A lignocellulose-degrading strain with high yield of beta-glucosidase and application thereof

Abstract

The application discloses a lignocellulose-degrading strain with high yield of beta-glucosidase and application thereof, the strain is named Trichoderma asperellum (T. Trichoderma asperellum ) ML02, has been preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20221877 and the preservation date is December 5, 2022. The strain grows rapidly, can cover a PDA plate in 72 h, and the mycelium is more dense, which is beneficial to the secretion of hydrolytic enzyme and the combination with a substrate, and the degradation performance is improved. The strain can be used for directly degrading lignocellulose such as corn cob and wheat straw without pretreatment, and the degradation rates are 54.6% and 56.8% respectively, indicating that the strain has excellent lignocellulose degradation capacity and a wide substrate spectrum. When the strain is mixed with lactic acid bacteria and cellulose is used as a carbon source, the lactic acid yield can reach 50 g / L in 168 h, and the fermentation period is shortened. The application provides a lignocellulose-degrading strain with excellent performance, and the strain has great industrial development potential.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a lignocellulose-degrading strain that produces high levels of β-glucosidase and its applications. Background Technology

[0002] Lignocellulose is the most abundant biomass on Earth. As a green and renewable resource, its full utilization can effectively alleviate environmental pollution and the energy crisis. Cellulose, as the main component of lignocellulose (40-50%), requires efficient conversion, which is a crucial prerequisite for the biorefining industry. Cellulose degradation requires the collaborative action of endoglucanases (endoglucanase, EG, EC 3.2.1.4), exoglucanases (exoglucanase, CBH, EC 3.2.1.74), and β-glucosidases (BG, EC 3.2.1.21). Endoglucanases randomly cleave the long chains within cellulose, producing oligosaccharides to varying degrees; exoglucanases hydrolyze the reducing and non-reducing ends of cellulose to produce cellobiose; finally, β-glucosidase attacks the glycosidic bonds of cellobiose to generate reducing sugars.

[0003] Filamentous fungi are commonly used strains for cellulose conversion due to their strong protein synthesis capabilities and relatively abundant and efficient secretion of cellulose-degrading enzyme systems. However, industrial model strains, such as *Trichoderma reesei*, generally suffer from insufficient β-glucosidase secretion, which significantly limits degradation efficiency, increases enzyme usage and cost, and hinders the industrialization of biorefining. On the other hand, mixed-culture systems have become a research hotspot due to their economic advantages, such as labor and energy saving, relatively simplified process equipment, and reduced intermediate product separation. The substrate utilization efficiency of the upstream strain (usually a degrading strain) and its ability to provide sufficient fermentable sugars to the downstream strain are key factors determining the overall quality of the system and its industrialization. Therefore, there is an urgent need to develop strains that produce high levels of β-glucosidase to compensate for the insufficient secretion of cellulose-degrading enzyme systems and improve cellulose degradation rates. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is that existing cellulose degrading strains have incomplete cellulase systems and mainly suffer from insufficient secretion of β-glucosidase. The present invention provides a strain that produces high levels of β-glucosidase, has a more complete enzyme system, and has high degradation performance.

[0005] Another technical problem that this invention aims to solve is to provide applications of the above-mentioned strains.

[0006] To further improve the efficiency of cellulose degradation, this invention involved two rounds of ethyl methanesulfonate (EMS) mutagenesis on an existing laboratory strain of *Trichoderma echinocandes* with good lignocellulose degradation performance. After initial screening using Congo red agar and esculin citrate plates, supplemented by shake-flask screening, a high-yielding BG-producing mutant strain was selected. Its BG enzyme activity reached 1.11 U / mL, three times that of the original strain, and was named ML02. Simultaneously, the endonuclease and exonuclease activity were 1.6 and 1.9 times that of the original strain, respectively. Stability testing showed that the enzyme activity remained stable even after six generations. Furthermore, its degradation efficiency was very high, achieving a degradation rate of over 40% for microcrystalline cellulose in just 8 days.

[0007] This invention discloses a strain that produces high levels of β-glucosidase, classified as *Trichoderma asperellum* ML02, which has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20221877 and deposit date of December 5, 2022.

[0008] The mutagenesis method for *Trichoderma asperellum* ML02 described in this invention is as follows: The starting strain *Trichoderma asperellum* LYS1 (deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20211179, accession date September 15, 2021) is activated on a PDA plate and cultured at 30℃ for 3-4 days. The PDA plate is then washed with 0.9% physiological saline to adjust the spore concentration to 1×10⁻⁶. 8 / mL. Take 1mL of spore solution into ten 2mL EP tubes, add 40μL of EMS, and incubate at 30℃ on a shaker for the appropriate time (20-180min, 180rpm). Remove the tubes and add 5% sodium thiosulfate antidote for 5min. Dilute 10 3 After doubling, 100 μL was spread onto a Congo red plate. The lethality curve indicated that 100-120 min was the appropriate mutagenesis time.

