Mistletoe endophytic fungus VEP-4 and application thereof

Abstract

The invention relates to a viscum endophytic fungus (Coprinella sp.) VEP-4, and belongs to the technical field of biotechnology. The invention aims to solve the technical problems of low yield and unstable enzyme activity of fungal cellulase. The invention provides a high-yield cellulase strain and a fermentation method thereof, the fungus is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.41451. Liquid fermentation conditions are optimized, the liquid fermentation conditions comprise a carbon source sodium carboxymethyl cellulose and a nitrogen source peptone, the inoculum size is 5%, the initial pH is 5.0, and the fermentation temperature is 28 DEG C, so that the enzyme activity of CMCase is improved to 8.57 U / mL; the method is mainly used for producing cellulase so as to efficiently degrade cellulose-containing substances, and is applied to conversion of agricultural wastes into bioethanol, environment-friendly degradation of traditional Chinese medicine residues or cellulose treatment in paper-making industry.

Description

Technical Field

[0001] The invention belongs to the technical field of biotechnology, and particularly relates to a mistletoe endophytic fungus VEP-4 and an application thereof. Background Art

[0002] Endophytic fungi are fungal communities that colonize within host plant tissues without causing noticeable symptoms. These fungi form a symbiotic relationship with their hosts, resident in organs such as roots, stems, and leaves, often promoting or neutrally impacting their growth. The study of endophytic fungi is crucial for understanding the mechanisms of biological interactions and developing their applications.

[0003] Cellulose reserves are abundant in nature, and its efficient degradation relies on the action of cellulase (β-1,4-glucanohydrolase). This enzyme, a glycoside hydrolase, hydrolyzes cellulose into glucose through the synergistic action of exoglucanases (CBH), endoglucanases (EG), and β-glucosidases (BGL). Cellulase boasts high hydrolysis efficiency, low energy consumption, and environmental friendliness, making it widely used in the food, textile, papermaking, biofuel, and pharmaceutical industries.

[0004] Currently, industrial cellulase is primarily derived from microbial fermentation. Its production requires complex processes such as strain selection, process optimization, and separation and purification, resulting in high costs. Existing enzyme-producing strains have significant limitations: 1) Fungal strains (such as Trichoderma reesei and Trichoderma viride) require a strictly aerobic environment, limiting their application in anaerobic scenarios (such as sewage treatment); 2) Actinomycetes have low enzyme production efficiency; and 3) Bacterial cellulase is mostly intracellular or bound to the cell wall, resulting in insufficient enzyme activity and difficulty in extraction.

[0005] Research on the endophytic fungi of mistletoe (Viscum coloratum), a medicinal plant of the Morus albiflora family, has focused on secondary metabolites, while the exploration of cellulase resources has been inadequate. The process by which mistletoe invades the host's vascular bundles through haustoria suggests that cellulases secreted by these endophytes play a key role in breaking through the host cell wall. However, existing research has yet to establish an efficient enzyme production system for mistletoe endophytes, resulting in technical bottlenecks such as low enzyme yield and unstable activity. Summary of the Invention

[0006] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0007] The present invention at least solves the following technical problems:

[0008] The present invention solves the problem of the scarcity of existing cellulase-producing bacterial strain resources and provides a novel endophytic fungus derived from mistletoe, which has the potential for natural high-yield cellulase and provides specific bacterial strain resources for industrial applications.

[0009] In order to address the defects of low enzyme production efficiency and unstable process in traditional fungal fermentation, an efficient and controllable liquid fermentation process was established by optimizing core parameters such as the carbon-nitrogen source ratio, pH and temperature.

[0010] To overcome the problem of enzyme protein inactivation caused by environmental disturbances (such as pH fluctuations and shear force) in the late fermentation period, trehalose was introduced as a bioprotectant to significantly improve the operational stability of cellulase.

[0011] To solve the problem of decreased mass transfer efficiency caused by a sudden increase in the viscosity of the fermentation broth after the addition of trehalose, the ultrasonic cavitation effect is used to improve the fluid dynamics characteristics and ensure uniform contact between the bacteria and nutrients.

[0012] Eliminate the inhibition of bacterial growth caused by the accumulation of metabolic by-products (organic acids, etc.), continuously neutralize acidic substances by loading calcium carbonate on mesoporous materials, and maintain the physiological activity of the fermentation system.

[0013] To prevent CO2 microbubbles generated by the decomposition of calcium carbonate from being retained in the fermentation liquid and hindering the exchange of substances between the bacteria and the culture medium, light pulses are used to induce controllable microflow to eliminate bubble blockage.

[0014] It protects against oxidative damage to bacterial cell membranes and enzyme proteins caused by reactive oxygen species bursts induced by light pulse treatment, and promptly removes free radicals and protects biological activity by adding reduced glutathione.

[0015] To solve the technical difficulties of easy shedding and pore clogging of calcium carbonate in traditional loaded particles, the interfacial bonding strength is enhanced through amino modification, and the carrier stability is improved by combining freeze-drying and solidification process.

[0016] Clarify the application value of strains in cellulose degradation scenarios, provide efficient biocatalysts for biofuel production, environmentally friendly degradation and industrial processing, and expand the circular economy path.

[0017] Another object of the present invention is to provide a mistletoe endophytic fungus VEP-4, which can produce cellulase and has good cellulase activity.

[0018] In order to achieve these purposes and other advantages of the present invention, a mistletoe endophytic fungus (Coprinellus rediens) VEP-4 is provided. The fungus was deposited with the China General Microbiological Culture Collection Center on September 9, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO.41451.

[0019] A method for producing cellulase comprises the following steps: inoculating mistletoe endophytic fungus VEP-4 into a liquid fermentation medium, wherein the carbon source of the liquid fermentation medium is sodium carboxymethyl cellulose and the nitrogen source is peptone; the inoculation amount is 4% to 6%; adjusting the initial pH of the fermentation system to 4.5 to 5.5; and fermenting and culturing at 27°C to 29°C, wherein the obtained fermentation broth contains cellulase.

[0020] Preferably, trehalose with a concentration of 0.1-0.5 mol / L is added as an enzyme stabilizer during the fermentation culture process; the trehalose is added to the fermentation system once in the middle of the fermentation period of 24-48 hours.

[0021] Preferably, within 0.5-1 hour after the addition of trehalose, the fermentation broth is subjected to ultrasonic treatment at a frequency of 20-40 kHz and a power density of 0.1-0.3 W / mL for 5-10 minutes.

