Talaromyces with multi-oxidase synergistic expression ability and fermentation method and application thereof

Abstract

The application discloses a Talaromyces with multi-oxidase synergistic expression capacity and a fermentation method and application thereof. Talaromyces sp. The Talaromyces (Talaromyces autumnalis) Ta-lac is preserved in the China General Microbiological Culture Collection Center on July 11, 2025, and the preservation number is CGMCC No. 42125. Compared with the engineering strain for biosynthesis of laccase, the strain has strong environmental adaptability, can naturally synthesize laccase, and has relatively stable genome, no potential risk of exogenous genes and relatively high safety. The Talaromyces has a short growth cycle, high yield of laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase and high lignin degradation activity in a fermentation process. The Talaromyces Ta-lac is applied to degradation of toxin dye wastewater, and has remarkable detoxification and decolorization effects.

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 produces laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase simultaneously.

[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 basket fungus colony 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 colony color 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 substrate is lignin or corn cob powder, and the laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase have high yield and high substrate degradation rate.

[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 achieved by the following technical scheme.

[0011] The application of the basket fungus in the fermentation production of laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase.

[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 achieved by the following technical scheme.

[0013] The application of the basket fungus in the degradation of environmental pollutants.

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

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

[0016] A method for the fermentation production of laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase by using the basket fungus, which comprises the following steps:

[0017] a. The basket fungus Ta-lac liquid stored at-80 DEG C is streaked on a CM culture medium, and cultured at 35-37 DEG C for 70-72 h;

[0018] b. Sterilized distilled water is added to the cultured plate, and spores are scraped to collect and prepare a spore suspension;

[0019] c. The spore suspension was inoculated into the fermentation medium at an inoculation amount of 5x10 5 -5x10 6 / mL, and cultured at 35-37 ℃, 180-200 rpm / min for 94-96 h.

[0020] Further, the formula of the CM medium is glucose 1 wt%, 1 mol / L MgSO4 mother liquor 0.2 wt%, casein hydrolysate 0.1 wt%, yeast extract 0.5 wt%, ABTS 0.08 wt%.

[0021] Further, the formula of the fermentation medium is lignin 1.7 wt%, potassium nitrate 0.7 wt%, casein hydrolysate 0.1 wt%, ABTS 0.08 wt%, and the K + concentration in the medium is adjusted to 0.3 wt%.

[0022] The application has the following beneficial effects.

[0023] 1. Based on the natural characteristics of the Talaromyces sp. in high-yield oxidation enzyme, the monooxygenase and dioxygenase produced in the fermentation process play a role together 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.

[0024] 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 resistance to antibiotics.

[0025] 3. The Talaromyces sp. naturally producing laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase has an effect on the degradation of different dyes, providing a new direction for dye decolorization and toxin degradation. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a gel map of the 18S rDNA PCR electrophoretic product of the Talaromyces sp. Ta-lac of the application (lanes 1-5 are all 18S rDNA PCR products);

[0027] Figure 2 is an evolutionary tree map constructed after sequence comparison of the 18S rDNA of the Talaromyces sp. Ta-lac of the application;

[0028] Figure 3 is a different temperature optimization result graph of the Tulasnella Ta-lac of the present application;

[0029] Figure 4 is a different pH optimization result graph of the Tulasnella Ta-lac of the present application;

[0030] Figure 5 is a different carbon source optimization result graph of the Tulasnella Ta-lac of the present application;

[0031] Figure 6 is a different nitrogen source optimization result graph of the Tulasnella Ta-lac of the present application;

[0032] Figure 7 is a different metal ion optimization result graph of the Tulasnella Ta-lac of the present application;

[0033] Figure 8 is a SDS-PAGE verification result graph of the fermentation liquid of the Tulasnella Ta-lac of the present application;

[0034] Figure 9 is a degradation result graph of the Tulasnella Ta-lac on crystal violet;

[0035] Figure 10 is a degradation result graph of the Tulasnella Ta-lac on malachite green;

[0036] Figure 11 is a degradation rate graph of the Tulasnella Ta-lac on crystal violet;

[0037] Figure 12 is a degradation rate graph of the Tulasnella Ta-lac on malachite green. DETAILED DESCRIPTION

[0038] The application will be further described below in conjunction with the drawings and examples. Unless otherwise specified, the experimental methods used in the application are conventional methods, and the experimental apparatus, materials, reagents, etc. used can be purchased from relevant material selling companies.

