Endoglucanase derived from phyllophaga dilata intestinal flora and application thereof

Abstract

The application discloses endoglucanases derived from the intestinal flora of Phyllobius oblongus and application thereof. Eight high-activity endoglucanases are obtained from the intestinal flora of Phyllobius oblongus by metagenomics technology, the amino acid sequences of the endoglucanases are respectively shown as SEQ ID NO:1, 3, 5, 7, 9, 11, 13 and 15, and the nucleotide sequences of the encoding genes are respectively shown as SEQ ID NO:2, 4, 6, 8, 10, 12, 14 and 16. The above-mentioned genes are heterologously expressed in Zymomonas mobilis, purified and subjected to enzymatic property determination, and it is confirmed that the endoglucanases have excellent enzymatic activity, and the specific enzyme activity of CSP2705 is as high as 22.1 U / mg. The endoglucanases can be efficiently expressed in Zymomonas mobilis, and provide key enzyme elements for constructing CBP strains with Zymomonas mobilis as a chassis and realizing direct conversion of lignocellulose into fuel ethanol, and have important industrial application value.

Description

Technical Field

[0001] This application relates to the field of biotechnology. Specifically, this application relates to endoglucanases derived from the intestinal flora of the leaf beetle, polynucleotides encoding these endoglucanases, recombinant vectors containing said polynucleotides, recombinant host cells, and the use of these endoglucanases in the degradation of cellulose and the production of fuel ethanol. Background Technology

[0002] Lignocellulose is the most abundant and cheapest renewable biomass resource on Earth, and is considered an ideal raw material for producing second-generation biofuels such as cellulosic ethanol. Efficiently converting lignocellulose into fuel ethanol is of great significance for alleviating energy shortages and ensuring energy security. However, lignocellulose has a complex structure, making it difficult for microorganisms to utilize directly. The cost of cellulase is one of the key factors restricting its industrial application. To reduce costs, researchers have proposed the concept of Consolidated Bioprocessing (CBP), which integrates cellulase production, cellulose hydrolysis, and ethanol fermentation into a single microbial cell, thereby simplifying the process and significantly reducing production costs.

[0003] Zymomonas mobilis is recognized as one of the most promising chassis cells for the direct cell production process (CBP). It is a naturally occurring, highly efficient ethanol-producing strain, second only to yeast, possessing excellent characteristics such as high glucose consumption, high ethanol yield, low byproduct production, strong alcohol tolerance, and the ability to grow over a wide temperature and pH range. Therefore, genetically engineering Zymomonas mobilis to express and secrete highly efficient cellulase is crucial for constructing CBP strains and achieving the direct conversion of cellulose to ethanol.

[0004] Cellulase is a complex enzyme system, typically composed of endoglucanase (EG), exoglucanase (CBH), and β-glucosidase (BGL) working synergistically. Among these, endoglucanase acts on the amorphous regions of cellulose molecules, randomly hydrolyzing β-1,4-glycosidic bonds, and is one of the initiating and rate-limiting steps in cellulose degradation. Although *Fermentomonas motilityis* carries endogenous endoglucanase genes (such as CelA, ZMO1086), their enzyme activity is extremely low, lacking the practical ability to degrade cellulose. Therefore, identifying and screening exogenous endoglucanases that can be efficiently expressed in *Fermentomonas motilityis* is one of the core tasks in optimizing cellulosic ethanol strains.

[0005] In existing technologies, there have been attempts to transfer exogenous cellulase genes into *Fermentomonas motilityis*. For example, one study transferred the endoglucanase gene *celZ* from *Erwinia chrysanthemi* into *Fermentomonas motilityis*. Although expression was achieved, the specific enzyme activity was only about 3.4 U / mg. Furthermore, this study indicated that most heterologous genes are expressed at very low levels in *Fermentomonas motilityis*, with high expression being an "exception" (Brestic-Goachet et al., 1988). In addition, researchers have also extracted endoglucanases from sources such as *Bacillus subtilis* and *Pseudomonas fluorescens* and expressed them in *Fermentomonas motilityis*, but the enzyme activity levels were not ideal, far below the requirements for industrial applications (Yoon et al., 1988; Lejeune et al., 1988). In recent years, although there have been reports of mining cellulases from insect guts (such as grasshoppers and beet armyworms) using metagenomics technology, most of these studies have remained at the level of enzyme gene discovery and basic characterization, without systematic functional screening and adaptability verification for specific CBP chassis cells (such as motile fermentum).