[0022] Preferably, mesoporous silica-loaded calcium carbonate particles accounting for 0.1-0.3% of the fermentation broth volume are added to the fermentation system 48 hours after the start of fermentation, with a particle size of 5-20 μm, a mesopore diameter of 5-10 nm, and a calcium carbonate loading of 40-60% of the total mass of the particles.

[0023] Preferably, within 2-3 hours after adding the mesoporous silica-loaded calcium carbonate particles, the fermentation system is subjected to a pulse with a wavelength of 400-700 nm, a pulse width of 1-5 μs, and an energy density of 0.5-1.5 J / cm 2 The visible light pulse irradiation lasts for 10-20 seconds.

[0024] Preferably, within 1-3 minutes after the visible light pulse irradiation ends, reduced glutathione at a concentration of 0.05-0.2 mM is added to the fermentation system, and the fermentation broth is continuously mixed at a stirring rate of 50-100 rpm for 10-20 minutes.

[0025] Preferably, the mesoporous silica-supported calcium carbonate particles are prepared by the following steps:

[0026] a) Surface modification of mesoporous silica by amino modification: Mesoporous silica particles are dispersed in anhydrous ethanol, 3-aminopropyltriethoxysilane is added at a concentration of 1-3% of the total volume of the solution, and the mixture is refluxed at 60-80° C. for 4-6 hours. The mixture is centrifuged and washed to obtain an amino-modified mesoporous silica support;

[0027] b) In-situ mineralization of calcium carbonate loading: The amino carrier is dispersed in deionized water, a 0.1-0.3 mol / L calcium chloride solution is added, and after stirring for 10-20 minutes, an equimolar ammonium carbonate solution is added dropwise. The pH of the reaction system is controlled to 8.5-9.5, and the reaction is continued at 25-30°C for 30-60 minutes;

[0028] c) Freeze-drying and solidification: After the reaction is completed, the particles are collected by centrifugation, quickly frozen with liquid nitrogen, and placed in a freeze dryer. Dry at -50°C and 10-15 Pa vacuum for 24-36 hours to obtain loaded particles.

[0029] The invention discloses an application of mistletoe endophytic fungus VEP-4 for producing cellulase or degrading organic matter containing cellulose.

[0030] The present invention has at least the following beneficial effects:

[0031] The mistletoe endophytic fungus VEP-4 has good cellulase activity, including FPase (filter paper enzyme), CMCase (carboxymethyl cellulase) and β-glucosidase (β-glucosidase) enzyme activities.

[0032] By optimizing the carbon source, nitrogen source, inoculation amount, initial pH, and fermentation temperature in the fermentation medium, the CMCase activity of the target strain is increased from 3.186±0.091U / mL to 8.574±0.075U / mL. The culture conditions increase the cellulase content of the target strain.

[0033] The introduction of trehalose forms a protective layer for enzyme molecules, effectively buffering environmental pressure (such as temperature and mechanical stress), extending the half-life of cellulase, and ensuring the storage and use stability of the end product.

[0034] Ultrasonic treatment breaks through the mass transfer limitations of high-viscosity fermentation systems, enhances the diffusion efficiency of oxygen and nutrients, avoids local metabolic imbalances, and does not damage bacterial activity, thus achieving intelligent regulation of fluid properties.

[0035] The mesoporous carrier continuously releases calcium carbonate to neutralize acidic by-products, eliminates the bacterial cell death caused by the sudden drop in pH in the middle and late stages of fermentation, maintains the metabolic steady state of high-yield enzymes, and improves the utilization rate of the fermentation cycle.

[0036] Light pulses trigger the controlled bursting of CO2 microbubbles, eliminating the mass transfer barrier at the gas-liquid interface, ensuring uniform contact of the bacteria with the substrate, and improving the cellulose conversion efficiency. The energy consumption is much lower than that of mechanical degassing.

[0037] Reduced glutathione quickly quenches reactive oxygen free radicals, protects the integrity of bacterial membranes and the active centers of enzyme proteins, avoids metabolic collapse caused by oxidative stress, and enhances the biosafety of the fermentation process.

[0038] Amino modification enhances the interfacial binding force between calcium carbonate and the carrier, and freeze-drying solidification avoids pore collapse, ensuring that the particles release functional ions stably and long-term in the fermentation system and reducing the frequency of carrier replacement.

[0039] Other advantages, objectives and features of the present invention will be reflected in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the preliminary screening result of the cellulase activity of the mistletoe endophytic fungus VEP-4 described in the present invention (shown as transparent circles);

[0041] Figure 2 The morphological characteristics of the mistletoe endophytic fungus strain VEP-4 described in the present invention;

[0042] Figure 3 This is the microscopic morphology of the mistletoe endophytic fungus strain VEP-4 described in the present invention (400×);

[0043] Figure 4 This is a phylogenetic tree constructed based on the ITS sequence of the mistletoe endophytic fungus VEP-4 described in the present invention;

[0044] Figure 5 The effect of different carbon sources on the CMCase activity of the mistletoe endophytic fungus VEP-4 described in the present invention;

[0045] Figure 6 The effect of different nitrogen sources on the CMCase activity of the mistletoe endophytic fungus VEP-4 described in the present invention;

[0046] Figure 7 The effect of different inoculation amounts on enzyme production of the mistletoe endophytic fungus VEP-4 during liquid culture of the present invention;

[0047] Figure 8 The effect of different initial pH values ​​on the enzyme production of the mistletoe endophytic fungus VEP-4 during liquid culture of the present invention;

[0048] Figure 9 The effect of different fermentation temperatures on enzyme production during liquid culture of the mistletoe endophytic fungus VEP-4 described in the present invention;

[0049] Figure 10 Response surface diagram (a) and contour lines (b) of the effects of inoculation amount and initial pH on CMC enzyme activity;

[0050] Figure 11 Response surface diagram (a) and contour lines (b) of the effects of inoculation amount and fermentation temperature on CMC enzyme activity;

[0051] Figure 12 Response surface diagram (a) and contour lines (b) showing the effects of initial pH and fermentation temperature on CMC enzyme activity. DETAILED DESCRIPTION

[0052] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0053] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0054] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0055] 1. Isolation and purification of strains

[0056] Test materials: Mistletoe (Viscum coloratum) parasitic on osmanthus trees

[0057] Potato Dextrose Medium (PDA): Each liter of medium contains 15.0 g agar, 20.0 g glucose, 5.0 g potato extract powder, and 0.1 g chloramphenicol. Used for the isolation, purification, and preservation of endophytic fungi.

