Exoglucanase-producing strain and application thereof

Abstract

This invention relates to a strain that produces exoglucanase, named *Aspergillus terreus* (…). Aspergillus terreus CB10, deposited on February 17, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 40080. Fermentation culture using this strain can produce highly active exoglucanase, thereby expanding the range of exoglucanase-producing strains. This invention also provides a method for preparing exoglucanase by fermentation using Aspergillus terreus strain. Specifically, Aspergillus terreus strain CB10 is subjected to liquid fermentation, and the fermentation supernatant obtained after centrifugation to remove the cell bodies is the exoglucanase. The enzyme activity reaches 110.7 U / ml under conditions of pH 5.0 and 50℃. This invention is of great significance for improving the degradation efficiency of natural cellulose.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain that produces exoglucanase and its applications. Background Technology

[0002] Exoglucanase (EC 3.2.1.91), also known as exoglucanase hydrolase or microcrystalline cellulase, is an important component of the cellulase system. During cellulose degradation, it cleaves the non-reduced and reduced ends of cellulose chains to generate soluble cellulose dextrin. The hydrolytic substrates of exoglucanase mainly include highly crystalline celluloses such as microcrystalline cellulose (Avicel), straw, or rice husk. Therefore, the activity of exoglucanase plays a crucial role in the degradation of natural cellulose substances. (Li Fangzheng, Tang Liang, Zhao Jianwen, Wang Wenjuan, Zhao Hongkun. Isolation, identification and phylogenetic analysis of a highly efficient straw cellulose-degrading fungus, Shandong Agricultural Sciences, 2011, (07): 5-8.)

[0003] Currently, there are few research reports on exoglucanase in China. The microbial strains that can produce exoglucanase are mainly concentrated in thermophilic Chaetomium, thermocellulose Clostridium, anomalae, as well as fungi such as Trichoderma and Penicillium. (Suhas, VK Gupta, PJ Carrott, R. Singh, M. Chaudhary, S. Kushwaha. Cellulose: Areview as natural, modified and activated carbon adsorbent, BioresourTechnol, 2016, 216(1066-1076.), but there are no related reports in Aspergillus terreus. In addition, some strains that can synthesize exoglucanase, including genetically engineered mutant strains, have not shown ideal exoglucanase activity, and the enzyme activity is generally low (Zhang Tong. Screening and identification of exoglucanase-producing Aspergillus niger and expression of its gene in Pichia pastoris [Master's thesis]. Sichuan Agricultural University, 2011.). Therefore, it is urgent to screen strains that can produce highly active exoglucan to expand the application of this enzyme in agriculture, industry, medicine and other fields. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a strain producing exoglucanase and its application, the strain being Aspergillus terreus CB10 (… Aspergillus terreusCB10) can produce highly active exoglucanase through fermentation, thereby expanding the types of exoglucanase-producing strains and solving the problem of low exoglucanase activity in existing genetically engineered strains.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0008] In a first aspect, the present invention provides a strain that produces exoglucanase, named Aspergillus terreus CB10 was deposited on February 17, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 40080.

[0009] Aspergillus terreus CB10 was isolated from the soil of the wetland ecosystem in Caofeidian District, Tangshan City, Hebei Province, after being cultured using microcrystalline cellulose as the sole carbon source.

[0010] Species identification of Aspergillus terreus CB10 includes:

[0011] (1) Identification of growth and morphological characteristics:

[0012] Strain CB10 grows rapidly on solid plates, forming flat colonies with radial wrinkles. The outer hyphae are white, while the center is yellowish-brown. The central aerial hyphae extend into the air. On the reverse side of the culture plate, the endophytic cells exhibit a radial pattern, with the center being yellow and gradually fading outwards. Based on these colony morphological and structural characteristics, and referring to the "Handbook of Fungal Identification," these features are consistent with those of Aspergillus terreus.