[0006] In summary, while existing technologies provide a technical approach for expressing heterologous cellulases in *Fermentomonas motilityis*, limitations in enzyme gene sources and enzyme-host system compatibility issues have resulted in a lack of endoglucanases that can be stably and efficiently expressed in *Fermentomonas motilityis* with significantly high specific activity. Therefore, it is urgent to discover and screen endoglucanases from new biological resources that are more suitable for functioning in *Fermentomonas motilityis*, providing key enzyme elements for constructing efficient CBP strains and promoting the efficient conversion and utilization of lignocellulose. Summary of the Invention

[0007] In view of this, the purpose of this application is to provide an endoglucanase with high enzymatic activity that can be efficiently expressed in *Fermentomonas motilityis*, thereby solving the technical problem of the lack of highly active cellulase elements suitable for CBP chassis cells in the prior art. This application, through metagenomic sequencing of the intestinal flora of *Plagiodera versicolora*, identified and screened eight novel endoglucanase genes. These genes were then heterologously expressed, purified, and their enzymatic properties compared in *Escherichia coli* and *Fermentomonas motilityis*, respectively. Ultimately, endoglucanases CSP2705, CSP44, CSP7688-48, CSP504-9, CSP6959, CSP7503, CSP4850, and CSP3788 with significantly high specific enzyme activity in *Fermentomonas motilityis* were obtained. The endoglucanase of this application exhibits excellent enzyme activity in *Fermentomonas motilityis*, laying a solid foundation for constructing CBP strains using *Fermentomonas motilityis* as chassis cells and realizing the direct conversion of lignocellulose into fuel ethanol, which has important industrial application value.

[0008] To achieve the above objectives, this application provides the following technical solution:

[0009] In a first aspect, this application provides an endoglucanase selected from at least one of the following (a) to (h):

[0010] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:1;

[0011] (b) A protein consisting of the amino acid sequence shown in SEQ ID NO:3;

[0012] (c) A protein consisting of the amino acid sequence shown in SEQ ID NO:5;

[0013] (d) A protein consisting of the amino acid sequence shown in SEQ ID NO:7;

[0014] (e) A protein consisting of the amino acid sequence shown in SEQ ID NO:9;

[0015] (f) A protein consisting of the amino acid sequence shown in SEQ ID NO:11;

[0016] (g) A protein consisting of the amino acid sequence shown in SEQ ID NO:13;

[0017] (h) A protein consisting of the amino acid sequence shown in SEQ ID NO:15.

[0018] Secondly, this application provides an isolated nucleic acid molecule that encodes the endoglucanase described in the first aspect.

[0019] In some preferred embodiments, the nucleic acid molecule is selected from at least one of the following (1)-(8):

[0020] (1) The nucleotide sequence is shown in SEQ ID NO:2, which encodes the amino acid sequence shown in SEQ ID NO:1;

[0021] (2) The nucleotide sequence is shown in SEQ ID NO:4, which encodes the amino acid sequence shown in SEQ ID NO:3;

[0022] (3) The nucleotide sequence is shown in SEQ ID NO:6, which encodes the amino acid sequence shown in SEQ ID NO:5;

[0023] (4) The nucleotide sequence is shown in SEQ ID NO:8, which encodes the amino acid sequence shown in SEQ ID NO:7;

[0024] (5) The nucleotide sequence is shown in SEQ ID NO:10, which encodes the amino acid sequence shown in SEQ ID NO:9;

[0025] (6) The nucleotide sequence is shown in SEQ ID NO:12, which encodes the amino acid sequence shown in SEQ ID NO:11;

[0026] (7) The nucleotide sequence is shown in SEQ ID NO:14, which encodes the amino acid sequence shown in SEQ ID NO:13;

[0027] (8) The nucleotide sequence is shown in SEQ ID NO:16, which encodes the amino acid sequence shown in SEQ ID NO:15.