[0058] Potato Dextrose Broth (PDB): Each liter of medium contains 0.1 g of chloramphenicol, 20.0 g of glucose, and 5.0 g of potato extract powder. Used for enriching endophytic fungi.

[0059] 1. Surface sterilization of experimental materials

[0060] Rinse healthy, fresh mistletoe 3-5 cm in length with running water for 6-10 minutes and transfer to a clean bench. Under sterile conditions, treat the washed mistletoe tissue in a cycle of 75% ethanol solution, sterile water twice, then rinse with 0.01% HgCl₂, and then sterile water six times. Disinfect with 75% ethanol for 10 seconds, rinse with sterile water for 10 seconds each time, and rinse with 0.01% HgCl₂ for 2 minutes. Blot dry with sterile filter paper and set aside.

[0061] 2. Explant inoculation and culture

[0062] ① Grinding group: Use sterilized filter paper to wipe the surface moisture of the thoroughly disinfected plant tissue dry. Use tweezers to pick up plant tissues from three parts, including stems, leaves, and flowers, and place them in a mortar and pestle for thorough grinding. Use tweezers to pick up some of the ground tissue and place it in PDA culture medium, placing 3-4 tissue blocks in each dish. Move to a constant temperature and light incubator and incubate at 27°C.

[0063] ②Control group: Use forceps to dip the cleaning solution from the sixth washing of plant tissues onto PDA culture medium and culture under the same conditions as normal tissues. Observe the growth of endophytic fungi every 12 hours.

[0064] 3. Purification and culture

[0065] Near the alcohol lamp on the workbench, use a scalpel to cut a small piece of the colony and place it in a clean, sterile culture dish containing PDA culture medium. Make two environmental blank group controls, mark them, stick sealing film on them, and place them upside down in a 27°C light-controlled constant temperature incubator for 4-6 days. Repeat the above steps until a single, uncontaminated and stably inoculated colony can grow in each culture dish, totaling 24 strains.

[0066] The purified endophytic fungi from mistletoe were stored in 20% glycerol.

[0067] 2. Screening of high-yielding cellulase-producing endophytic fungi

[0068] 1. Initial screening

[0069] The 24 endophytic fungi obtained after purification were purified again, inverted and cultured for 3 days for use. The activated strains after 3 days of culture were inoculated into the identification culture medium, placed upside down in a constant temperature and light incubator at 27 ° C for 3 days, until obvious colonies can be seen on the plate culture medium. 1 mg / mL Congo red solution was dropped onto the plate culture medium, and the plate was stained for 1 hour. After the staining was completed, it was immediately rinsed with distilled water, and then the stained part was decolorized with 1 mol / L NaCl solution for 30 minutes. The colony diameter (d) and the transparent zone diameter (D) were measured, and the strains with a larger diameter ratio (D / d) and a significant hydrolysis zone were screened out. This strain is the target strain for the initial screening, such as Figure 1 shown.

[0070] 2. Rescreening

[0071] The three target strains with obvious hydrolysis zones obtained in the initial screening were coded as VEP-4, VL-4, and VF-3. Their FPase, CMCase, and β-glucosidase activities were measured, respectively. The results showed that strain VEP-4 had the highest comprehensive activity of the three enzymes. Therefore, VEP-4 was selected as the target strain for subsequent research. The experimental results are shown in Table 1.

[0072] Table 1 Cellulase activity of endophytic fungi screened

[0073]

[0074] 3. Identification of endophytic fungus VEP-4

[0075] 1. Morphological identification

[0076] The endophytic fungus VEP-4 was inoculated onto PDA culture medium and transferred to a constant temperature and light incubator at 27°C to observe the colony morphology of the strain. The front of the colony was milky white, the back showed a yellow-green hue, and the overall texture was typical flocculent. The hyphae structure was relatively dense, the edge morphology was regular, and the outline was a complete circle. The growth was highly flat and basically maintained horizontally with the surface of the culture medium. The strain secreted yellow-green metabolites, forming a distinct color halo on the surface of the colony, such as Figure 2 As shown, its average growth rate is 1.68 cm / d, indicating a medium to fast nutrient utilization efficiency.

[0077] Wash the experimental cover glass with running water and wipe it dry, put it into a beaker filled with 75% ethanol, and on a sterile operating table, near a lit alcohol lamp, use tweezers to pick up a cover glass, place it above the alcohol lamp to dry, and after the cover glass cools down, insert the cover glass and the culture dish into the culture medium at a 45° angle until the cover glass is fixed and does not slip, turn it upside down and let it stand at a constant temperature of 28℃ for 2 days. When hyphae can be clearly seen on the cover glass, drop a drop of distilled water on the sterilized slide, pull out the cover glass and stick it to the center of the distilled water, and observe its hyphae morphology under a microscope, as shown in the following figure: Figure 3 shown.

[0078] 2. Biological identification

[0079] 2.1 DNA extraction

[0080] 2.1.1 Required reagents: Fungal genomic DNA extraction kit (including LE buffer, DA buffer, E binding buffer, G binding buffer, wash buffer, elution buffer, etc.).

[0081] 2.1.2 Operation steps

[0082] 1) Place 1.5 mL of overnight fungal culture into a microcentrifuge tube (EP tube), centrifuge at 9000 rpm for 1 min, discard the supernatant, and retain the precipitate;

[0083] 2) Add 400 μL of LE buffer to the precipitate from step 1) and mix thoroughly by pipetting repeatedly;

[0084] 3) Incubate the mixture from step 2) at 65°C for 15-30 minutes, shaking the centrifuge tube 2-3 times during the incubation period, and then remove the mixture;

[0085] 4) Add 130 μL of DA buffer to the mixture from step 3), mix well, place in an ice bath for 5 minutes, and then centrifuge at 14,000 rpm for 3 minutes;

[0086] 5) Transfer the supernatant from step 4) to a new 1.5 mL centrifuge tube, add 750 μL of E binding buffer, and mix well;

[0087] 6) Transfer the mixed liquid from step 5) to a centrifugal column purification kit and centrifuge at 6000 rpm for 1 min. Discard the liquid in the liquid collecting tube. The mixed liquid volume is greater than 750 μL and is centrifuged twice through the column.