[0009] The screening method for *Trichoderma asperellum* ML02 described in this invention is as follows: The original fungal spore solution is mutagenized at a suitable mutagenesis time, detoxified and diluted, then spread onto a ferric citrate aescinate plate. After incubation at 30℃ for 1-2 days, the blacker spots on the ferric citrate aescinate plate are selected and inoculated onto a Congo red plate (e.g.,...). Figure 2 (As shown). Next, spots on the Congo red plate with a clear zone / diameter ratio larger than the original strain were selected for shake-flask rescreening. This screened out the high-β-glucosidase-producing mutant strain (Trichoderma asperellum) ML02.

[0010] Screening plates: The screening plates were ferric citrate aescinate plate and Congo red plate.

[0011] Congo Red Plate: 2 g / L sodium carboxymethyl cellulose (CMC-Na), 0.5 g / L magnesium sulfate heptahydrate, 1.0 g / L potassium dihydrogen phosphate, 1.1 g / L peptone, 1.0 g / L lactose, 1.0 g / L sodium deoxycholate, 0.1 g / L Congo Red, 20.0 g / L agar, the remainder being water.

[0012] Ferric citrate aescin plate: 0.5 g / L magnesium sulfate heptahydrate, 1.0 g / L potassium dihydrogen phosphate, 1.1 g / L peptone, 1.0 g / L lactose, 1.0 g / L sodium deoxycholate, 2.0 g / L aescin, 0.5 g / L ferric citrate, 20.0 g / L agar. The remainder is water; the incubation temperature is 28-30℃, preferably 30℃; the incubation time is 48-72 h, preferably 48 h.

[0013] Properties of the selected strain: The colony characteristics of the *Trichoderma elatior* ML02 strain are as follows: mycelia can fully cover the plate after 3 days of cultivation on potato dextrose (PDA) medium at 30°C, and its growth is more rapid than that of the original strain. Its colony color remains white (the original strain's colonies are initially white, then green, and turn yellow upon aging). The colony structure is felt-like with rounded, serrated edges, relatively regular, with low spore production and dense mycelia, which is conducive to the secretion of hydrolytic enzymes and their binding to substrates, promoting degradation.

[0014] The culture conditions for the strain are as follows:

[0015] Plate culture: Trichoderma elatior ML02 was inoculated onto plate culture medium and cultured at 28-30℃ for 72-96 h. The plate culture medium was potato dextrose agar (PDA), yeast peptone agar (YPD), or malt extract agar (MEA). Potato dextrose agar (PDA) was preferred in this experiment, with the carbon source being 200-300 g / L potato, 15-20 g / L glucose, 15-20 g / L agar powder, and the remainder being water. The preferred culture temperature was 30℃, and the preferred culture time was 72 h.

[0016] Seed culture: Inoculate the plate-cultured strains into seed culture medium, and culture at a temperature of 28-30℃ for 24-48 hours at a rotation speed of 160-200 rpm.

[0017] The components of the seed culture medium are as follows: 0.1-0.5 g / L urea, 0.5-1.0 g / L peptone, 0.2-0.5 g / L yeast extract, 1.0-2.0 g / L ammonium sulfate, 1.5-2.5 g / L potassium dihydrogen phosphate, 0.1-0.5 g / L calcium chloride, 0.1-0.5 g / L magnesium sulfate heptahydrate, 10.0-20.0 g / L glucose, 0.002-0.008 g / L ferrous sulfate heptahydrate, 0.001-0.003 g / L manganese sulfate tetrahydrate, 0.001-0.003 g / L zinc sulfate heptahydrate, 0.001-0.003 g / L cobalt chloride hexahydrate, and the remainder is water.

[0018] More preferably, the seed culture medium consists of 0.3 g / L urea, 0.75 g / L peptone, 0.25 g / L yeast extract, 1.4 g / L ammonium sulfate, 2.0 g / L potassium dihydrogen phosphate, 0.3 g / L calcium chloride, 0.3 g / L magnesium sulfate heptahydrate, 10.0 g / L glucose, 0.002-0.008 g / L ferrous sulfate heptahydrate, 0.001-0.003 g / L manganese sulfate tetrahydrate, 0.001-0.003 g / L zinc sulfate heptahydrate, 0.001-0.003 g / L cobalt chloride hexahydrate, with the remainder being water; the preferred culture temperature is 30℃; the preferred culture time is 48 h; and the preferred culture rotation speed is 180 rpm.

