagram of malachite green by the Talaromyces of the application Ta-lac. DETAILED DESCRIPTION

[0024] The application will be further described below in combination with the drawings and examples. Unless otherwise specified, the experimental methods used in the application are conventional methods, and the experimental apparatus, materials and reagents used in the application can be purchased from relevant material selling companies.

[0025] The application provides a Talaromyces capable of simultaneously producing laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase, which is screened from a sample collected from a fir tree in Mangshan National Forest Park in Yizhang County, Chenzhou City, Hunan Province, and preserved in China General Microbiological Culture Collection Center on July 11, 2025, with a preservation number of CGMCC No.42125.

[0026] Specifically, the screening method of the Talaromyces capable of simultaneously producing laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase comprises the following steps: I. Isolation and screening of strains 2 g of the sample of the pulverized fir tree in Mangshan National Forest Park in Yizhang County, Chenzhou City, Hunan Province is added into 50 mL of sterilized LB medium (1 wt% of tryptone, 0.5 wt% of yeast extract and 1 wt% of NaCl) for enrichment, and the enrichment is cultured at 37 ℃ and 180 rpm / min of oscillation for 24 h; 1 mL of the bacterial suspension is taken for enrichment culture, and the enrichment liquid is subjected to 10 -1 ~10 -7Gradient dilution, 100 μL of each dilution was spread on PDA medium containing 0.08wt% ABTS, and incubated at 37℃ for 72 h; well-grown, moderate-sized and obvious greenish-blue hydrolysis ring were selected and purified by streaking several times to obtain strain Ta-lac.

[0027] II. Identification of the strain 1. Physiological and biochemical characteristics The colony of strain Ta-lac on CM medium [glucose 1wt%, 1 mol / L MgSO4mother liquor 0.2wt%, casein hydrolysate 0.1wt%, yeast extract 0.5wt%, 2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) 0.08wt%] was round or irregular in shape, with dense cord-like mycelium on the surface, smooth and moist overall, neat edge, sticky texture, and the color of the colony was bean green with certain luster and elasticity. The carbon sources that could be utilized included fructose, maltose, sucrose, starch, arabinose, cellulose, etc., and the nitrogen sources included ammonium sulfate, ammonium chloride, potassium nitrate, urea, and yeast powder. The strain could produce laccase, various monooxygenases and dioxygenases, and catalase.

[0028] 2. 18S rRNA sequence identification The activated strain Ta-lac was inoculated in liquid CM medium and cultured at 37℃ with 180 rpm shaking for 24 h. 1 μL of the bacterial solution was used as a template to amplify the 18S rDNA gene of the strain with universal primers 18S-F: 5'-CCAGTAGTCATATGCTTGTCT-3' (SEQ ID NO. 8) and 18S-R: 5'-ACCTTGTTACGACTTTTACTTCC-3' (SEQ ID NO. 9). After 2 μL of PCR product was verified by 1% agarose gel electrophoresis, Figure 1 , the product was sent to Beijing Huada Gene Company.

[0029] Based on the identification results and sequence alignment, it was determined that strain Ta-lac belonged to Talaromyces sp. Talaromyce sp. , and was identified as Talaromyces sp. by comparing with NCBI data, with 99% coverage, and finally named Talaromyces sp. Ta-lac. The phylogenetic tree of Talaromyces sp. Ta-lac is shown in Figure 2 .

[0030] III. Optimization of seed culture The Ta-lac bacteria liquid stored at -80 °C was streaked on CM medium and cultured in a 37 °C incubator for 72 h. Sterilized distilled water was added to the medium, spores were scraped and collected to prepare a spore suspension, which was diluted after microscopic counting. The diluted spore suspension was spread on CM plates at an inoculum of 10 6 / mL, and placed in 25 °C, 28 °C, 30 °C, 37 °C and 42 °C incubators for 96 h. The growth state was observed and recorded every 24 h. The results, as shown in Figure 3 , showed that a small amount of mycelium germination could be seen at 25 and 28 °C after 48 h of plate culture, white mycelium was obviously visible at 30 and 37 °C, and no germination was observed at 42 °C. Therefore, the optimum growth temperature of the Talaromyces Ta-lac was 30-37 °C, and the best growth was observed at 37 °C.