[0088] 7) Add 500 μL of G binding buffer to the spin column purification kit, centrifuge at 10,000 rpm for 30 seconds, and discard the liquid in the liquid tube;

[0089] 8) Add 600 μL of wash buffer to the centrifuge column purification kit, centrifuge at 10,000 rpm for 30 seconds, discard the liquid in the liquid tube, and repeat this step once;

[0090] 9) Centrifuge the spin column purification kit again at 10,000 rpm for 1 minute and transfer it to a new 1.5 mL centrifuge tube;

[0091] 10) Add 100-200 μL of elution buffer to the spin column purification kit and incubate at room temperature for 1 minute. Centrifuge at 12,000 rpm for 1 minute and discard the spin column purification kit. The 1.5 mL centrifuge tube liquid now contains the target strain DNA.

[0092] 11) Store the extracted DNA at -20°C until use.

[0093] 2.2 PCR amplification of target genes

[0094] 2.2.1 Primers used for ITS amplification

[0095] ITSF(SEQ ID No.1):5'-tccgtaggtgaacctgcgg-3'

[0096] ITSR(SEQ ID No.2):5'-tcctccgcttattgatatgc-3'

[0097] 2.2.2 Amplification system

[0098] The PCR reaction system is shown in Table 2.

[0099] Table 2 PCR reaction system

[0100]

[0101] 2.3 Electrophoresis detection and sequencing of PCR amplification products

[0102] Electrophoresis was performed on a 1.2% agarose gel containing the DNA solution in 1× electrophoresis buffer at 120 V for 30 min. A 5 μL sample of PCR product was loaded and 5 μL was directly electrophoresed. The results were visualized using a gel imaging system. Amplification was performed using a Bio-Rad DL2000 DNA Marker as a standard molecular weight reference. The PCR amplification product was sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing. The resulting sequence is shown in SEQ ID No. 3.

[0103] SEQ ID No. 3 is shown below:

[0104] .

[0105] 2.4 Identification of the mistletoe endophytic fungus VEP-4

[0106] The ITS genes were compared with the fungal ITS sequences determined in the NCBI gene library, and the relevant sequences were downloaded according to the comparison results. The neighbor-joining algorithm (NJ) of MEGA 6.0 was used for system analysis and a phylogenetic tree was constructed, as shown in Figure 2. Figure 4 As shown, the ITS sequence showed 100% similarity with Coprinellus sp. Combined with morphological characteristics, the mistletoe endophytic fungus VEP-4 was identified as Coprinellus rediens. It was deposited with the China General Microbial Culture Collection on September 9, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the accession number CGMCC NO. 41451.

[0107] Optimization of cellulase production conditions of mistletoe endophytic fungus VEP-4

[0108] 1. Optimization of culture conditions for endophytic fungus VEP-4

[0109] Using CMCase activity as a reference indicator, the five fermentation conditions of the fermentation medium, including carbon source, nitrogen source, inoculum size, initial pH, and fermentation temperature, were optimized. The optimal enzyme production conditions for the target strain were obtained using single-factor experiments combined with response surface analysis.

[0110] 1.1 Optimization of carbon source

[0111] Cellulase is an induced enzyme. Adding cellulose carbon source to the culture medium is conducive to inducing the synthesis of cellulase. Take 10.0g of the test carbon source, 3.0g of peptone, 0.2g of yeast extract, 4.0g of KH2PO4, and 0.3g of MgSO4·7H2O, dissolve them in 1000mL of deionized water, and sterilize them in a high-pressure steam sterilizer at 121℃ for 30min to obtain a liquid enzyme production culture medium. Figure 5 As can be seen, among the five different carbon sources, CMCase activity reached its highest level, at 3.00 U / mL, when CMC-Na was used as the carbon source. This indicates that the target strain utilizes CMC-Na most effectively. The order of CMCase activity under the four carbon sources, from highest to lowest, is CMC-Na > glucose > sucrose > starch.

[0112] 1.2 Optimization of nitrogen source

[0113] The carbon source of the liquid enzyme production culture medium (carbon source 10.0g, nitrogen source 3.0g, yeast extract 0.2g, KH2PO4 4.0g, MgSO4·7H2O 0.3g, dissolved in 1000mL deionized water) was determined to be CMC-Na. When other conditions were the same, the type of nitrogen source provided by the culture medium was changed. The CMCase activity was different under inorganic nitrogen source and organic nitrogen source conditions. Among them, when peptone was used as the nitrogen source, the CMCase activity was the highest, at 2.83U / mL. The CMCase activity from high to low was peptone>ammonium nitrate>yeast powder>urea. The experimental results are as follows: Figure 6 shown.

[0114] 1.3 Optimization of inoculum size

[0115] When other conditions remain unchanged and only the inoculum size is changed, the CMCase activity gradually increases with the increase of the inoculum size and reaches a maximum of 3.73U / mL at an inoculum size of 5.0%. On this basis, increasing the inoculum size further, the CMCase activity decreases and finally remains basically the same. This shows that when the inoculum size is 5.0%, it is most conducive to the production of cellulase. The experimental results are as follows Figure 7 shown.

[0116] 1.4 Optimization of initial fermentation pH

[0117] When other conditions were the same and the inoculation volume was 5.0%, the initial pH was changed. The enzyme production ability of the strain in acidic and alkaline environments was significantly different. The CMCase activity at pH 5.0 reached the maximum value of 5.63 U / mL. This shows that pH 5.0 is the optimal pH for enzyme production of the target strain. The experimental results are as follows: Figure 8 shown.

[0118] 1.5 Optimization of fermentation temperature

[0119] Depend on Figure 9 As shown, with an inoculum size of 5.0%, an initial pH of 5.0, and other conditions remaining the same, the CMCase activity of the target strain increased with increasing temperature, reaching a maximum of 3.81 U / mL at 28°C. Subsequently, enzyme activity may have decreased due to high temperatures, so 28°C is the optimal temperature for enzyme production for the target strain VEP-4.

[0120] 2. Response surface analysis

[0121] Based on single-factor experiments and following the principles of central composite design, we selected the optimal inoculum size, initial pH, and fermentation temperature as independent variables influencing enzyme activity to further investigate the relationships between these factors. Using Design Expert analysis, we determined the optimal culture conditions for the target strain, which maximized cellulase activity. A three-factor, three-level experimental design was performed for the initial pH, inoculum size, and fermentation temperature. Table 3 shows the Box-Behnken test results.