[0028] Thirdly, this application provides a recombinant vector containing the nucleic acid molecules described in the second aspect.

[0029] Fourthly, this application provides a recombinant host cell containing the nucleic acid molecule described in the second aspect or the recombinant vector described in the third aspect.

[0030] In some embodiments, the host cell is Zymomonas mobilis or Escherichia coli.

[0031] Fifthly, this application provides a method for preparing the endoglucanase described in the first aspect, comprising the following steps:

[0032] (1) Culturing the recombinant host cells described in the fourth aspect to express the endoglucanase; and

[0033] (2) The endoglucanase was isolated and purified from the culture.

[0034] In a sixth aspect, this application provides a cellulase preparation comprising the endoglucanase described in the first aspect.

[0035] In a seventh aspect, this application provides the use of the dextranase described in the first aspect or the recombinant host cell described in the fourth aspect in the degradation of cellulose or the production of glucose.

[0036] Eighthly, this application provides the use of the dextranase described in the first aspect or the recombinant host cell described in the fourth aspect in the production of fuel ethanol.

[0037] Compared with the prior art, this application has the following beneficial effects:

[0038] 1. This application marks the first time that eight novel endoglucanase genes have been discovered and obtained from the gut microbiota of the specific herbivorous insect *Plagiodera versicolora* using metagenomics technology. *Plagiodera versicolora* feeds exclusively on cellulose-rich plants such as willows, and its gut microbiota has developed a unique cellulose-degrading enzyme system over a long period of evolution, providing valuable genetic resources for the discovery of novel, highly active cellulases.

[0039] 2. The eight endoglucanases (CSP504-9, CSP6959, CSP7503, CSP2705, CSP7688-48, CSP3788, CSP4850, and CSP44) screened in this application all exhibited high enzyme activity in *Fermentomonas motilityis*. Among them, CSP2705 showed a specific enzyme activity as high as 22.1 U / mg, far exceeding the expression levels of exogenous endoglucanases in *Fermentomonas motilityis* reported in the prior art, indicating that the enzymes in this application have a significant advantage in catalytic efficiency.

[0040] 3. By comparing the specific enzyme activities after dual-host expression and purification in *E. coli* and *Fermentomonas motilityis*, this application is the first to discover and verify that the specific enzyme activities of these eight endoglucanases are generally higher in *Fermentomonas motilityis* than in *E. coli*. This finding indicates that the enzymes in this application have a natural compatibility with *Fermentomonas motilityis*, the core chassis cell of the CBP process, laying a solid foundation for constructing highly efficient CBP strains.

[0041] 4. This application employs a strategy of fusion expression of INP-anchored proteins and truncated signal peptides, combined with trypan blue plate hydrolysis zone screening, to achieve high-throughput, visualized initial screening of candidate endoglucanases in *Fermentosum motilityis*. Subsequently, reliable specific enzyme activity data were obtained through dual-host expression, affinity purification, and comparison of enzymatic properties, demonstrating rigorous and credible screening results.

[0042] 5. The eight endoglucanases and their encoding genes of this application can serve as key enzyme elements for constructing genetically engineered bacteria capable of directly utilizing cellulose substrates, particularly CBP strains based on *Fermentomonas motilityis*. Heterologous expression of these enzymes in *Fermentomonas motilityis* holds promise for achieving an integrated bioprocessing workflow combining cellulase production, cellulose hydrolysis, and ethanol fermentation, thereby significantly reducing the production cost of cellulosic ethanol and promoting the industrial application of lignocellulose biorefining technology. Attached Figure Description

[0043] Figure 1 The results of trypan blue plate screening for the expression of 18 endoglucanases from the leaf beetle ZMNP in *Zygomyces molluscum*.

[0044] Figure 2 SDS-PAGE analysis of CSP2705 expression in ZM4-T7 of *Morphozoa motifacica*.

[0045] Figure 3 SDS-PAGE analysis of CSP2705 expression in Escherichia coli BL21(DE3).