[0019] Fermentation culture: Inoculate the seed culture strain into the fermentation medium, culture at a temperature of 28-30℃ for 168-192 hours, and rotate at a speed of 160-200 rpm.

[0020] The fermentation medium comprises the following components: 0.1-0.5 g / L urea, 0.5-1.0 g / L peptone, 0.2-0.5 g / L yeast extract, 1.0-2.0 g / L ammonium sulfate, 1.5-2.5 g / L potassium dihydrogen phosphate, 0.1-0.5 g / L calcium chloride, 0.1-0.5 g / L magnesium sulfate heptahydrate, 10.0-20.0 g / L glucose, 0.002-0.008 g / L ferrous sulfate heptahydrate, 0.001-0.003 g / L manganese sulfate tetrahydrate, 0.001-0.003 g / L zinc sulfate heptahydrate, 0.001-0.003 g / L cobalt chloride hexahydrate, with the remainder being water.

[0021] More preferably, the fermentation medium consists of 0.3 g / L urea, 0.75 g / L peptone, 0.25 g / L yeast extract, 1.4 g / L ammonium sulfate, 2.0 g / L potassium dihydrogen phosphate, 0.3 g / L calcium chloride, 0.3 g / L magnesium sulfate heptahydrate, 60.0 g / L microcrystalline cellulose (MCC), 0.005 g / L ferrous sulfate heptahydrate, 0.0016 g / L manganese sulfate tetrahydrate, 0.0014 g / L zinc sulfate heptahydrate, 0.002 g / L cobalt chloride hexahydrate, with the remainder being water; the preferred culture temperature is 30℃; the preferred culture time is 192 h; and the preferred culture rotation speed is 180 rpm.

[0022] The application of Trichoderma asperellum ML02 in the fermentation production of β-glucosidase.

[0023] The above seed culture was inoculated into a fermentation medium for liquid fermentation.

[0024] The inoculation amount of the seed liquid into the fermentation medium is 1%-10% v / v, preferably 10%.

[0025] All culture media were sterilized at 115°C for 20 minutes under high temperature and pressure.

[0026] The culture mentioned above is a shaker culture.

[0027] During the fermentation process, starting from the second day, the pH of the fermentation broth was adjusted to stabilize at 5.5 using 3 mol / L sodium hydroxide and 1 mol / L dilute hydrochloric acid, and samples were taken on the eighth day to measure enzyme activity.

[0028] Application of the strain that produces high levels of β-glucosidase in the degradation of lignocellulose.

[0029] Replace the 60.0 g / L microcrystalline cellulose in the above fermentation medium with 60.0 g / L of untreated corn cob / corn stalk / wheat straw. The remaining operations are the same as those described above for the fermentation production of β-glucosidase. After fermentation, centrifuge the fermentation broth at 8000 rpm for 5 min, collect the solids, dry them at 70℃ to constant weight, and calculate the degradation rate.

[0030] Application of the strain that produces high levels of β-glucosidase in lactic acid production in a mixed bacterial system based on cellulose substrate.

[0031] The strain that produces high levels of β-glucosidase was cultured in combination with lactic acid bacteria to produce lactic acid.

[0032] Lactobacillus paracasei MRS medium: 10.0 g / L peptone, 5.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 1.0 g / L Tween-80, 2.0 g / L dipotassium hydrogen phosphate, 5.0 g / L sodium acetate, 2.0 g / L triammonium citrate, 0.2 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate tetrahydrate, 20.0 g / L agar, the remainder being water, pH 6.0.

[0033] Lactobacillus paracasei seed culture medium: 5.0 g / L yeast extract, 10.0 g / L sodium bicarbonate, 10.0 g / L sodium dihydrogen phosphate dihydrate, 15.0 g / L dipotassium hydrogen phosphate trihydrate, 3.0 g / L corn steep liquor powder, 10.0 g / L glucose, with water as the solvent.

[0034] Lactic acid concentration determination: 1 mL of fermentation broth was centrifuged at 12000 rpm for 1 min. The supernatant was diluted 20-100 times with the mobile phase and then filtered twice through a 0.22 μm membrane. The concentration of organic acid was detected by high-performance liquid chromatography (HPLC) under the following conditions: 0.25 mmol / L H₂SO₄ solution as the mobile phase, flow rate of 0.5 mL / min. An organic acid column was used.