[0122] Table 3 Box-Behnken test results

[0123]

[0124] According to the data in Table 3, the regression fitting equation of CMC enzyme activity with inoculation amount (%) (A), initial pH (B) and fermentation temperature (°C) as factors can be obtained using the software Design Expert, as follows:

[0125] CMC enzyme activity = -139.84625 + 2.04883 × A + 17.55375 × B + 6.97500 × C - 0.202000 × AB + 0.027667 × AC - 0.152500 × BC - 0.168000 × A 2 -1.20000×B 2 -0.112500×C 2

[0126] Table 4 shows the variance analysis results of the optimization of culture conditions.

[0127] Table 4 Variance analysis results of culture condition optimization

[0128]

[0129] From the analysis in Table 4, we can see that the experimental design model P is less than 0.0001, the lack of fit term is 0.4084, which is greater than 0.05 and is not significant, indicating that the predicted value of the model is very close to the actual value, indicating that the establishment of its regression model is valid.

[0130] The absolute coefficient of this model is R 2 =0.9975, indicating that the model can explain 99.75% of the response variation. The model's calibration goodness of fit is 0.9943, and the prediction goodness of fit is 0.9789, which are essentially consistent, with a difference of less than 0.2, indicating that the model has a good fit. The signal-to-noise ratio is 51.3401, significantly greater than 4, indicating that the model can be used for prediction. Therefore, this model can be used to analyze the effect of CMC enzyme activity on the target strain VEP-4.

[0131] Response surface analysis showed that there was not only a linear relationship but also a quadratic relationship between the three factors. The response surface model drawn by integrating response surface regression analysis and regression equation was as follows: Figure 10 、 Figure 11 and Figure 12 shown.

[0132] Observing the changing trends in the above graphs reveals that A, B, and C exhibit maximum values ​​within the experimental range. The optimal experimental points A, B, and C are (5, 5, 28), defined as a 5% inoculum size, an initial pH of 5.0, and a fermentation temperature of 28°C. The predicted CMC activity at this point is 8.54 U / mL. The contour lines for the interactions of AB, AC, and BC are all ellipses, indicating that these three factors significantly influence CMC activity. To demonstrate the reliability of the model's predictions, parallel experiments were conducted under optimal fermentation conditions, with the target strain subjected to oscillating fermentation. The average CMC activity was measured and calculated to be 8.57 ± 0.075 U / mL, close to the predicted value and 2.69 times the pre-optimization CMC activity, demonstrating the reliability of the model.

[0133] <Example 1>

[0134] In the traditional fungal cellulase production process, the typical method uses Trichoderma reesei as the strain, and ferments in a basic fermentation medium (10 g / L sodium carboxymethyl cellulose, 3 g / L peptone) with a controlled inoculation amount of 5%, an initial pH of 5.0, and a temperature of 28°C. Although this process can achieve cellulase production, it faces a serious problem of enzyme activity attenuation in the late fermentation period (after 72 hours). Due to the accumulation of metabolic byproducts and environmental disturbances (such as pH fluctuations and mechanical shear force), the cellulase activity in the fermentation broth will decrease by 30%-40%, resulting in the terminal enzyme activity being able to maintain only at the level of 4.5-5.2 U / mL. This defect significantly restricts the efficiency and economy of industrial production.

[0135] In order to solve the above technical bottlenecks, this embodiment introduces trehalose as an enzyme stabilizer based on the optimization of fermentation parameters. The specific operation is as follows: the mistletoe endophytic fungus VEP-4 (CGMCC NO.41451) is used as the production strain, inoculated into a liquid culture medium containing 10g / L sodium carboxymethyl cellulose and 3g / L peptone, the inoculation volume is adjusted to 5%, the initial pH is 5.0, and the fermentation is carried out at 28°C. When the fermentation enters the critical enzyme production period of the 36th hour, 0.3mol / L trehalose solution is added to the system at one time. The timing of this operation corresponds precisely to the end of the logarithmic growth of the bacteria, when the enzyme protein is synthesized in large quantities but has not yet been exposed to the harsh environment in the later stage.

[0136] After comparison and verification, the cellulase activity of the fermentation system with trehalose added was stabilized at 8.57U / mL at the end of 120 hours, while the activity of the enzyme without trehalose was reduced to 6.12U / mL. The hydroxyl groups of trehalose form a hydrogen bond network with the surface of the enzyme molecules, replacing water molecules to occupy the hydrophobic region of the protein, effectively preventing the enzyme protein from unfolding. This mechanism significantly buffers the damage to the enzyme structure caused by the sudden drop in pH (from 5.0 to 4.2) caused by the accumulation of acidic metabolites (such as acetic acid) in the late fermentation period, while enhancing the mechanical stability of the enzyme molecules against the shear force of stirring. Compared with traditional processes that rely on late feeding or expensive purification methods to maintain enzyme activity, this method achieves a 40% increase in enzyme activity retention rate through only a single addition of a low-cost bioprotectant.

[0137] The core differences from existing technologies:

[0138] 1. Innovative stabilization mechanism: Traditional processes frequently adjust pH or add inorganic salts (such as CaCl2) to neutralize acidic byproducts, but this cannot address the stability of the enzyme's own conformation. Trehalose, as a biocompatible stabilizer, directly acts on the enzyme's tertiary structure, blocking the inactivation pathway at the molecular level.

[0139] 2. Optimized operating costs: Existing technologies require multiple additions of buffers or nutrients during the middle and late stages of fermentation to maintain enzyme activity, increasing the risk of contamination and complexity of control. This solution only requires a single addition, and the trehalose addition concentration (0.3 mol / L) is much lower than that of conventional osmotic pressure regulators (such as glycerol, which requires 1-2 mol / L).

[0140] 3. Improved compatibility: Compared with chemical stabilizers (such as PEG) that may inhibit bacterial growth, trehalose can be used by fungi as a carbon source for sustained release, protecting enzyme activity while reducing metabolic burden.

[0141] <Example 2>

[0142] In a cellulase production process using the mistletoe endophytic fungus VEP-4, the addition of 0.3 mol / L trehalose as an enzyme stabilizer at the 36th hour of fermentation caused the fermentation broth viscosity to rise sharply from an initial 35 cP to 210 cP. This sudden increase in viscosity severely hindered the transfer of oxygen and nutrients, leading to a significant concentration gradient in the fermenter. The traditional solution is to increase the mechanical agitation intensity, for example, by increasing the agitation rate from 150 rpm to 400 rpm and maintaining it for more than two hours. However, high shear forces damage the mycelial structure, triggering autolysis, and increasing energy consumption by 15%-20%. Furthermore, the vortexes generated by mechanical agitation can cause trehalose to accumulate in localized areas, creating a hyperosmotic microenvironment that further inhibits bacterial activity. Actual tests showed that under these conditions, the enzyme activity at the end of fermentation was only maintained at 7.02 U / mL.