[0046] Figure 4 SDS-PAGE analysis of CSP3788 expression in ZM4-T7 of *Morphozoa motilityis*.

[0047] Figure 5 SDS-PAGE analysis of CSP3788 expression in Escherichia coli BL21(DE3).

[0048] Figure 6 SDS-PAGE analysis of CSP4850 expression in *Zygomycosis motility fermentation monocytogenes* ZM4-T7.

[0049] Figure 7 SDS-PAGE analysis of CSP4850 expression in Escherichia coli BL21(DE3).

[0050] Figure 8 SDS-PAGE analysis of CSP504-9 expression in *Zygomycosis fungoides* ZM4-T7.

[0051] Figure 9 SDS-PAGE analysis of CSP504-9 expression in Escherichia coli BL21(DE3).

[0052] Figure 10 SDS-PAGE analysis of CSP6959 expression in *Zygomycosis fungi* ZM4-T7.

[0053] Figure 11SDS-PAGE analysis of CSP6959 expression in Escherichia coli BL21(DE3).

[0054] Figure 12 SDS-PAGE analysis of CSP7503 expression in ZM4-T7 of *Mortrophilia motilityis*.

[0055] Figure 13 SDS-PAGE analysis of CSP7503 expression in Escherichia coli BL21(DE3).

[0056] Figure 14 SDS-PAGE analysis of CSP7688-48 expression in ZM4-T7 of *Mortrophilia motilityis*.

[0057] Figure 15 SDS-PAGE analysis of CSP7688-48 expression in Escherichia coli BL21(DE3).

[0058] Figure 16 SDS-PAGE analysis of CSP44 expression in *Zygomycosis fungoides* ZM4-T7.

[0059] Figure 17 SDS-PAGE analysis of CSP44 expression in Escherichia coli BL21(DE3).

[0060] Figure 18 SDS-PAGE analysis of CSP CelA expression in *Zygomycosis fungoides* ZM4-T7.

[0061] Figure 19 Comparison of the enzyme activities of eight recombinant endoglucanases in *Zygomorpha motileis* ZM4-T7 and *Escherichia coli* BL21.

[0062] Figure 20 Comparison of specific enzyme activities of eight recombinant endoglucanases in *Zygomorpha motileis* ZM4-T7 and *Escherichia coli* BL21. Detailed Implementation

[0063] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0064] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0065] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying common rounding techniques.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before providing a detailed description of this application, the following terms and definitions are provided to better understand it.

[0067] 1. Endoglucanase (EG): An enzyme with endo-1,4-β-glucanase (EC3.2.1.4) activity, capable of randomly hydrolyzing the β-1,4-glycosidic bonds within cellulose molecules to generate low-polymerization-degree small cellulose molecules with reducing ends such as cellodextrin, cellobiose, and cellotriose. This activity can be determined by methods known in the art, such as using sodium carboxymethyl cellulose (CMC) as a substrate and determining the amount of reducing sugar generated using the 3,5-dinitrosalicylic acid (DNS) method.

[0068] 2. Zymomonas mobilis: refers to a Gram-negative, facultative anaerobic ethanol-producing strain, which is one of the core chassis cells in the CBP process. In this application, the Zymomonas mobilis includes, but is not limited to, strains ZMNP (see Chinese Patent ZL202211138020.6), ZM4-T7 (see Chinese Patent CN114774453B), and their derivative strains.

[0069] 3. Metagenomics: This refers to the technique of directly extracting total DNA from all microorganisms in environmental samples (such as the intestinal contents of the leaf beetle) and obtaining the gene sequence information of all microorganisms through high-throughput sequencing and bioinformatics analysis. This application identified 18 potential endoglucanase genes through metagenomic sequencing of the intestinal flora of the leaf beetle.

[0070] 4. INP Fusion Expression: This refers to a strategy of fusing a target protein (such as an endoglucanase) with an ice nucleation protein (INP). As an anchoring protein, the INP can display the fusion protein on the cell surface or anchor it to the cell membrane, facilitating enzyme-substrate contact and enabling activity screening via plate hydrolysis. In this application, to promote the secretion of the enzyme protein extracellularly, an endoglucanase with a truncated signal peptide is fused with an INP for expression.