[0035] Mixed culture for lactic acid production: Colonies of *Lactobacillus paracasei* MRS solid medium were rinsed with sterile water and inoculated into *Lactobacillus paracasei* seed medium, and cultured at 37°C and 180 rpm for 12-18 h. Simultaneously, *Trichoderma echinococcus* ML02 was cultured under aerobic conditions at 30°C and 180 rpm for 48 h, following the same procedure as for β-glucosidase production (except that the substrate concentration was changed from 60 g / L MCC to 80 g / L MCC). Next, Lactobacillus paracasei LYS2 activated for 18 hours (accessed at the China Center for Type Culture Collection, accession number CCTCC NO: M 20211178, accession date September 15, 2021) was inoculated at a rate of 10% (v / v) into Trichoderma echinococcus fermentation medium that had been cultured for 48 hours. At the same time, 7.5 g / L corn steep liquor powder and 15 g / L CaCO3 were added, and the medium was transferred to anaerobic conditions and cultured at 37°C for several days.

[0036] In this invention, *Trichoderma echinosporum* ML02 achieved an 8-day degradation rate of over 40% for microcrystalline cellulose, a 33.3% increase compared to the original strain's 30%. Furthermore, its 8-day degradation rates for untreated corn cobs and wheat straw reached 54.6% and 56.8%, respectively. This represents a significant advantage compared to the 56.3% degradation rate of straw by *Penicillium expansum* reported by Fan Bingquan et al. This strain can be used as a high-performance upstream strain in mixed microbial systems, reducing costs and promoting the industrialization of biomass refining.

[0037] The strain *Trichoderma asperellum* ML02 described in this invention grows rapidly, covering PDA plates within 72 hours. Simultaneously, its mycelial density is higher, which facilitates the secretion of hydrolytic enzymes and their binding to substrates, thus enhancing degradation performance. Furthermore, when this strain is used for fermentation with microcrystalline cellulose as a substrate, the β-glucosidase activity reaches 1.1 U / mL on day 8, and the degradation rate of microcrystalline cellulose can reach over 40%, a 33.3% increase compared to the original strain's 30% degradation rate. Moreover, it can be used to directly degrade lignocellulose from untreated corn cobs and wheat straw, achieving degradation rates of 54.6% and 56.8%, respectively, demonstrating its excellent lignocellulose degradation ability and broad substrate spectrum. When mixed with lactic acid bacteria and cultured using cellulose as a carbon source, the lactic acid yield reached 50 g / L in 168 hours, while the original bacteria required nearly 240 hours to reach the same yield. This shortened the fermentation cycle and resulted in a final yield of 72.5 g / L, which is 28.9% higher than the final yield of 57.6 g / L of the original bacteria.

[0038] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0039] This invention uses an existing *Trichoderma echinococcus* strain in the laboratory as the starting strain. After two rounds of EMS mutagenesis, and initial screening using ferric citrate aescinate and Congo red plates, supplemented by shake-flask screening, a high-yielding β-glucosidase mutant strain was selected. This strain not only exhibits 3 times the β-glucosidase activity of the starting strain, but also has 1.6 times and 1.9 times the endonuclease and exonuclease activity, respectively. The strain provided by this invention effectively compensates for the imbalance of cellulose-degrading enzyme systems and possesses high degradation efficiency. It also grows rapidly, with denser hyphae, which facilitates the secretion of hydrolases and their binding to substrates, thus improving degradation performance. When ML02 was used as the upstream degrading strain and co-cultured with lactic acid bacteria, it was found that compared with the original strain, it had the advantages of a shorter fermentation cycle and increased yield and productivity, demonstrating high development potential and application value in industry. Attached Figure Description

[0040] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0041] Figure 1 This is the mutagenic lethal curve;

[0042] Figure 2 The plate for screening bacteria for ferric citrate aescin (left side is the starting bacteria, right side is ML02);

[0043] Figure 3This is a plate image of the mutagenic fungus Trichoderma asperellum ML02;

[0044] Figure 4 Stability passaging of ML02β-glucosidase was performed (original bacterial enzyme activity was set at 100%).

[0045] Figure 5 Stability passaging of ML02 endonuclease was performed (original bacterial enzyme activity was set at 100%).

[0046] Figure 6 Stability passaging of ML02 exonuclease was performed (original bacterial enzyme activity was set at 100%).

[0047] Figure 7 The degradation rate of the original bacteria and ML02 after 8 days;

[0048] Figure 8 The degradation of ML02 on different lignocellulose substrates;

[0049] Figure 9 This refers to the lactic acid production and the accumulation of glucose and cellobiose in the original bacteria.

[0050] Figure 10 This refers to the lactic acid production and the accumulation of glucose and cellobiose in ML02. Detailed Implementation

[0051] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0052] Lactobacillus paracasei LYS2 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20211178 and deposit date of September 15, 2021.