[0013] (2) 18S rDNA sequence sequencing of the strain: Total DNA of Aspergillus terreus CB10 was extracted using a fungal genomic DNA extraction kit (Solarbio, D2300), and its 18S rDNA sequence was amplified. Sequencing was performed by a third-party company to obtain the 18S rDNA sequence of this strain, as shown in SEQ ID NO.1. The sequenced 18S rDNA nucleic acid sequence was submitted online to the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) and compared and analyzed using BLAST (Basic Local Alignment Search Tool). The 18S rDNA sequence of strain CB10 of this invention was compared with that of the reference strain. Aspergillus terreus ATE 1 (Genbank No. KM462826.1) and Aspergillus terreusATCC1012 (Genbank No. NG064804.1) showed a similarity of over 99%, and based on the above morphological characteristics, it was identified as Aspergillus terreus. Aspergillus terreus ).

[0014] In a second aspect, the present invention provides a method for producing exoglucanase, comprising: inoculating the above-mentioned Aspergillus terrestris CB10 into a liquid culture medium for fermentation culture to produce exoglucanase.

[0015] Preferably, the liquid culture medium contains a carbon source, a nitrogen source, inorganic salts, and water.

[0016] Preferably, the carbon source is one or a combination of two or more of the following substances: sodium carboxymethylcellulose (CMC), avicel, cellobiose, glucose, sucrose, lactose, glycerol, starch, corn stalk, and wheat straw.

[0017] Preferably, the nitrogen source is one or a combination of two or more of the following substances: ammonium nitrogen, nitrate nitrogen, ammonium nitrate nitrogen, acyl ammonium nitrogen, whey powder, peptone, and yeast extract.

[0018] Preferably, ammonium nitrogen is ammonium sulfate, ammonium chloride, ammonium bicarbonate, ammonia water, and liquid ammonia, etc.; nitrate nitrogen is potassium nitrate, calcium nitrate, and other nitrates; ammonium nitrate nitrogen is ammonium nitrate, calcium ammonium nitrate, and ammonium thionitrate, etc.; and acyl ammonium nitrogen is urea, etc.

[0019] Preferably, the water in the liquid culture medium is tap water or distilled water.

[0020] Preferably, the inorganic salts contained in the liquid culture medium include KH2PO4, (NH4)2SO4,

[0021] MgSO4, CaCl2, MnSO4, FeSO4, ZnSO4 and CoCl2.

[0022] Preferably, the fermentation temperature is 28-30℃ and the fermentation time is 2-10 days.

[0023] Preferably, the fermentation culture is carried out in a constant temperature shaker with a rotation speed of 150-220 rpm for 2-10 days.

[0024] After the culture was completed, the supernatant was obtained by centrifugation, and a crude extract rich in exoglucanase was obtained.

[0025] (III) Beneficial Effects

[0026] Aspergillus terreus strain CB10 of the present invention ( Aspergillus terreus CB10 has the following advantages: rapid growth and reproduction; a wide range of carbon sources that can be effectively utilized; a simple and highly operable enzyme production fermentation process; and high enzyme activity in the production of exoglucanase, with the highest enzyme activity reaching 110.7 U / mL. Attached Figure Description

[0027] Figure 1 This is a comparative diagram showing the exoglucanase activity of the 25 screened strains of this invention.

[0028] Figure 2 The enzyme activity value of exoglucanase in Aspergillus terrestris CB10 cultured for 4 days is shown in this invention.

[0029] Figure 3 Aspergillus terreus CB10 of the present invention Aspergillus terreus CB10 plate colony morphology diagram.

[0030] Figure 4 Aspergillus terreus CB10 of the present invention Aspergillus terreus Microscopic image of mycelial characteristics of CB10.

[0031] Figure 5 Aspergillus terreus CB10 of the present invention Aspergillus terreus Phylogenetic tree diagram of CB10 and reference strain.

[0032] Figure 6 Aspergillus terreus CB10 ( Aspergillus terreus (CB10) Comparison of the utilization of different carbon source substances. Detailed Implementation

[0033] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Example 1

[0034] This example illustrates the preliminary screening process for exoglucanase-producing strains:

[0035] (1) Sample collection: Soil samples were collected from the campus of North China University of Technology (Caofeidian Campus) in Tangshan City, Hebei Province in September 2021 and stored in sterile bottles.