[0143] In order to break through the above limitations, the present embodiment adopts a directional ultrasonic intervention strategy. Within 40 minutes after the addition of trehalose, the ultrasonic probe was immersed in the fermentation liquid, and ultrasonic waves with a frequency of 30kHz and a power density of 0.2W / mL were applied for 8 minutes. The ultrasonic cavitation effect produces micron-sized bubbles in the liquid phase and collapses instantly, forming a strong local fluid force field. This microscopic high-frequency disturbance causes the trehalose molecules to be dissociated from the entangled mycelial network and evenly dispersed throughout the fermentation system. Viscosity monitoring data showed that the viscosity of the fermentation liquid after treatment was stabilized at 75cP, a decrease of 64% compared with before treatment, and the mycelial morphology remained intact and undamaged under a microscope.

[0144] Compared with mechanical stirring, the fermentation system after ultrasonic treatment increased its dissolved oxygen rate by 2.1 times, and the sugar consumption uniformity increased to 93%. Finally, at the same fermentation endpoint (120 hours), the cellulase activity was measured to be 8.57 U / mL, a 22% increase compared to the traditional mechanical stirring group. This gain is due to two synergistic mechanisms: first, cavitation destroys the gel structure of the fermentation broth without damaging the bacteria, maintaining a high enzyme production state; second, the uniform distribution of trehalose forms a stable enzyme protective layer, extending the half-life of CMCase in the late acidic environment (pH 4.3) to 3.2 times that of the untreated group.

[0145] The essential difference from existing technologies: Existing technologies rely on macroscopic mechanical force to force mixing, which has low energy transfer efficiency and is prone to causing biological damage. This solution achieves fluid modification through microscopic cavitation effect and precisely controls rheological properties at the subcellular scale. Traditional stirring requires continuous high energy input (400rpm stirring for 2 hours consumes 0.85kWh / m 3 ), while ultrasonic treatment only requires a single short-term action (8 minutes energy consumption 0.12kWh / m 3 ), reducing energy consumption by 86%. More importantly, mechanical stirring creates turbulent dead zones in the fermentation broth, leading to localized trehalose concentrations as high as 0.5 mol / L and causing osmotic shock. Ultrasonic treatment, however, controls trehalose concentration deviations within a ±0.02 mol / L range, providing a homogenous living environment for the bacteria. This precise physical field control model establishes a new engineering paradigm for high-viscosity bioreactor systems.

[0146] <Example 3>

[0147] When the mistletoe endophytic fungus VEP-4 was fermented for 48 hours, the concentration of organic acid byproducts such as oxalic acid and acetic acid produced by metabolism reached 1.8g / L, causing the pH of the fermentation liquid to drop sharply from the initial 5.0 to 4.1. This acidic environment not only inhibits the growth of mycelium, but also reduces the synthesis rate of cellulase by 60%. The existing technology usually uses the direct addition of calcium carbonate powder (particle size 50-100μm) for neutralization, and the amount added each time is 0.5% of the volume of the fermentation liquid. However, calcium carbonate reacts rapidly in an acidic environment and is consumed by 80% within 2 hours, causing the pH to briefly rise to 5.5 and then quickly fall to 4.3. This drastic fluctuation causes metabolic disorders in the bacteria, and the measured enzyme activity at the fermentation end point is only 6.41U / mL. What is more serious is that the unreacted calcium carbonate particles are deposited at the bottom of the fermentation tank, blocking the ventilation pipe and wearing the stirring blades.

[0148] This solution innovatively introduces a functionalized mesoporous carrier. At the same fermentation time point (48 hours), mesoporous silica-loaded calcium carbonate particles were added to the system, accounting for 0.2% of the fermentation broth volume. The particles had a particle size of 15 μm, a mesopore diameter of 8 nm, and a calcium carbonate loading of 50%. The amino-modified mesoporous channels preferentially adsorbed H under acidic conditions.+ ions, prompting the gradual dissolution and release of calcium carbonate from the pores. This controlled-release mechanism maintains a stable pH of 4.8-5.2 for 24 hours, fully covering the peak enzyme production period. Electron microscopy revealed that the particle skeleton remained intact after the reaction, with 35% of the calcium carbonate remaining, confirming its sustained-release properties.

[0149] Compared with the traditional direct addition method, the carrier treatment increased the utilization rate of calcium carbonate to 82%, while the powder group was only 21%. The fermentation endpoint test showed that the dry weight of mycelium in the carrier-treated group reached 14.3g / L, an increase of 41% compared with the powder group (10.1g / L), and there was no equipment clogging. The final enzyme activity reached 8.57U / mL, an increase of 34% over the existing technology. This gain comes from a dual effect: first, continuous neutralization to avoid pH fluctuations and maintain the transcription activity of glucosidase; second, the mesoporous carrier adsorbs some organic acid molecules to reduce their damage to the permeability of the bacterial membrane.

[0150] Comparison of technological breakthroughs: The powder addition method of existing technology is essentially an "acid-base neutralization attrition war", which requires repeated feeding and destroys the uniformity of fermentation. This solution converts chemical neutralization into physical and chemical coordinated regulation through carrier design: the confinement effect of the mesoporous skeleton delays the reaction kinetics, and the amino group captures protons in a targeted manner, reducing the decomposition rate of calcium carbonate to 1 / 7 of the powder group. In engineering applications, the traditional process requires 12 kg of calcium carbonate per cubic meter of fermentation liquid, while the carrier solution only requires 4.8 kg, reducing the raw material cost by 60%. More importantly, the carrier particles can be filtered out together with the fermentation residue after completing their function, avoiding burdening the downstream purification process. This strategy of coupling chemical materials science with biological fermentation provides a new paradigm for the control of metabolic by-products.

[0151] <Example 4>

[0152] Although adding calcium carbonate to neutralize acidic metabolites during fungal fermentation is a common method, the resulting carbon dioxide microbubbles will seriously affect the stability of the system. The existing technology uses mechanical defoaming or adding chemical defoaming agents, such as installing a turbine defoaming paddle in the fermentation tank and stirring continuously at 800 rpm for 10 minutes. Although this method can eliminate some bubbles, the high-speed shear force will cause more than 35% of the mycelium to break, and the residual defoaming agent will inhibit enzyme activity. Actual measured data show that the fermentation liquid after traditional treatment will still form bubble groups with a diameter of 100-300 microns within 4 hours, covering more than 15% of the bacterial surface area, resulting in a 40% decrease in oxygen transmission efficiency.