[0053] Example 1

[0054] Mutagenesis, selection, and identification of *Trichoderma asperellum* ML02, a high-β-glucosidase producer:

[0055] The starting strain *Trichoderma asperellum* LYS1 was activated on PDA plates and incubated at 30°C for 3-4 days. The PDA plates were then washed with 0.9% physiological saline to adjust the spore concentration to 1×10⁻⁶. 8 / mL. Take 1mL of spore solution into ten 2mL EP tubes, add 40μL of EMS, place in a shaker at 30℃ and 180rpm, and incubate for the appropriate time (20-180min). Remove and add 5% sodium thiosulfate antidote for 5min. Dilute 10 3 After doubling, 100 μL was plated on a Congo red plate. The lethality curve indicated that 100-120 min was the appropriate mutagenesis time (e.g., ...). Figure 1 (As shown).

[0056] The screening method for *Trichoderma asperellum* ML02 described in this invention is as follows: The original fungal spore solution is mutagenized at a suitable mutagenesis time, detoxified and diluted, then spread onto a ferric citrate aescinate plate. After incubation at 30℃ for 1-2 days, the blacker spots on the ferric citrate aescinate plate are selected and inoculated onto a Congo red plate (e.g.,...). Figure 2 (As shown). Next, spots on the Congo red plate with a clear zone / diameter ratio larger than the original strain were selected for shake-flask rescreening. This screened out the high-β-glucosidase-producing mutant strain (Trichoderma asperellum) ML02.

[0057] Finally, the 18S rDNA of the mutant strain was determined using primers ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3'.

[0058] PCR amplification was performed, and the strain ML02 was identified as belonging to the genus Trichoderma asperellum at the molecular level after comparison with the NCBI database. Its 18S rDNA nucleotide sequence is shown in SEQ ID NO.1.

[0059] The colony characteristics of the *Trichoderma elatior* strain ML02 described above are as follows: On potato dextrose (PDA) medium, mycelia can completely cover the plate within 3 days at 30°C, showing faster growth than the original strain. Its colony color remains white (the original strain's colonies are initially white, then green, and turn yellow / yellow-green upon aging). The colony structure is felt-like with rounded, serrated edges, relatively regular, and produces few spores. The mycelia are numerous and dense, which is conducive to the secretion of hydrolytic enzymes and their binding to substrates, promoting degradation (e.g., ...). Figure 3 (As shown).

[0060] The PDA plate culture medium used in the above culture process is: potato 200g / L, glucose 20g / L, agar powder 20g / L, and the remainder is water;

[0061] Congo red plate medium: 2 g / L sodium carboxymethyl cellulose (CMC-Na), 0.5 g / L magnesium sulfate heptahydrate, 1.0 g / L potassium dihydrogen phosphate, 1.1 g / L peptone, 1.0 g / L lactose, 1.0 g / L sodium deoxycholate, 0.1 g / L Congo red, 20.0 g / L agar, the remainder being water.

[0062] Ferric citrate aescin plate medium: 0.5 g / L magnesium sulfate heptahydrate, 1.0 g / L potassium dihydrogen phosphate, 1.1 g / L peptone, 1.0 g / L lactose, 1.0 g / L sodium deoxycholate, 2.0 g / L aescin, 0.5 g / L ferric citrate, 20.0 g / L agar. The remainder is water.

[0063] The PCR reaction system consisted of 10 μL fungal genomic DNA, 25 μL 2×Rapid Taq Master Mix, 1 μL each of 10 μmol / L forward and reverse primers, and then sterile water was added to a final volume of 50 μL. PCR conditions were as follows: 95℃ pre-denaturation for 8 min, 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 1 min, for a total of 30 cycles; 72℃ repair extension for 10 min, and the reaction was terminated at 4℃.

[0064] Example 2

[0065] Study on enzyme production characteristics and stability of liquid fermentation using microcrystalline cellulose as a substrate.

[0066] The strain *Trichoderma asperellum* ML02 was inoculated onto PDA culture plates (200g potato, 20g glucose, 20g agar, 1000mL distilled water, sterilized at 115℃ for 20min) and incubated at 30℃ for 72h. The strain was then transferred to seed culture medium using a sterilized pipette tip at a concentration of 10 g / mL seed culture. 8 One spore was collected and cultured at 30°C and 180 rpm for 48 hours. Then, the mycelial solution was aspirated with a pipette tip and inoculated into the fermentation medium at a 10% v / v inoculum. After inoculation, the bottle mouth was sealed with sealing film to prevent contamination, and the bottle was placed in a shaker at 30°C and 180 rpm for incubation. From day 2, the pH of the fermentation broth was adjusted to a stable level of 5.5 using 3 mol / L sodium hydroxide and 1 mol / L dilute hydrochloric acid. On day 8, samples were taken to measure endonuclease activity, exonuclease activity, and β-glucosidase activity to assess its stability after six generations.