[0031] A 100 mmol / L phosphoric acid-citric acid buffer with a pH of 4.0 was prepared, and 2 mmol / L ABTS solutions with pH values of 3-9 were prepared using the buffer. The OD values of the 2 mmol / L ABTS solutions with pH values of 3-9 were measured, and then 200 μL of the crude enzyme solution (the spore suspension of Talaromyces Ta-lac was inoculated in CM liquid medium at an inoculum of 10 6 / mL, and cultured at 37 °C and 180 rpm / min for 96 h. The resulting fermentation broth was centrifuged at 4500 rpm to obtain the crude enzyme solution) was added to 800 μL of the above solution, and the reaction was carried out at 37 °C for 1 min. The total oxidase activity under different pH conditions was measured, and an equal volume of distilled water was used as a blank control. The amount of enzyme required to oxidize 1 μmol of ABTS in 1 min was defined as 1 enzyme activity unit (U). The culture solution of the heat-inactivated strain was used as a control, and all experiments were repeated three times. The total oxidase activity calculation formula was: enzyme activity (U / L) = N ×V1× (A3- A0) / (ε × L × V2× t), where N is the dilution factor; V1 is the total reaction volume; V2 is the total enzyme volume in the reaction system; ε is the extinction coefficient, which is 36000; A0 is the initial absorbance; A3 is the final absorbance; L is the optical path, and the width of all cuvettes in this experiment was 1 cm; t is the reaction time, which is 1 min. The experimental results are shown in Figure 4 , which showed that the optimum pH of Talaromyces Ta-lac was 2.5-3.5, and the best growth was observed at pH 3.

[0032] Four, optimization of fermentation medium Ta-lac bacterial suspension stored at -80℃ was streaked onto CM medium and incubated at 37℃ for 72 h. Sterile distilled water was added to the medium, spores were scraped off and collected to prepare a spore suspension. After microscopic counting, the suspension was diluted to a spore count of 102. 7 / mL, using an inoculation loop, spore suspension was collected and inoculated into fermentation media (containing 0.08 wt% ABTS) with different carbon sources, nitrogen sources, and metal ions. Fermentation was carried out at 37 ℃ and pH=3 for 96 h. The optimized carbon sources for the fermentation media were: glucose, fructose, galactose, arabinose, sucrose, maltose, starch, xylan, mannan, dextrin, cellulose, and lignin; the optimized nitrogen sources were: ammonium sulfate, ammonium chloride, potassium nitrate, urea, yeast extract, tryptone, and soybean meal; the optimized chloride metal ions were: Fe 2+ Fe 3+ Cu 2+ K + Na + Mn 2+ Mg 2+ Co 2+ Zn 2 + The experimental results are as follows: Figures 5-7 As shown, Figure 5 It can be seen that the ABTS carbon source of polysaccharides has a significantly better color development effect (showing blue and / or red during the culture process, and finally showing blue-black) than disaccharides and monosaccharides. Among polysaccharides, cellulose and xylan have obvious color development effects, while lignin has a significant decolorization effect. Moreover, lignin is composed of phenylpropane derivatives, which are the basic structural units, and requires a polyoxidase system to be effectively degraded. Therefore, lignin was selected as the best carbon source for subsequent experiments. Figure 6 The results show that ABTS exhibits the most significant colorimetric effect when potassium nitrate is used as the sole nitrogen source, therefore potassium nitrate is the optimal nitrogen source. Figure 7 The results showed that iron ions had a significant inhibitory effect on ABTS degradation, K + It has a significant promoting effect on the degradation of ABTS, increasing enzyme activity by 59%, therefore the optimal metal ion is K. + .

[0033] Ta-lac bacterial suspension stored at -80℃ was streaked onto CM medium and incubated at 37℃ for 72 h. Sterile distilled water was added to the medium, spores were scraped and collected to prepare a spore suspension. After microscopic counting, the suspension was diluted, and the spore suspension was divided into 5 × 10⁻⁶ spore suspensions. 6 Inoculation was performed at a rate of / mL on the optimized culture medium (lignin 1.7 wt%, potassium nitrate 0.7 wt%, casein hydrolysate 0.1 wt%, ABTS 0.08 wt%, KCl supplemented with K). +Fermentation was carried out in a medium with a final concentration of 0.3 wt%. The total oxidase activity in the crude enzyme solution was measured to increase from 21.7 U / L before the medium optimization to 126.9 U / L, reaching the highest level.