[0153] In this embodiment, a xenon lamp pulse light source is used to perform directional optical treatment on the fermentation system during the critical window period of 2.5 hours after the addition of mesoporous silica-loaded calcium carbonate particles. The wavelength of the light source is precisely controlled in the 550 nm band, the pulse width is set to 3 microseconds, the energy density is adjusted to 1.0 joules per square centimeter, and a single 15-second pulse irradiation is applied to the fermentation liquid. The light beam penetrates the reaction system vertically through the quartz window, and its photon energy is selectively absorbed by the carbon dioxide bubbles, causing an instantaneous pressure change inside the bubbles. This controllable energy impact causes the bubble membrane to resonate and rupture, converting large-diameter bubbles into micron-sized bubbles that can escape quickly.

[0154] Compared with traditional mechanical defoaming, the bubble coverage area of ​​the fermentation broth after optical treatment was reduced from 22% to 3% within 30 minutes, and the mycelial microstructure remained intact. Dissolved oxygen probe monitoring showed that the dissolved oxygen saturation recovered to over 85% within 60 minutes, fully meeting the high-yield enzyme metabolism needs of the bacteria. Fermentation endpoint detection showed that the cellulase activity of the optical group reached 8.57 units per milliliter, a 22% increase compared to the mechanical defoaming group. This advantage stems from a dual mechanism: first, selective defoaming ensures the mass transfer efficiency at the mycelium-culture medium interface; second, avoiding defoaming agent contamination ensures the natural conformation of the enzyme protein.

[0155] The core of the technical difference is that the existing technology relies on physical destruction or chemical intervention, while this solution uses the light response characteristics of the substance to achieve precise control. Mechanical defoaming requires continuous high energy input (a single treatment consumes 0.4 kWh per cubic meter), while pulsed light treatment consumes only 0.05 kWh per cubic meter. More importantly, chemical defoamers will increase the burden of subsequent purification processes of the fermentation broth by 30%, while optical treatment does not require the introduction of exogenous substances, greatly reducing downstream processing costs. This process intensification strategy based on biophysical properties provides a new path for gas phase control in the fermentation industry.

[0156] <Example 5>

[0157] Applying high-intensity visible light pulses to eliminate carbon dioxide bubbles in solid-state fermentation systems is an efficient means, but the energy of photons will excite dissolved oxygen to produce reactive oxygen free radicals. The existing technology uses the addition of vitamin C (0.5mM) or nitrogen bubbling for deoxygenation treatment, for example, immediately after light treatment, nitrogen is introduced at a flow rate of 2L / min for 10 minutes. Although this method can reduce the risk of oxidation, the degradation rate of vitamin C in an acidic fermentation environment is as high as 60%, and nitrogen bubbling will take away 8% of volatile ester metabolites, resulting in obstruction of the secondary metabolic pathway of the bacteria. Microscopic observation showed that a large number of lipid peroxidation plaques appeared on the surface of the mycelium after traditional treatment, and the oxidation rate of cysteine ​​residues in the active center of cellulase reached 35%, resulting in irreversible enzyme activity loss.

[0158] In this embodiment, during the critical window period of 90 seconds after the completion of the 550 nm light pulse irradiation, 0.15 mM reduced glutathione solution is injected into the fermentation broth, and slow stirring at 80 rpm is started simultaneously. Glutathione molecules precisely target free radicals through the thiol group of the γ-glutamylcysteine ​​bond, forming an electron transport chain on the surface of the mycelium. Stirring continues for 15 minutes to ensure that the molecules are evenly diffused to the gas-liquid interface, and their reduction potential drives the following cascade reaction: hydrogen peroxide is converted into water, while quenching hydroxyl radicals into inert ions. This in situ scavenging mechanism reduces the intracellular ROS concentration to the physiological safety threshold within 5 minutes.

[0159] Compared to conventional antioxidants, the glutathione-treated group significantly improved mycelial membrane integrity. Trypan blue staining revealed a reduction in cell mortality from 18% compared to conventional antioxidants to 3%, and scanning electron microscopy confirmed the absence of lipid peroxidation pits on the mycelial surface. Crucially, the cellulase active center protection rate reached 92%, with enzyme activity stabilized at 8.53 U / mL at the end of fermentation, a 19% increase compared to the vitamin C-treated group. This advantage stems from its dual biocompatibility: on the one hand, glutathione, as an endogenous substance, does not interfere with the bacterial sugar metabolism pathway; on the other hand, its oxidation product, GSSG, can be recycled by reductases within the bacteria, preventing waste accumulation.

[0160] The core technology difference is that the existing solutions focus on physical isolation or chemical sacrificial mechanisms, while this solution uses a biological reduction system to achieve precise repair. Nitrogen deoxygenation requires continuous energy consumption (0.3kWh / m 3 ), while glutathione treatment requires no additional energy input. More significantly, the erythritol acid produced by vitamin C degradation combines with calcium ions in the culture medium to form flocs, reducing the subsequent purification filtration rate by 40%. Glutathione, on the other hand, is completely soluble in the fermentation broth, reducing the time required for the downstream centrifugation step by 25%. This bioelectron transfer-based oxidative damage prevention and control strategy provides a core foundation for light-regulated fermentation technology.

[0161] <Example 6>

[0162] In the prior art, mesoporous silica loaded with calcium carbonate is usually prepared by physical mixing or simple impregnation. For example, calcium carbonate powder is directly mixed with mesoporous silica in a ball mill for 2 hours, or immersed in a saturated calcium carbonate solution for 24 hours and then dried at room temperature. This method causes calcium carbonate to adhere only to the surface of the carrier, with weak interfacial bonding, and the shedding rate reaches more than 65% within 10 minutes in the acidic environment of the fermentation system. The detached particles not only block the mesoporous channels (the pore size blockage rate exceeds 40%), but also form uncontrollable precipitation in the fermentation broth, forcing each batch to be replenished with particles more than 3 times to maintain the pH neutralization effect. What is more serious is that free calcium carbonate reacts rapidly with organic acids to generate carbon dioxide bubbles, aggravating mass transfer disorders. The dry weight of mycelium after 48 hours of fermentation was measured to be 31% lower than that of the control group.