[0067] The seed culture medium consisted of 0.3 g / L urea, 0.75 g / L peptone, 0.25 g / L yeast extract, 1.4 g / L ammonium sulfate, 2.0 g / L potassium dihydrogen phosphate, 0.3 g / L calcium chloride, 0.3 g / L magnesium sulfate heptahydrate, 10.0 g / L glucose, 0.005 g / L ferrous sulfate heptahydrate, 0.0016 g / L manganese sulfate tetrahydrate, 0.0014 g / L zinc sulfate heptahydrate, 0.002 g / L cobalt chloride hexahydrate, with the remainder being water.

[0068] The fermentation medium formula is as follows: 0.3 g / L urea, 0.75 g / L peptone, 0.25 g / L yeast extract, 1.4 g / L ammonium sulfate, 2.0 g / L potassium dihydrogen phosphate, 0.3 g / L calcium chloride, 0.3 g / L magnesium sulfate heptahydrate, 60.0 g / L microcrystalline cellulose (MCC), 0.005 g / L ferrous sulfate heptahydrate, 0.0016 g / L manganese sulfate tetrahydrate, 0.0014 g / L zinc sulfate heptahydrate, 0.002 g / L cobalt chloride hexahydrate, with the remainder being water.

[0069] The preparation method for the reagents used to determine enzyme activity is as follows.

[0070] Preparation of pH 4.8, 50mM citrate buffer: 210g citric acid monohydrate, 750mL sterile water, add sodium hydroxide (50-60g), dilute to 1000mL to obtain a 1mol / L citrate buffer stock solution with pH 4.5. (When diluted 20 times to 50mM, the pH value is 4.8).

[0071] 1% sodium carboxymethyl cellulose solution: Weigh 1g of sodium carboxymethyl cellulose into a 100mL volumetric flask, add pH 4.8, 50mM citrate buffer, bring to volume, and heat until completely dissolved.

[0072] 2% microcrystalline cellulose suspension: Weigh 2g of microcrystalline cellulose into a 100mL volumetric flask, add pH 4.8, 50mM citrate buffer, bring to volume, and sonicate.

[0073] 1mM p-nitrophenyl-β-D-glucopyranoside (1mM pNPG): Weigh 0.0301g of p-nitrophenyl-β-D-glucopyranoside into a 100mL volumetric flask, add 50mM citrate buffer (pH 4.8), and bring the volume to the mark.

[0074] The methods for measuring the activity of the enzymes mentioned above are as follows.

[0075] Method for determining endonuclease activity (endoglucanase activity): Take fermentation broth and centrifuge at 12000 rpm for 5 min. In a 2 mL centrifuge tube, add 50 μL of appropriately diluted supernatant, 250 μL of pH 4.8, 50 mM citrate buffer, and 100 μL of 1% sodium carboxymethyl cellulose solution. Incubate at 50 °C for 10 min. Then add 400 μL of DNS solution, incubate in a boiling water bath for 5 min, and immediately cool. Measure the absorbance at OD 540 nm to calculate enzyme activity. Each treatment was performed in triplicate, with inactivated enzyme solution serving as a control. Endonuclease activity is defined in SI units as the amount of enzyme that catalyzes the hydrolysis of glucose to produce 1 μmol per minute at pH 4.8 and 50 °C; one enzyme activity unit (IU) is defined as this amount of enzyme that catalyzes the hydrolysis of glucose to produce 1 μmol per minute.

[0076] Method for determining exonuclease activity (exoglucanase activity): Take fermentation broth and centrifuge at 12000 rpm for 5 min. Add 50 μL of appropriately diluted supernatant, 250 μL of pH 4.8, 50 mM citrate buffer, and 100 μL of 2% microcrystalline cellulose solution to a 2 mL centrifuge tube. Incubate at 50 °C for 30 min. Then add 400 μL of DNS solution, incubate in a boiling water bath for 5 min, and immediately cool. Measure the absorbance at OD 540 nm and calculate the enzyme activity. Each treatment was performed in triplicate, with an inactivated enzyme solution as a control. Exonuclease activity is defined in SI units as the amount of enzyme that catalyzes the hydrolysis of glucose to produce 1 μmol per minute at pH 4.8 and 50 °C.

[0077] β-glucosidase activity assay: Fermentation broth was centrifuged at 12000 rpm for 5 min. In a 2 mL centrifuge tube, 100 μL of appropriately diluted supernatant and 900 μL of 1 mM p-nitrophenyl-β-D-glucopyranoside were added, and the mixture was incubated at 50 °C for 10 min. The reaction was then terminated by adding 500 μL of 10% sodium carbonate solution. The absorbance was measured at OD 400 nm to calculate enzyme activity. The control was an inactivated enzyme solution. Each treatment was repeated three times. β-glucosidase activity is defined as the amount of enzyme that catalyzes the hydrolysis of pNPG to produce 1 μmol of p-nitrophenol (pNP) per minute at pH 4.8 and 50 °C, defined as one unit (IU).