[0039] The application provides a Tulasnella capable of simultaneously producing laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase, which is screened from a sample collected from a pine tree in the Mangshan National Forest Park in Yizhang County, Chenzhou City, Hunan Province, and preserved in the China General Microbiological Culture Collection Center on July 11, 2025, with a preservation number of CGMCC No.42125.

[0040] Specifically, the screening method of the Tulasnella capable of simultaneously producing laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase and squalene monooxygenase includes the following steps:

[0041] I. Isolation and screening of the strain

[0042] Take 2 g of the pulverized sample of the fir tree from the Mangshan National Forest Park in Yizhang County, Chenzhou City, Hunan Province, and add it to 50 mL of sterilized LB medium (1 wt% of tryptone, 0.5 wt% of yeast extract, and 1 wt% of NaCl) for enrichment, and cultivate it at 37 °C and 180 rpm / min for 24 h. Take 1 mL of the bacterial suspension for enrichment cultivation, and perform 10 -1 ~10 -7 Gradient dilution, take 100 μL of the dilution liquid and spread it on PDA medium containing 0.08 wt% of ABTS, and cultivate it at 37 °C for 72 h. Select single colonies with good growth morphology, moderate size, and obvious dark green hydrolysis rings, and purify and isolate them by streaking multiple times to obtain the strain Ta-lac.

[0043] II. Identification of the strain

[0044] 1. Physiological and biochemical property identification

[0045] The strain Ta-lac has round or irregular-shaped colonies on CM medium [1 wt% of glucose, 0.2 wt% of 1 mol / L MgSO4 stock solution, 0.1 wt% of casein hydrolysate, 0.5 wt% of yeast extract, and 0.08 wt% of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)], with dense rope-shaped mycelium on the surface, smooth and moist overall, neat edges, viscous texture, and a bean green color with certain gloss and elasticity. The carbon sources that can be utilized include fructose, maltose, sucrose, starch, arabinose, and cellulose, and the nitrogen sources that can be utilized include ammonium sulfate, ammonium chloride, potassium nitrate, urea, and yeast powder. The strain can produce laccase, various monooxygenases and dioxygenases, and catalase.

[0046] 2. 18S rRNA sequence identification

[0047] Inoculate the activated strain Ta-lac in liquid CM medium, and cultivate it at 37 °C and 180 rpm for 24 h. Take 1 μL of the bacterial liquid as a template, and use the universal primers 18S-F: 5'-CCAGTAGTCATATGCTTGTCT-3' (SEQ ID NO. 8) and 18S-R: 5'-ACCTTGTTACGACTTTTACTTCC-3' (SEQ ID NO. 9) to amplify the 18S rDNA gene of the strain. Take 2 μL of the PCR product, and perform 1% agarose gel electrophoresis for verification (Fig. 2) before sending the product to Beijing Huada Gene Company. Figure 1

[0048] Based on the identification results and sequence alignment, it is determined that the strain Ta-lac belongs to the genus Talaromyces.​Talaromyce sp. ), and compared with NCBI data, it was determined to be a basket fungus with 99% query coverage, and finally named Talaromyces sp. Ta-lac. The phylogenetic tree of the basket fungus Ta-lac is shown in Figure 2 .

[0049] III. Seed culture optimization

[0050] The Ta-lac bacterial solution stored at -80 ℃ was streak inoculated on CM medium and cultured in a 37 ℃ incubator for 72 h. Sterilized distilled water was added to the above-mentioned 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 a 10 6 / mL inoculum and placed in 25 ℃, 28 ℃, 30 ℃, 37 ℃ and 42 ℃ incubators for 96 h. The growth state was observed and recorded every 24 h. The results are shown in Figure 3 At 48 h of plate culture, a small amount of mycelium germination was observed at 25 and 28 ℃, white mycelium was clearly visible at 30 and 37 ℃, and no germination was observed at 42 ℃. Therefore, the optimum growth temperature of the basket fungus Ta-lac was 30-37 ℃, and the best growth was observed at 37 ℃.

[0051] 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 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 the basket fungus Ta-lac was inoculated in CM liquid medium at a 10 6 / mL inoculum, cultured at 37 ℃ and 180 rpm / min for 96 h, and the resulting fermentation broth was centrifuged at 4500 rpm to obtain the crude enzyme solution) was added to 800 μL of the above-mentioned solution, and the total oxidase activity under different pH conditions was measured after 1 min of reaction at 37 ℃. An equal volume of distilled water was used as a blank control instead of the enzyme solution. 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 3 times. The total oxidase activity calculation formula is: 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 is 1 cm; t is the reaction time, 1 min. The experimental results are shown inFigure 4 The results showed that the optimal pH for *Ta-lac* was 2.5-3.5, with the best growth observed at pH 3.