[0036] A screening medium with microcrystalline cellulose as the sole carbon source was prepared as follows: KH2PO4 15 g / L, (NH4)2SO4 5 g / L, MgSO4 0.6 g / L, CaCl2 0.8 g / L, MnSO4·H2O 0.0016 g / L, FeSO4·7H2O 0.0005 g / L, ZnSO4·7H2O 0.0014 g / L, CoCl2 0.0002 g / L, microcrystalline cellulose 10 g / L, agar powder 20 g / L, pH 4.8.

[0037] Prepare PDA enrichment medium: 200 g potato chunks, add appropriate amount of distilled water, boil for 30 minutes, filter with gauze to remove residue, add 20 g glucose, 20 g agar powder, and finally add water to make up to 1 L.

[0038] Screening method: Soil samples were soaked in sterile water, and the supernatant was serially diluted and plated onto the screening medium containing microcrystalline cellulose as the sole carbon source. The samples were then incubated at 30°C for 7 days. Single colonies on the initial screening plates were selected and passaged and rescreened on the microcrystalline cellulose-only carbon source screening medium. After rescreening and removal of duplicate strains, a total of 25 fungi capable of growing in the microcrystalline cellulose-only carbon source medium were obtained. These 25 fungi were transferred to PDA agar plates for enrichment, preparing for subsequent fermentation and enzyme activity assays. Example 2

[0039] In this embodiment, the target strain was screened by measuring the exoglucanase activity, as follows:

[0040] 1. Culture medium and fermentation process

[0041] Seed culture medium: KH2PO4 15 g / L, (NH4)2SO4 5 g / L, MgSO4 0.6 g / L, CaCl2 0.8 g / L, MnSO4·H2O 0.0016 g / L, FeSO4·7H2O 0.0005 g / L, ZnSO4·7H2O 0.0014 g / L, CoCl2 0.0002 g / L, glucose 20 g / L, peptone 1 g / L, pH 4.8.

[0042] Fermentation flask culture medium: KH2PO4 15 g / L, (NH4)2SO4 5 g / L, MgSO4 0.6 g / L, CaCl2 0.8 g / L, MnSO4·H2O 0.0016 g / L, FeSO4·7H2O 0.0005 g / L, ZnSO4·7H2O 0.0014 g / L, CoCl2 0.0002 g / L, microcrystalline cellulose 20 g / L, wheat bran 20 g / L, peptone 1 g / L, urea 0.3 g / L, pH 4.8.

[0043] Twenty-five fungal spores obtained from screening in Example 1 were collected and serially diluted with sterile water to a concentration of 1.0 × 10⁻⁶. 6 The spore suspension was prepared at 100 μl / ml. The fermentation process is as follows: 100 μl of spore suspension (1.0 × 10⁻⁶ spores / ml) was fermented. 6 The inoculum was inoculated into seed culture bottles (50 ml of culture medium in a 250 ml Erlenmeyer flask) and cultured at 30°C with shaking at 200 rpm for 2 days. Then, the inoculum was transferred into fermentation culture medium at a 5% inoculation rate (50 ml of culture medium in a 250 ml Erlenmeyer flask). Fermentation broth was collected every 2 days, centrifuged at 8000 rpm, and the supernatant crude enzyme solution was collected.

[0044] 2. Glucose standard curve and exoglucanase activity determination: The determination method is as follows:

[0045] (1) Preparation of DNS reagent: Solution A: Weigh 10g of 3,5-dinitrosalicylic acid and 10g of NaOH, mix and dissolve in water; Solution B: Dissolve 200g of potassium sodium tartrate in 250ml of water, add 2.0g of phenol and 5.0g of Na2SO3, dissolve and cool. Mix solutions A and B and make up to 1000ml. Let stand in a brown bottle for 7 days before use. DNS reagent is used to determine the reducing sugar content in substances.