[0078] from Figure 4 As can be seen from Figures 5 and 6, this mutant strain has high passage stability. After multiple rounds of passage, it still maintains relatively high β-glucosidase, endonuclease, and exonuclease activities, and can maintain strong degradation performance.

[0079] Example 3

[0080] To verify the high degradation efficiency of the mutant Trichoderma ML02, the degradation rate was measured using microcrystalline cellulose as a substrate.

[0081] The strain *Trichoderma asperellum* ML02 was inoculated onto a PDA plate (200g potato, 20g glucose, 20g agar, 1000mL distilled water, sterilized at 115℃ for 20min) and incubated at 30℃ for 72h. Using a sterilized pipette tip, the strain was then transferred from the plate culture to the seed culture medium at a concentration of 10 g / mL seed culture. 8 1 spore. Incubate at 30℃ and 180 rpm for 24-48 h. Then, using a pipette tip, inoculate the mycelial solution at a 10% v / v rate into 70 mL of 60 g / L MCC Trichoderma echinocandi fermentation medium (0.3 g / L urea, 0.75 g / L peptone, 0.25 g / L yeast extract, 1.4 g / L ammonium sulfate, 2.0 g / L potassium dihydrogen phosphate, 0.3 g / L calcium chloride, 0.3 g / L magnesium sulfate heptahydrate, 60.0 g / L microcrystalline cellulose (MCC), 0.005 g / L ferrous sulfate heptahydrate, 0.0016 g / L manganese sulfate tetrahydrate, 0.0014 g / L... Zinc sulfate heptahydrate, cobalt chloride hexahydrate (0.002 g / L), and water were used as the remainder. After inoculation, the bottle mouth was sealed with sealing film to prevent contamination, and the bottle was placed in a shaker at 30°C and 180 rpm for incubation. Starting from day 2, the pH of the fermentation broth was adjusted to a stable level of 5.5 using 3 mol / L sodium hydroxide and 1 mol / L dilute hydrochloric acid. On day 8, the fermentation broth was centrifuged at 8000 rpm for 5 min, the solids were collected, dried at 70°C, and weighed to constant weight. The degradation rate was then calculated.

[0082] The procedure for the original bacteria is the same as above.

[0083] like Figure 7 As shown, the degradation rate of ML02 was 33.3% higher than that of the original bacteria, indicating that this mutant bacteria has a stronger degradation efficiency, can achieve efficient biomass conversion, and has broad application prospects.

[0084] Example 4

[0085] The specific operation for the degradation of microcrystalline cellulose by ML02 is the same as in Example 3, with the addition of 60 g / L of lignocellulose such as corn cob and wheat straw as substrates. Figure 8 As shown, the mutagenic Trichoderma ML02 achieved 8-day degradation rates of 54.6% and 56.8% for corn cobs and wheat straw, respectively. Moreover, these substrates were not pretreated, indicating that it has good degradation ability for lignocellulose and a broad degradation substrate spectrum.

[0086] Example 5

[0087] To verify the practical application of the highly efficient degradation ability of the mutant Trichoderma ML02, it was cultured in a mixed culture with lactic acid bacteria.

[0088] Lactobacillus paracasei MRS medium: 10.0 g / L peptone, 5.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 1.0 g / L Tween-80, 2.0 g / L dipotassium hydrogen phosphate, 5.0 g / L sodium acetate, 2.0 g / L triammonium citrate, 0.2 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate tetrahydrate, 20.0 g / L agar, the remainder being water, pH 6.0.

[0089] Lactobacillus paracasei seed culture medium: 5.0 g / L yeast extract, 10.0 g / L sodium bicarbonate, 10.0 g / L sodium dihydrogen phosphate dihydrate, 15.0 g / L dipotassium hydrogen phosphate trihydrate, 3.0 g / L corn steep liquor powder, 10.0 g / L glucose, with water as the solvent.

[0090] Lactic acid concentration determination: 1 mL of fermentation broth was centrifuged at 12000 rpm for 1 min. The supernatant was diluted 20-100 times with the mobile phase and then filtered twice through a 0.22 μm membrane. The concentration of organic acid was detected by high-performance liquid chromatography (HPLC) under the following conditions: 0.25 mmol / L H₂SO₄ solution as the mobile phase, flow rate of 0.5 mL / min. An organic acid column was used.

[0091] Trichoderma culture was performed as shown in Example 3 (except that the substrate concentration of 60 g / L MCC was replaced with 80 g / L MCC).