[0052] IV. Fermentation Culture Medium Optimization

[0053] 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. + .

[0054] The Ta-lac bacterial liquid stored at -80°C was streak inoculated in CM medium and cultured in a 37°C incubator for 72 h. Sterilized distilled water was added to the above-mentioned medium, spores were scraped and collected to prepare a spore suspension, which was diluted after microscopic counting. The spore suspension was inoculated in an optimized medium (lignin 1.7 wt%, potassium nitrate 0.7 wt%, casein hydrolysate 0.1 wt%, ABTS 0.08 wt%, KCl to supplement K 6 final concentration to 0.3 wt%) at an inoculation amount of 5x10 + / mL, and fermented at 37°C and 180 rpm / min for 96 h. The fermented liquid was concentrated and denatured, and then subjected to SDS-PAGE protein gel verification. After staining and decolorizing, different oxidase molecular weights were compared. The experimental results are shown in

[0055] V. SDS-PAGE and mass spectrometry verification of the fermented liquid

[0056] The Ta-lac bacterial liquid stored at -80°C was streak inoculated in CM medium and cultured in a 37°C incubator for 72 h. Sterilized distilled water was added to the above-mentioned medium, spores were scraped and collected to prepare a spore suspension, which was diluted after microscopic counting. The spore suspension was inoculated in an optimized medium (lignin 1.7 wt%, potassium nitrate 0.7 wt%, casein hydrolysate 0.1 wt%, ABTS 0.08 wt%, KCl to supplement K 6 final concentration to 0.3 wt%) at an inoculation amount of 5x10 + / mL, and fermented at 37°C and 180 rpm / min for 96 h. The fermented liquid was concentrated and denatured, and then subjected to SDS-PAGE protein gel verification. After staining and decolorizing, different oxidase molecular weights were compared. The experimental results are shown in Figure 8 .

[0057] The protein gel map of the fermented liquid was subjected to mass spectrometry analysis (the mass spectrometry analysis was completed by the public technical center of the Chinese Academy of Sciences), and the experimental results are shown in Table 1. The experimental results show that the Talaromyces Ta-lac fermentation of the present application can simultaneously produce laccase, nitropropane dioxygenase, hydrogenase coenzyme dioxygenase, squalene monooxygenase and catalase.

[0058] Table 1

[0059]

[0060] The laccase 1 gene encodes a 562-amino-acid sequence as follows (SEQ ID NO. 1)

[0061] MVLRGILLLHYFLPGLHMPFGAAVEPDSGNTAADRSKWCTYNIHTDYYNVVPDTGVTREYWFDLRDRVLAPDGIRPYTQSINGSIPGPTIRANWGDEVVVHVQNNFQNTSNGTSLHFHGIRQNYTNQNDGVVSVTECPTAPHGRTTYKRWAAQYGTTFYHSHFSLQAHQGVFGAIVIDGPASANYDVDQGALILTDWGHQTPDEIFYHESRHQLPTYLENGLINGMNIYNIGDKQVTYRFTIEFQPGTSYRLRLIADALDTHFTFDTDHHTMTVIAADLVPLEEPTTNVITIGIGQRYDVIVKADQGIAHEFWMRAVPQKLCSRNNNTENIRGIVYYTDTVQGQPSTSGYNTTTGCVDQTDIAQPVSKSVSEDFVYYAVAALTIGKNTAGLFVWQVNDVSMQVEVVNPTLLQIYNNVTDWSSTEGFFQLDEVDKWIYVLIQSSLPISHPMHLHGHDFEELAQGTGAYEPSAAVSNFDNPPRRDTGLLLAGGYLLLAFQTDNPGAWLLHCHVGFHATCCLAVQFVERYDEIRDLELYESLSQTCKAWDSWEKSKGLVEVDSGI

[0062] The laccase 2 gene encodes a 680 amino acid sequence as follows (SEQ ID NO. 2)