[0046] (2) Preparation of glucose standard curve: Prepare 1.0 mg / ml glucose standard solution: After drying an appropriate amount of glucose at 60℃ for 24 hours, weigh 1.0 g of glucose and dissolve it in water to make up to 1000 ml. Prepare glucose standard curve: Add 0, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, 1.0 ml, 1.2 ml and 1.5 ml of 1.0 mg / ml glucose standard solution to 8 test tubes respectively. Make up to 1.5 ml of total volume in each tube with distilled water. Add 2.0 ml of DNS solution to each test tube, boil for 10 min, cool and make up to 10 ml of volume with distilled water. Measure the absorbance at OD 540 nm. Plot the standard curve with the mass of standard glucose as the x-axis and the absorbance value as the y-axis.

[0047] (3) Procedure for determining exoglucanase activity: Add 1.0 ml of 1% microcrystalline cellulose solution and 0.5 ml of diluted fermentation enzyme solution to a test tube, react thoroughly in a water bath for 30 minutes, add 2.0 ml of DNS reagent, and add 6.5 ml of distilled water to make up to 10 ml. Measure the absorbance (OD) at 540 nm. Enzyme activity unit (U) definition: The amount of enzyme required to catalyze the production of 1 μg of glucose from salicin within 1 min at 50℃ and pH 5.0.

[0048] (4) The exoglucanase activity in the crude enzyme solution of the 25 strains in Example 1 was determined and evaluated using the DNS method described above. For example... Figure 1 As shown, after 4 days of cultivation and three parallel tests, it was found that among the 25 strains, strain CB10 had the highest exoglucanase activity in its crude enzyme solution, and its enzyme activity was the most significant. Figure 2 As shown, after 4 days of shaking culture, the enzyme activity of strain CB10 reached a peak of 110.7 U / ml, so CB10 was selected as the target strain for screening.

[0049] The above results represent enzyme activity measured at pH 5.0 and 50°C. Example 3

[0050] This example illustrates the identification of strain CB10, including morphological characteristics and phylogenetic analysis:

[0051] (1) Observation of morphological characteristics of strain CB10 on PDA plates

[0052] Spores of strain CB10 were enriched on PDA plates to prepare a concentration of 1.0 × 10⁻⁶. 6 A spore suspension of 1 spore / ml was prepared, and 2 μl of the suspension was inoculated into a PDA solid culture plate and incubated statically at 30 ℃. Figure 3 As shown in Figure A, strain CB10 grows rapidly, reaching a colony diameter of approximately 37±2 mm after 6 days. The colony on the front of the plate is flat with radial wrinkles; the outer edge hyphae are white, while the center is yellowish-brown. The central aerial hyphae extend into the air. Mature spores are yellow, plump, and easily detached. On the back of the culture plate, the endophytic cells exhibit a radial pattern, with the center being yellow and gradually fading outwards. Figure 3 B).

[0053] Using a sterile toothpick, pick a small amount of mycelium from the plate (for easy observation, the incubation time should not be too long; 3-5 days of growth is suitable) and place it on a glass slide. Add lactic acid cotton blue and heat slightly to stain and fix. Microscopic characteristics are as follows: Figure 4 As shown, the conidiophores are dense, the apical sacs are hemispherical, the conidiophores are relatively thick, and the scattered conidia are spherical, smooth, and mostly clustered together.

[0054] Based on the colony morphology and structural characteristics, and referring to the "Handbook of Fungal Identification", these characteristics are consistent with those of Aspergillus terreus.

[0055] (2) Phylogenetic analysis of strain CB10

[0056] Genomic DNA was extracted from strain CB10 and used as a template to amplify the 18S rDNA sequence using primers NS1 and NS8.

[0057] NS1: 5'-GTAGTCATATGCTTGTCTC-3';

[0058] NS8: 5'-TCCGCAGGTTCACCTACGGA-3'.