[0071] In this application, 18 potential endoglucanase genes were mined from the gut microbiota of *Plagiodera versicolora* using metagenomics technology. After initial screening with trypan blue plates, eight endoglucanases with high enzyme activity in *Zymomonas mobilis* were obtained. Their amino acid sequences are shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, and 15, and their nucleotide sequences encoding these amino acid sequences are shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, and 16 (correspondence shown in Table 1). These eight genes were heterologously expressed, purified, and their enzyme activity was measured in *Escherichia coli* BL21(DE3) and *Zymomonas mobilis* ZM4-T7, respectively. The results confirmed their excellent enzyme activity in *Zymomonas mobilis*, providing key enzyme elements for constructing CBP strains based on *Zymomonas mobilis*.

[0072] Table 1. Correspondence between amino acid sequences and nucleotide sequences of endoglucanases

[0073]

[0074] The following examples will illustrate in detail: metagenomic sequencing and candidate gene mining of the intestinal flora of *Leptochloa chinensis*, preliminary screening of candidate genes in *Fermentomonas motilityis*, gene sequences and amplification primers of 8 endoglucanases obtained from screening, heterologous expression and purification of 8 genes in *Escherichia coli* and *Fermentomonas motilityis*, and enzyme activity determination and comparison of specific enzyme activities of purified endoglucanases, etc.

[0075] Main materials and methods used in the embodiments:

[0076] 1. Strains and plasmids.

[0077] Zymomonas mobilis ZMNP strain: See Chinese Patent ZL202211138020.6.

[0078] ZM4-T7 strain of *Morphoblastic fermentum*: See Chinese Patent CN114774453B.

[0079] Escherichia coli BL21(DE3): purchased from Shanghai Sangon Biotech, B528414.

[0080] Escherichia coli DH5α: purchased from Daling, DZC101-96B100 Trelief® 5α.

[0081] Expression vector pEZ15A: See

[0082] html .

[0083] Expression vector pTZ28a: See Chinese Patent CN114774453B.

[0084] 2. Main reagents

[0085] Sodium carboxymethyl cellulose (CMC): Purchased from Sinopharm, 30036328.

[0086] Trypan Blue: Purchased from McLean, T6275-2g.

[0087] 3,5-Dinitrosalicylic acid (DNS): Purchased from Krypton, 22502862.

[0088] Ni-NTA agarose affinity chromatography medium: purchased from Cytiva, 10353996.

[0089] Bradford reagent kit: purchased from Beyotime, P0006-1.

[0090] Restriction endonuclease (NEB, R3733V), T4 DNA ligase (Takara, 2011A), T5 exonuclease (NEB, M0663S).

[0091] IPTG: Purchased from Fonsber, 24N05601.

[0092] Arabic sugar: purchased from (Maclean's, C17007641).

[0093] Primer synthesis: Shanghai Sangon Biotech

[0094] 3. Culture medium

[0095] RMG5 medium: 50 g / L glucose (Sinopharm), 10 g / L yeast extract (Shanghai Sangon Biotech), 2 g / L KH2PO4 (Sinopharm), sterilized at 108℃ for 30 min.

[0096] RMG2-Spe medium: 20 g / L glucose (Sinopharm), 10 g / L yeast extract (Shanghai Sangon Biotech), 2 g / L KH2PO4 (Sinopharm), 100 µg / mL spectinomycin, with an additional 15 g / L agar added when preparing the solid medium, sterilized at 108 ℃ for 30 min.

[0097] LB medium: 10 g / L peptone (OXOID, LP0024B), 5 g / L yeast extract (OXOID, LP0021B), 10 g / L sodium chloride (Sinopharm), with an additional 15 g / L agar added when preparing the solid medium, sterilized at 121℃ for 30 min.