[0063] MMHRVVVSLCCYGGYFTCCYPTAWFNNHMLKTLISMAINPIKVNLGRKPRMLQQVQQRSHLLTLLNGSKQSQAQVLVLSMVLVATGLAFTSLDIPALYLFFDNVSLKLQGTKTLDQPTILEYQTAICQOSLGQYHSGRKKSWPRHVIPDINLERSSGVKSMSILNDRTCLLLTDTSPVIAINGVWPAPMIRGTVNDTVVVKVHPPLGNQSLSIHWSSLHMVVSVLVMHCMQLDEPGTCCYHSHSPSQYVDGIRGPILVDDPFSPYADQDWGELVVTLSDWYHVCMLTRGIIALPGNPTALEPIPMSALMNDQLWPTLSVLPNKTYFLRNVNIAGYAQFYFHIDAQHMTIIQADGVYTDLQLVQDLYLATGQRYGVLLHTLPTPCQNYVMLGAMDIAGFPGSAPTPVSPNVTGVLVYDPYLSIPSTPLVEQFIAFDDFCLTPIDSNILLDPPNKEIVLNLSTFSQVVPLNEQNRGGFNNITYITQRVPSLYTALSGGLYALNPIVYGNHSNAFVLNLNDIVETTIYNYDTGLHPIHTHGHNVQLIYRTGSSCKNYRIPMRRDTWMEPRWNSLDPNHPSTVVRFVADNPGIWFLHCHMEWHLVAGVLIILVEDPLQIQRGQRDIPLSMKLICLDQKIPLKGNAAGNWINFLDLTGEVNVAPMECGSLSSTTIFDWEDLSTAP

[0064] The laccase 3 gene encodes a 691 amino acid sequence as follows (SEQ ID NO. 3)

[0065] MQRRLSKRQQQQDATSRPDATAQPVEQQQKTEANDKRRGSKTTLHTPSILIALVCILPFLGSVVLFVRYYYESNNDDSINRLSNSLHDHESISSTLKYDNRIHENGRLRPEDHIHVAAITQTLNWSVTAGQRRPDGVDKRIYLINDMFPGPSIEARSGDTLQILVHNNLEDEQISLHWHGLNMRGANTMDGVIGVTQCGIQPGQSFWYNFTISETQSGTFWYVKHSAVQRADGLYGSLVVHRPDSTLVSPLLRSEMVSDSVKYGYDKEIITMIGDWSPRTATDVASWYLWWGSMGYEPVPDSLLVNGAGRFNCSRAVRARPLDCIGSADEMPPLILDGNSSYRVRVVNTGSLTGIILGFAPGTSIQVITIDGGNPVEMEAVDGDVSSVGILFPGQRVDFVLRPLKGQTSWMTVKMDDSYVSPYTLYKTAELSVSSDFTIGNPALVPQQSFPIFQTPLPSDSLPSSPTAQSNNTNDIEEDDIININTLPSTRTLLSSLPPKSEQTHIVYTKVEKLSRLDNIPHGFFNRTTWKIQSDPPYPLLGLPRNQWDKHQFAVSTGIHNGWVDLVVNNLDEGAHPFHLHGYNFYVVDIYESPEGSGRWGSYNPWTSPSFNENIDPYDLTKAVVLDTVQIPRRGYAVLRFKADNPGVWLFHCHVMWHLAFGMAMVIDSGSGDDSVAHEPWLAEEGMECRV

[0066] The nitropropane dioxygenase gene encodes a 353 amino acid sequence as follows (SEQ ID NO. 4)

[0067] MASPQNIRTPVTDLLKINHPILLDFMNVAAGPKLAAAVTMKGGLGVIGGIGYTPEMLQWQLNELKGYLNDKNAPFGVDLLLPQVGAFARKTNHDYTKGKLNELIDIIIESGAKLFVWVGVPPKHVVEKLHKAGVLYMNMIGHPKHVKQLELGVDIICAQGGEGGGHTGDVPTTVLIPTVAKLVQGHKSPLTGAPVQVIAAGGLFNGQSVAAALOLGASAVWIGTRFILSEEAGASEAOKEAVRTAGFDDNIRTIIFTGKPLRVRNNATITNWEENRSQEIKDLTSKGIIPVEHDFENLPDDVDEEVLENARPFLMGKVAAVLTEKKPAKAIVDELVTDASGHLQKGNKMIAKL

[0068] Hydrogenase coenzyme dioxygenase gene encodes 303 amino acid sequence as follows (SEQ ID NO. 5)