[0059] PCR reaction conditions: 98℃ pre-denaturation for 30 sec; 98℃ denaturation for 10 sec, 54℃ annealing for 30 sec, 72℃ extension for 1 min, 30 cycles; final extension at 72℃ for 10 min; storage at 4℃. The PCR product was purified using an agarose gel extraction kit, ligated with the pMD™19-T Vector, and transformed into *E. coli* DH5α. Positive clones (containing plasmid pMD19T-18S rDNA) were selected by blue-white screening and sequenced by Beijing Qingke Biotechnology Co., Ltd. to obtain the 18S rDNA sequence. A 1770 bp 18S rDNA sequence (SEQ ID NO. 1) was obtained and uploaded to the Genbank database, obtaining sequence number NO. OM250078.

[0060] The 18S rDNA gene sequence of strain CB10 was submitted to the NCBI database for online BLAST nucleic acid sequence alignment. Published reference sequences were selected from the database, and multiple alignments were performed using MEGA software (version X). Phylogenetic analysis and phylogenetic tree construction were then performed using the MEGA X software package. Neighbor-joining (NJ) was used for evolutionary distance analysis, and the bootstrap method (repeated 1000 times) was used to test the statistical significance of differences between branches of the phylogenetic tree. The results are as follows: Figure 5 As shown, the screened strain CB10 and Aspergillus terreus ATE 1 (Genbank No. KM462826.1) and Aspergillus terreus ATCC1012 (Genbank No. NG064804.1) is the most closely related. (Referencing relevant literature, and combining...) Figure 3 and Figure 4The morphological characteristics of strain CB10 are basically consistent with those of Aspergillus terreus, and strain CB10 is identified as belonging to the Aspergillus terreus species. Example 4

[0061] This embodiment focuses on Aspergillus terreus CB10 ( Aspergillus terreus Fermentation culture was carried out on strain CB10, and the utilization of different carbon sources by strain CB10 was investigated. The experimental method was as follows:

[0062] (1) Prepare a basic medium lacking carbon source: KH2PO4 15 g / L, (NH4)2SO4 5 g / L, MgSO4 0.6 g / L, CaCl2 0.8 g / L, MnSO4·H2O 0.0016 g / L, FeSO4·7H2O 0.0005 g / L, ZnSO4·7H2O 0.0014 g / L, CoCl2 0.0002 g / L, agar powder 20 g / L, pH 4.8.

[0063] (2) Add sodium carboxymethylcellulose (CMC), avicel, cellobiose, glucose, sucrose, lactose, glycerol and starch to the basic culture medium lacking carbon source at a final concentration of 1%.

[0064] (3) Take equal amounts of CB10 spores (2 μl, 1.0 × 10⁻⁶) 6 ( / ml) was inoculated into the culture media with different carbon sources mentioned above and incubated statically for 5 days.