[0092] Mixed culture for lactic acid production: Colonies of *Lactobacillus paracasei* MRS solid medium were rinsed with sterile water and inoculated into *Lactobacillus paracasei* seed medium, and cultured at 37°C and 180 rpm for 12-18 h. Simultaneously, *Trichoderma echinococcus* ML02 was cultured under aerobic conditions at 30°C and 180 rpm for 48 h, following the same procedure as for β-glucosidase fermentation. Next, *Lactobacillus paracasei* LYS2 activated for 18 h was inoculated at a rate of 10% (v / v) into the *Trichoderma echinococcus* fermentation medium cultured for 48 h, with the addition of 7.5 g / L corn steep liquor powder and 15.0 g / L CaCO3, and then transferred to anaerobic conditions and cultured at 37°C for several days.

[0093] from Figure 10 It can be seen that the lactic acid production of the mutated strain ML02 reached 50 g / L in 168 h, while the original strain required nearly 240 h to reach this production, effectively shortening the fermentation cycle. The final yield reached 72.5 g / L, which is 28.9% higher than the final yield of 57.6 g / L of the original strain. This effectively optimized the multicellular system for lignocellulose biorefining and has great potential for industrial development.

[0094] The above embodiments are merely for analyzing and understanding the preparation method and application scope of the present invention, but the present invention is not limited to the above examples. If those skilled in the art are inspired by these embodiments and make direct changes, substitutions, modifications, etc., to the present invention, all such changes should fall within the protection scope of this patent.

Claims

1. A lignocellulose-degrading strain that produces a high level of β-glucosidase is classified as *Trichoderma echinosporum* (…). Trichoderma asperellum ML02 has been deposited at the China Center for Type Culture Collection (CCTCCNO: M 20221877) on December 5, 2022.

2. The application of the lignocellulose-degrading strain with high β-glucosidase production as described in claim 1 in the fermentation production of β-glucosidase.

3. The application of the lignocellulose-degrading strain with high β-glucosidase production as described in claim 1 in the degradation of lignocellulose.

4. The application according to claim 2, characterized in that, The strain Trichoderma ML02 was subjected to plate culture, seed culture and fermentation culture to obtain the corresponding enzyme activity; The plate culture was carried out by inoculating Trichoderma elatior ML02 onto a plate culture medium, and the culture temperature was 28-30 ℃ for 72-96 h. Seed culture: The strain cultured on plates is inoculated into seed culture medium, and the culture temperature is 28-30 ℃, the culture time is 24-48 h, and the culture speed is 160-200 rpm; Fermentation culture: The strain cultured in seed liquid is inoculated into fermentation medium, and the culture temperature is 28-30 ℃, the culture time is 168-192 h, and the culture speed is 160-200 rpm.

5. The application according to claim 4, characterized in that, The strain cultured on the plate was inoculated into the seed culture medium at a concentration of 10 μL per mL of seed culture. 7 -10 9 One spore.

6. The application according to claim 4, characterized in that, The components of the seed culture medium are as follows: 0.1-0.5 g / L urea, 0.5-1.0 g / L peptone, 0.2-0.5 g / L yeast extract, 1.0-2.0 g / L ammonium sulfate, 1.5-2.5 g / L potassium dihydrogen phosphate, 0.1-0.5 g / L calcium chloride, 0.1-0.5 g / L magnesium sulfate heptahydrate, 10.0-20.0 g / L glucose, 0.002-0.008 g / L ferrous sulfate heptahydrate, 0.001-0.003 g / L manganese sulfate tetrahydrate, 0.001-0.003 g / L zinc sulfate heptahydrate, 0.001-0.003 g / L cobalt chloride hexahydrate, and the remainder is water.

7. The application according to claim 4, characterized in that, The inoculation amount of the strain cultured in the seed culture medium is 1%-10% v / v.

8. The application according to claim 4, characterized in that, The fermentation medium comprises the following components: 0.1-0.5 g / L urea, 0.5-1.0 g / L peptone, 0.2-0.5 g / L yeast extract, 1.0-2.0 g / L ammonium sulfate, 1.5-2.5 g / L potassium dihydrogen phosphate, 0.1-0.5 g / L calcium chloride, 0.1-0.5 g / L magnesium sulfate heptahydrate, 10.0-20.0 g / L glucose, 0.002-0.008 g / L ferrous sulfate heptahydrate, 0.001-0.003 g / L manganese sulfate tetrahydrate, 0.001-0.003 g / L zinc sulfate heptahydrate, 0.001-0.003 g / L cobalt chloride hexahydrate, with the remainder being water.

9. The application according to claim 4, characterized in that, The seed culture was conducted at a temperature of 30 ℃ for 48 h at a rotation speed of 180 rpm, and the fermentation culture was conducted at a temperature of 30 ℃ for 192 h at a rotation speed of 180 rpm.

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