[0069] MKTVDEFDRTTSPYADEIVASOIRNGGCVIRNMITDQMIODTLERDIRPHIEADRPWVHHDFFPPETRRVNGLVGKSRTFSENIPANKLYLGECSRLLSSTHSAWLGYQLNTTVSEPVLSNTIVFSIGPGAKRQELHRDDSIHHNTLIELKSHDQYRIGRDTSVGLFVAGKKTMRANGATRFIPGSHLWGDARCPDEEITYYAELEPGEAFLMLASCYHGGSAMMTPDQERLVYSCFMTKGYLRQEENQYLANTIQQVKLYPTELQRLIGYSVSKPFLGWVNLEDPIKLLHDDPETVGDFGMR

[0070] Squalene monooxygenase gene encodes 2072 amino acid sequence as follows (SEQ ID NO. 6)

[0071]

[0072] The catalase gene encodes a sequence of 734 amino acids as follows (SEQ ID NO. 7)

[0073] MRGLYSLGAFASLIAAASAACPMLTGEIPAGSVANPHHHGKRDDSNASSETEAFLSEFYLNDNDAYLTTDVGGPIEDQNSLKAGIRGSTLLEDFIFRQKIQHFDHERVPERAVHARGAGAHGVFTSYADWSNITAASFLGASGKETPTFVRFSTVAGSRGSADTARDVHGFATRFYTDEGNYDIVGNNIPVFFIQDAILFPDLIHSVKPQPANEIPQAATAHDTAYDFFGQQPSTLHTLFWAMAGHGIPRSFRHVDGFGVHTYRFVTDDGSSKLVKFHWTSLQGRASLVWEEAQATAGKNADFMRQDLYDSIEAGRYPEWELGVQIIEESDVLSYGFDLLDPTKILPVEKVPITALGKMQLNRNPLNYFAETEQVMFQPGHIVRGIDFTEDPLLQGRLFSYLDTQLNRNGGPNFEQIPINRPRVPIHNNNRDGFAQMFIPLNQAAYSPNTLNNGSPRQANETVGNGFFTAPGRSADGHLVRATSPTFADVWSQPGLFYNSLTATEQQFVINALRFELSNVKSEDVKSNFITQINRVNNTLATLVASAIGVSAPEPDSTYYHSNKTSNVGTFGTPLKKLDGLKVGVLASVNGESSIAEGQALAQSLAGSNVDVVIVAEHLTSNVSATYSGSDATNFDAVIVSSGAEGLFGPQTFTAESNTTLYPAGRPSQILVDAFRFGKPVGAVGGASAALSAVDISTDRSGVITGDSVSDDFVKQLTEDLATFKFLDRFAVDE

[0074] Six, application of optimized laccase, monooxygenase, dioxygenase and other oxidase system strains

[0075] 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.

[0076] 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.

[0077] 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 use of the basket-shaped bacteria of claim 1 in the fermentation production of laccase, nitropropane dioxygenase, hydrogenase-coenzyme dioxygenase and squalene monooxygenase.

3. The application of the basket-shaped bacteria of claim 1 in the degradation of environmental pollutants, characterized in that: The environmental pollutants mentioned are crystal violet, malachite green, and lignin.

4. A method for producing laccase, nitropropane dioxygenase, hydrogenase-coenzyme dioxygenase, and squalene monooxygenase by fermentation using the basket bacteria described in claim 1, characterized in that: Includes the following steps: a. Streak the Ta-lac bacterial suspension stored at -80℃ onto CM medium and incubate at 35-37℃ for 70-72h; b. Add sterile distilled water to the cultured plate, scrape off the spores, and collect them to prepare a spore suspension; c. Prepare the spore suspension at a ratio of 5 × 10 5 -5×10 6 Inoculate the culture medium with an inoculum of 1 / mL and incubate at 35-37 ℃ and 180-200 rpm / min for 94-96 h.

5. The method according to claim 4, characterized in that: The formulation of CM medium is as follows: 1 wt% glucose, 0.2 wt% 1 mol / L MgSO4 stock solution, 0.1 wt% casein hydrolysate, 0.5 wt% yeast extract, and 0.08 wt% ABTS.

6. The method according to claim 4, characterized in that: The fermentation medium was formulated with 1.7 wt% lignin, 0.7 wt% potassium nitrate, 0.1 wt% casein hydrolysate, and 0.08 wt% ABTS. The potassium content in the medium was adjusted... + The final concentration was 0.3 wt%.

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