[0065] The results are as follows Figure 6 As shown, strain CB10 can grow and ferment using a variety of carbon sources under various conditions. It not only grows well in media using sodium carboxymethyl cellulose, microcrystalline cellulose, and cellobiose as the sole carbon source, but also fully utilizes sucrose and lactose disaccharides. In media using glycerol as the sole carbon source, strain CB10 has a relatively small colony size, but the colony morphology is quite dense. Strain CB10 can also utilize wheat straw and microcrystalline cellulose to synthesize exonucleases. Plates using microcrystalline cellulose as a substrate have a larger diameter than those using other carbon sources, but the density is somewhat lower.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. sequence list <110> Zhongrong Technology Co., Ltd. <120> A strain producing exoglucanase and its application <141> 2022-03-17 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1770 <212> DNA / RNA <213> Aspergillus terreus <400> 1 gtagtcatat gcttgtctca aagattaagc catgcatgtc taagtataag cactttatac 60 tgtgaaactg cgaatggctc attaaatcag ttatcgttta tttgatagta ccttaactaca 120 tggatacctg tggtaattct agagctaata catgctaaaa accccgactt cggaaggggt 180 gtatttatta gataaaaaac caatgccctt cggggctcct tggtgattca taataactta 240 acgaatcgca tggccttgcg ccggcgatgg ttcattcaaa tttctgccct atcaactttc 300 gatggtagga tagtggccta ccatggtggc aacgggtaac ggggaattag ggttcgattc 360 cggagaggga gcctgagaaa cggctaccac atccaagga ggcagcaggc gcgcaaatta cccaatcccg acacggggag gtagtgacaa taaatactga tacggggctc tttcgggtct cgtaattgga atgagtacaa tctaaatccc ttaacgagga acaattggag ggcaagtctg gtgccagcag ccgcggtaat tccagctcca atagcgtata ttaaagttgt tgcagttaaa aagctcgtag ttgaaccttg ggtctggctg gccggtccgc ctcaccgcga gtactggtcc 660 ggctggacct ttccttctgg ggccct ggccttcact ggctgtgggg ggccgg 720 cttttactgt gaaaaaatta gagtgttcaa agcaggcctt tgctcgaata cattagcatg 840. 840. 840. 840. 840. 840. 840. 840. aatagggata gtcggggggcg tcagtattca gctgtcagag gtgaaattct tggatttgct gaagactaac tactgcgaaa gcattcgcca aggatgtttt cattaatcag ggaacgaaag ttaggggatc gaagacgatc agataccgtc gtagtcttaa ccataaacta tgccgactag ggatcgggcg gtgtttctat gatgacccgc tcggcacctt acgagaaatc aaagtttttg 1080. ggttctgggg ggagtatggt cgcaaggctg aaacttaaag aaattgacgg aagggcacca 1140 caaggcgtgg agcctgcggc ttaatttgac tcaacacggg gaaactcacc aggtccagac 1200 aaaataagga ttgacagatt gagagctctt tcttgatctt ttggatggtg gtgcatggcc 1260 gttcttagtt ggtggagtga tttgtctgct taattgcgat aacgaacgag acctcggccc 1320 ttaaatagcc cggtccgcat ttgcgggccg ctggcttctt agggggacta tcggctcaag 1380 ccgatggaag tgcgcggcaa taacaggtct gtgatgccct tagatgttct gggccgcacg 1440 cgcgctacac tgacagggcc agcgagtaca tcaccttggc cgagaggtct gggtaatctt 1500 gttaaaccct gtcgtgctgg ggatagagca ttgcaattat tgctcttcaa cgaggaatgc 1560 ctagtaggca cgagtcatca gctcgtgccg attacgtccc tgccctttgt acacaccgcc 1620 cgtcgctact accgattgaa tggctcggtg aggccttcgg actggctcca gggggttggc 1680 aacgaccccc cagagccgga aagttggtca aacccggtca tttagaggaa gtaaaagtcg 1740 taacaaggtt tccgtaggtg aacctgcgga 1770

Claims

1. A strain producing exoglucanase was named *Aspergillus terreus*. Aspergillus terreus CB10 was deposited on February 17, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 40080.

2. A method for producing exoglucanase, characterized in that, The strain described in claim 1 is inoculated into a liquid culture medium for fermentation to produce exoglucanase.

3. The method according to claim 2, characterized in that, The carbon source in the liquid culture medium is one or a combination of two or more of the following substances: sodium carboxymethyl cellulose, microcrystalline cellulose, cellobiose, glucose, sucrose, lactose, glycerol, starch, corn straw, and wheat straw.

4. The method according to claim 2, characterized in that, The nitrogen source in the liquid culture medium is one or a combination of two or more of the following substances: ammonium sulfate, ammonium chloride, ammonium bicarbonate, ammonia, potassium nitrate, calcium nitrate, ammonium nitrate, calcium ammonium nitrate, ammonium thionitrate, urea, whey powder, peptone, and yeast extract.

5. The method according to claim 2, characterized in that, The inorganic salts contained in the liquid culture medium include KH2PO4, (NH4)2SO4, MgSO4, CaCl2, MnSO4, FeSO4, ZnSO4 and CoCl2.

6. The method according to claim 2, characterized in that, The fermentation temperature is 28-30℃, and the fermentation time is 2-10 days.

7. The method according to claim 2, characterized in that, Fermentation is carried out in a constant temperature shaker at a speed of 150-220 rpm for 2-10 days.

8. The use of the strain according to claim 1 in the preparation of exoglucanase.

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