[0034] V. SDS-PAGE and Mass Spectrometry Verification of Fermentation Broth Ta-lac bacterial suspension stored at -80℃ was streaked onto CM medium and incubated at 37℃ for 72 h. Sterile distilled water was added to the medium, spores were scraped and collected to prepare a spore suspension. After microscopic counting, the suspension was diluted, and the spore suspension was divided into 5 × 10⁻⁶ spore suspensions. 6 Inoculate at a rate of / mL into lignin-based carbon source medium (lignin 1.7 wt%, potassium nitrate 0.7 wt%, casein hydrolysate 0.1 wt%, ABTS 0.08 wt%, KCl supplemented with K). + The mixture was cultured at 37 °C and 180 rpm / min for 96 h in a final concentration of 0.3 wt%. The fermentation broth was then concentrated and denatured, followed by SDS-PAGE verification. After staining and destaining, the results were compared based on the molecular weight of different oxidases. The experimental results are as follows: Figure 8 As shown.

[0035] The protein gel image of the fermentation broth was analyzed by mass spectrometry (mass spectrometry analysis was completed by the Institute-level Public Technology Center of the Institute of Microbiology, Chinese Academy of Sciences). The experimental results are shown in Table 1. The experimental results show that the fermentation of *Ta-lac* strain of *Bacillus basiliformes* can simultaneously produce laccase, nitropropane dioxygenase, hydrogenase-coenzyme dioxygenase, squalene monooxygenase, and catalase.