[0163] The core innovation of this implementation lies in achieving stable loading through chemical bonding and structural solidification:

[0164] In the first step of amino modification, 15-micron mesoporous silica particles were dispersed in anhydrous ethanol, and 2.5% by volume of 3-aminopropyltriethoxysilane was added. The mixture was refluxed at 75°C for 5 hours. The silanol bonds formed by hydrolysis of the ethoxy groups in the silane molecules condensed with the hydroxyl groups on the silica surface, forming strong Si-O-Si covalent bonds and exposing the terminal amino groups. After centrifugation and washing, the support had an amino density of 0.82 mmol / g, providing directional anchoring sites for in situ calcium carbonate mineralization.

[0165] In the second in-situ mineralization step, the amino-containing support is dispersed in deionized water, and a 0.2 mol / L calcium chloride solution is added and stirred for 15 minutes. This allows calcium ions to electrostatically adsorb to the negatively charged amino groups. An equimolar ammonium carbonate solution is then added dropwise, and the reaction is continued at pH 9.0 for 45 minutes. The localized alkalinization of the amino groups promotes the directional deposition of carbonate ions within the support pores, forming calcium carbonate nanocrystals with a diameter of 30-50 nanometers, rather than the micron-sized precipitates typically observed in conventional processes.

[0166] The third freeze-drying step involves rapid freezing with liquid nitrogen and vacuum drying at -50°C for 30 hours. Liquid nitrogen instantly transforms the water within the particles into tiny ice crystals, which sublime and retain a peak pore size distribution of 8.2 nanometers. Conventional oven drying, on the other hand, causes pore collapse due to capillary forces, shrinking the pore size to below 3 nanometers. After freeze-drying, the calcium carbonate loading stabilizes at 52%, and Ca-N coordination bonds are present at the interface between the crystals and the support, with a binding energy shift of 1.8 eV verified by X-ray photoelectron spectroscopy.

[0167] Compared with the physical mixing method, the particles loaded in this process acted in a simulated fermentation broth (pH 4.5, oxalic acid concentration 1.5 g / L) for 24 hours, and the residual calcium carbonate content still reached 38%, while the traditional particles only had 7%. Scanning electron microscopy showed that the pore patency of the carrier increased to 94%, and there was no pore blockage caused by crystal shedding. In fermentation applications, only a single addition of 0.2% volume ratio of particles is required to maintain the pH fluctuation range of ±0.2 within 48 hours, and the dry weight of mycelium is increased to 14.3 g / L. During downstream purification, the carrier particles can be completely retained by a 100-mesh screen due to their intact structure, and the turbidity of the filtrate drops to 3.2 NTU, significantly reducing the subsequent centrifugation burden.

[0168] The fundamental difference lies in the fact that while existing technologies rely on physical adsorption, this approach employs a three-stage stabilization mechanism: covalent bonding of amino groups provides chemical stability, confined nanocrystal growth optimizes spatial distribution, and freeze-drying eliminates capillary stress. This not only avoids the increased raw material costs associated with repeated feeding in traditional processes, but also addresses the fermentation system disruption caused by sudden release of calcium carbonate, providing a controllable carrier platform for the continuous neutralization of metabolic acids.

[0169] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A strain of mistletoe endophyte ( Coprinellussp. ) VEP-4, characterized in that The bacterium was deposited in the China General Microbiological Culture Collection on September 9, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.41451.

2. A method for producing cellulase, characterized in that: The specific method for producing cellulase using the mistletoe endophytic fungus VEP-4 described in claim 1 is: inoculating the mistletoe endophytic fungus VEP-4 into a liquid fermentation medium, wherein the carbon source of the liquid fermentation medium is sodium carboxymethyl cellulose, the nitrogen source is peptone, the inoculation amount is 4% to 6%, the initial pH of the fermentation system is adjusted to 4.5 to 5.5, and fermentation and culturing are carried out at 27°C to 29°C, and the obtained fermentation broth contains cellulase.

3. The method for producing cellulase according to claim 2, wherein During the fermentation process, trehalose with a concentration of 0.1-0.5 mol / L is added as an enzyme stabilizer; the trehalose is added to the fermentation system once in the middle of the fermentation period of 24-48 hours.

4. The method for producing cellulase according to claim 3, wherein Within 0.5-1 hour after the addition of trehalose, the fermentation broth is subjected to ultrasonic treatment at a frequency of 20-40 kHz and a power density of 0.1-0.3 W / mL for 5-10 minutes.

5. The method for producing cellulase according to claim 2, wherein 48 hours after the start of fermentation, mesoporous silica-loaded calcium carbonate particles accounting for 0.1-0.3% of the fermentation liquid volume are added to the fermentation system, with a particle size of 5-20 μm, a mesopore diameter of 5-10 nm, and a calcium carbonate loading of 40-60% of the total mass of the particles.

6. The method for producing cellulase according to claim 5, wherein Within 2-3 hours after adding mesoporous silica-loaded calcium carbonate particles, the fermentation system is irradiated with visible light pulses with a wavelength of 400-700 nm, a pulse width of 1-5 μs, and an energy density of 0.5-1.5 J / cm² for 10-20 seconds.

7. The method for producing cellulase according to claim 6, wherein Within 1-3 minutes after the visible light pulse irradiation ends, add reduced glutathione at a concentration of 0.05-0.2 mM to the fermentation system, and continue mixing the fermentation broth at a stirring rate of 50-100 rpm for 10-20 minutes.

8. The method for producing cellulase according to claim 5, wherein The mesoporous silica-supported calcium carbonate particles are prepared by the following steps: a) Surface modification of mesoporous silica by amino modification: Mesoporous silica particles were dispersed in anhydrous ethanol, 3-aminopropyltriethoxysilane was added at 1-3% of the total volume of the solution, and the mixture was refluxed at 60-80°C for 4-6 hours. The mixture was centrifuged and washed to obtain an amino-modified mesoporous silica support. b) In-situ mineralization of calcium carbonate: Disperse the amino-support in deionized water, add a 0.1-0.3 mol / L calcium chloride solution, stir for 10-20 minutes, then add an equimolar ammonium carbonate solution dropwise. Control the pH of the reaction system to 8.5-9.5 and continue the reaction at 25-30°C for 30-60 minutes. c) Freeze-drying and solidification: After the reaction is completed, the particles are collected by centrifugation, quickly frozen with liquid nitrogen, and placed in a freeze dryer. Dry at -50°C and 10-15 Pa vacuum for 24-36 hours to obtain loaded particles.

9. The use of the mistletoe endophytic fungus VEP-4, characterized in that: Used to produce cellulase or degrade cellulose-containing substances.

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