[0036] Table 1

[0037] The laccase 1 gene encodes a 562-amino acid sequence as shown below (SEQ ID NO.1). MVLRGILLLHYFLPGLHMPFGAAVEPDSGNTAADRSKWCTYNIHTDYYNVVPDTGVTREYWFDLRDRVLAPDGIRPYTQSINGSIPGPTIRANWGDEVVVHVQNNFQNTSNGTSLHFHGIRQNYTNQNDGVVSVTECPTAPHGRTTYKRWAAQYGTTFYHSHFSLQAHQGVFGAIVIDGPASANYDVDQGALILTDWGHQTPDEIFYHESRHQLPTYLENGLINGMNIYNIGDKQVTYRFTIEFQPGTSYRLRLIADALDTHFTFDTDHHTMTVIAADLVPLEEPTTNVITIGIGQRYDVIVKADQGIAHEFWMRAVPQKLCSRNNNTENIRGIVYYTDTVQGQPSTSGYNTTTGCVDQTDIAQPVSKSVSEDFVYYAVAALTIGKNTAGLFVWQVNDVSMQVEVVNPTLLQIYNNVTDWSSTEGFFQLDEVDKWIYVLIQSSLPISHPMHLHGHDFEELAQGTGAYEPSAAVSNFDNPPRRDTGLLLAGGYLLLAFQTDNPGAWLLHCHVGFHATCCLAVQFVERYDEIRDLELYESLSQTCKAWDSWEKSKGLVEVDSGI The laccase 2 gene encodes a 680 amino acid sequence as follows (SEQ ID NO. 2) MMHRVVVSLCCYGGYFTCCYPTAWFNNHMLKTLISMAINPIKVNLGRKPRMLQQVQQRSHLLTLLNGSKQSQAQVLVLSMVLVATGLAFTSLDIPALYLFFDNVSLKLQGTKTLDQPTILEYQTAICQOSLGQYHSGRKKSWPRHVIPDINLERSSGVKSMSILNDRTCLLLTDTSPVIAINGVWPAPMIRGTVNDTVVVKVHPPLGNQSLSIHWSSLHMVVSVLVMHCMQLDEPGTCCYHSHSPSQYVDGIRGPILVDDPFSPYADQDWGELVVTLSDWYHVCMLTRGIIALPGNPTALEPIPMSALMNDQLWPTLSVLPNKTYFLRNVNIAGYAQFYFHIDAQHMTIIQADGVYTDLQLVQDLYLATGQRYGVLLHTLPTPCQNYVMLGAMDIAGFPGSAPTPVSPNVTGVLVYDPYLSIPSTPLVEQFIAFDDFCLTPIDSNILLDPPNKEIVLNLSTFSQVVPLNEQNRGGFNNITYITQRVPSLYTALSGGLYALNPIVYGNHSNAFVLNLNDIVETTIYNYDTGLHPIHTHGHNVQLIYRTGSSCKNYRIPMRRDTWMEPRWNSLDPNHPSTVVRFVADNPGIWFLHCHMEWHLVAGVLIILVEDPLQIQRGQRDIPLSMKLICLDQKIPLKGNAAGNWINFLDLTGEVNVAPMECGSLSSTTIFDWEDLSTAP The laccase 3 gene encodes a 691 amino acid sequence as follows (SEQ ID NO. 3) MQRRLSKRQQQQDATSRPDATAQPVEQQQKTEANDKRRGSKTTLHTPSILIALVCILPFLGSVVLFVRYYYESNNDDSINRLSNSLHDHESISSTLKYDNRIHENGRLRPEDHIHVAAITQTLNWSVTAGQRRPDGVDKRIYLINDMFPGPSIEARSGDTLQILVHNNLEDEQISLHWHGLNMRGANTMDGVIGVTQCGIQPGQSFWYNFTISETQSGTFWYVKHSAVQRADGLYGSLVVHRPDSTLVSPLLRSEMVSDSVKYGYDKEIITMIGDWSPRTATDVASWYLWWGSMGYEPVPDSLLVNGAGRFNCSRAVRARPLDCIGSADEMPPLILDGNSSYRVRVVNTGSLTGIILGFAPGTSIQVITIDGGNPVEMEAVDGDVSSVGILFPGQRVDFVLRPLKGQTSWMTVKMDDSYVSPYTLYKTAELSVSSDFTIGNPALVPQQSFPIFQTPLPSDSLPSSPTAQSNNTNDIEEDDIININTLPSTRTLLSSLPPKSEQTHIVYTKVEKLSRLDNIPHGFFNRTTWKIQSDPPYPLLGLPRNQWDKHQFAVSTGIHNGWVDLVVNNLDEGAHPFHLHGYNFYVVDIYESPEGSGRWGSYNPWTSPSFNENIDPYDLTKAVVLDTVQIPRRGYAVLRFKADNPGVWLFHCHVMWHLAFGMAMVIDSGSGDDSVAHEPWLAEEGMECRV The nitropropane dioxygenase gene encodes a 353 amino acid sequence as follows (SEQ ID NO. 4) MASPQNIRTPVTDLLKINHPILLDFMNVAAGPKLAAAVTMKGGLGVIGGIGYTPEMLQWQLNELKGYLNDKNAPFGVDLLLPQVGAFARKTNHDYTKGKLNELIDIIIESGAKLFVWVGVPPKHVVEKLHKAGVLYMNMIGHPKHVKQLELGVDIICAQGGEGGGHTGDVPTTVLIPTVAKLVQGHKSPLTGAPVQVIAAGGLFNGQSVAAALOLGASAVWIGTRFILSEEAGASEAOKEAVRTAGFDDNIRTIIFTGKPLRVRNNATITNWEENRSQEIKDLTSKGIIPVEHDFENLPDDVDEEVLENARPFLMGKVAAVLTEKKPAKAIVDELVTDASGHLQKGNKMIAKL Hydrogenase coenzyme dioxygenase gene encodes 303 amino acid sequence as follows (SEQ ID NO. 5) MKTVDEFDRTTSPYADEIVASOIRNGGCVIRNMITDQMIODTLERDIRPHIEADRPWVHHDFFPPETRRVNGLVGKSRTFSENIPANKLYLGECSRLLSSTHSAWLGYQLNTTVSEPVLSNTIVFSIGPGAKRQELHRDDSIHHNTLIELKSHDQYRIGRDTSVGLFVAGKKTMRANGATRFIPGSHLWGDARCPDEEITYYAELEPGEAFLMLASCYHGGSAMMTPDQERLVYSCFMTKGYLRQEENQYLANTIQQVKLYPTELQRLIGYSVSKPFLGWVNLEDPIKLLHDDPETVGDFGMR Squalene monooxygenase gene encodes 2072 amino acid sequence as follows (SEQ ID NO. 6) The catalase gene encodes a 734 amino acid sequence as follows (SEQ ID NO. 7) MRGLYSLGAFASLIAAASAACPMLTGEIPAGSVANPHHHGKRDDSNASSETEAFLSEFYLNDNDAYLTTDVGGPIEDQNSLKAGIRGSTLLEDFIFRQKIQHFDHERVPERAVHARGAGAHGVFTSYADWSNITAASFLGASGKETPTFVRFSTVAGSRGSADTARDVHGFATRFYTDEGNYDIVGNNIPVFFIQDAILFPDLIHSVKPQPANEIPQAATAHDTAYDFFGQQPSTLHTLFWAMAGHGIPRSFRHVDGFGVHTYRFVTDDGSSKLVKFHWTSLQGRASLVWEEAQATAGKNADFMRQDLYDSIEAGRYPEWELGVQIIEESDVLSYGFDLLDPTKILPVEKVPITALGKMQLNRNPLNYFAETEQVMFQPGHIVRGIDFTEDPLLQGRLFSYLDTQLNRNGGPNFEQIPINRPRVPIHNNNRDGFAQMFIPLNQAAYSPNTLNNGSPRQANETVGNGFFTAPGRSADGHLVRATSPTFADVWSQPGLFYNSLTATEQQFVINALRFELSNVKSEDVKSNFITQINRVNNTLATLVASAIGVSAPEPDSTYYHSNKTSNVGTFGTPLKKLDGLKVGVLASVNGESSIAEGQALAQSLAGSNVDVVIVAEHLTSNVSATYSGSDATNFDAVIVSSGAEGLFGPQTFTAESNTTLYPAGRPSQILVDAFRFGKPVGAVGGASAALSAVDISTDRSGVITGDSVSDDFVKQLTEDLATFKFLDRFAVDE Six, application of optimized laccase, monooxygenase, dioxygenase and other oxidase system strains The Ta-lac bacterial liquid stored at -80℃ was streak inoculated in the CM culture medium and cultured in a 37℃ incubator for 72 h. Sterilized distilled water was added to the above culture medium, and spores were scraped and collected to prepare a spore suspension. After microscopic counting, the spore suspension was diluted to 5×10 6 / mL of inoculum was inoculated into lignin carbon source medium (lignin 1.7 wt%, potassium nitrate 0.7 wt%, casein hydrolysate 0.1 wt%, ABTS 0.08 wt%, KCl to supplement K + The final concentration was 0.3 wt%) and cultured at 37°C, 180 rpm / min for 96 h.

[0038] After centrifugation of the 96 h fermentation broth at 4500 rpm, 0, 200, 400, 600, 800 μL of crude enzyme solution was added into 20 mg·L -1 of crystal violet (maximum absorption wavelength 591 nm) and malachite green (maximum absorption wavelength 616 nm) solution, respectively, and the absorbance was measured after 96 h of reaction at 30°C. The experimental results are shown in Table 1. Figures 9-12 The degradation results show that when the crude enzyme solution addition is 600 μL or more, both crystal violet and malachite green have very significant degradation effect (the color of the sample in the cuvette becomes lighter), and the crude enzyme solution shows obvious dose-dependent degradation effect on both dyes. With the increase of the amount of crude enzyme solution, the degradation effect is more obvious. The degradation rate of crystal violet is 25.7%, 35.8%, 71.4% and 78.9%, respectively, and the degradation rate of malachite green is 9.6%, 10.2%, 24.8% and 33.8%, respectively. Among them, the degradation effect of the crude enzyme solution on crystal violet is significantly better than that on malachite green, and in both dye systems, the higher the enzyme addition, the higher the degradation rate.

[0039] The embodiments of the specific embodiment are the preferred embodiments of the present application, and do not limit the protection scope of the present application. Therefore, any equivalent changes made according to the structure, shape, principle of the present application should be covered within the protection scope of the present application.

Claims

1. A basket bacterium with the ability to co-express multiple oxidases ( Talaromyces sp. Ta-lac, a basket-shaped bacterium, was deposited on July 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 42125.

2. The basket fungus according to claim 1, characterized in that: The Talaromyces sp. can simultaneously produce laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase.

3. Use of the Talaromyces sp. in claim 1 in fermentation for producing laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase.

4. Use of the Talaromyces sp. in claim 1 in degrading environmental pollutants.

5. Use according to claim 4, characterized in that: The environmental pollutants include dyes and lignin.

6. A method for producing laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase using the fermentation of the Talaromyces of claim 1, characterized by: The method comprises the following steps: a. inoculating the Talaromyces sp. Ta-lac preserved at -80℃ into CM culture medium by streaking and culturing at 35-37℃ for 70-72h; b. adding sterilized distilled water to the culture plate, scraping the spores and collecting the spore suspension; c. The spore suspension was inoculated in fermentation medium at an inoculum of 5 x 10 5 -5 x 10 6 mL / mL and incubated at 35-37 °C, 180-200 rpm / min for 94-96 h.

7. The method of claim 6, wherein: The formula of the CM culture medium is: glucose 1 wt%, 1 mol / L MgSO4 mother liquor 0.2 wt%, casein hydrolysate 0.1 wt%, yeast extract 0.5 wt% and ABTS 0.08 wt%.

8. The method of claim 6, wherein: The recipe of the fermentation medium was lignin 1.7 wt%, potassium nitrate 0.7 wt%, casein hydrolysate 0.1 wt%, ABTS 0.08 wt%, adjusted to a K + Final concentration 0.3 wt%.

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