[0098] Example 1: Metagenomic sequencing of the intestinal flora of the blue leaf beetle and mining of endoglucanase genes

[0099] Fresh adult Plagiodera versicolora leaf beetles were collected, and intestinal tissue was obtained under aseptic conditions. Total DNA was extracted from the intestinal contents. Metagenomic sequencing was performed using the Illumina NovaSeq platform to obtain raw sequencing data. The metagenomic data were analyzed using the MajorBio Cloud Platform (https: / / www.majorbio.com / ). The BLASTP algorithm of Diamond software (version 2.0.13, available at https: / / github.com / bbuchfink / diamond) was used to align the amino acid sequences of the non-redundant gene set with the KEGG database. The expected value (e-value) parameter was set to 1e-5 to ensure high reliability of the alignment results. Through bioinformatics analysis, 18 potential endoglucanase genes were assembled, predicted, and annotated, and named CSP504-9, CSP6959, CSP7503, CSP2705, CSP7688-48, CSP3788, CSP4850, CSP44, CSP504-19, CSP2970, CSP3110, CSP3491, CSP34-48, CSP1029, CSP7032, CSP7688-38, CSP225, and CSP88.

[0100] Example 2: Preliminary screening of endoglucanase in *Mammotrophic motility*

[0101] 2.1 Cloning of cellulase, anchoring protein, and signal peptide genes

[0102] First, the Peno promoter, INP gene, and 18 candidate endoglucanase gene fragments were amplified separately for subsequent construction of the INP fusion expression plasmid.

[0103] (1) Peno promoter amplification: Using wild-type strain ZM4 (ATCC 31821) of *Bacillus motilityis* as a template, PCR amplification was performed using primers Peno-F and Peno-R to obtain the Peno promoter fragment.

[0104] Peno-F:5'-TGTCTATACTCCAGTTACTCAATACG-3' (SEQ ID NO:17);

[0105] Peno-R: 5'-ATCGAAACCTTTCTTAAAATCTTTTAGAC-3' (SEQ ID NO: 18).

[0106] (2) INP gene amplification: Using the plasmid Ex-construct-1 (CN118652307B) containing the INP gene as a template, PCR amplification was performed using primers INP-F and INP-R to obtain the INP gene fragment.

[0107] INP-F:5'-ATGCTGGATAAAGCGTTGG-3' (SEQ ID NO:19);

[0108] INP-R: 5'-GGTTTTGCAGATTTTGTGGTGTTGTGA-3' (SEQ ID NO: 20).

[0109] (3) Using the 18 synthesized candidate gene fragments (CSP504-9, CSP3788, CSP6959, CSP7503, CSP4850, CSP504-19, CSP2970, CSP2705, CSP3110, CSP3491, CSP34-48, CSP1029, CSP7032, CSP7688-38, CSP7688-48, CSP44, CSP225, CSP88) as templates, PCR amplification was performed using the corresponding primers to obtain the target gene fragments.

[0110] 2.2 Preparation of linearized carriers

[0111] Using pEZ15A plasmid as a DNA amplification template, reverse PCR amplification was performed using primers V-pEZ15A-F and V-pEZ15A-R to obtain the linearized vector fragment.

[0112] V-pEZ15A-F:5'-GGATCCAAACTCGAGTAAGGATCTCCAG-3' (SEQ ID NO:21);

[0113] V-pEZ15A-R: 5'-ACGGTGAGCTGGTGAC-3' (SEQ ID NO: 22).

[0114] The PCR reaction system (50 μL) consisted of: 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), 10 μL 2×T5 Super PCR Mix (Beijing Qingke), 15 μL PrimeSTAR® Max DNA Polymerase (takara), 20 μL ddH2O, and 1 μL bacterial culture template.

[0115] The PCR reaction program was as follows: 98 °C pre-denaturation for 5 min; 32 cycles: 98 °C denaturation for 10 s, 55 °C annealing for 10 s, 72 °C extension for 40 s; final extension: 72 °C for 3 min.

[0116] The Peno promoter fragment, INP gene fragment, 18 target gene fragments and linearized vector fragments obtained from the above amplification were recovered by gel extraction (gel extraction was performed according to the standard procedure of the gel extraction kit).

[0117] 2.3 Construction of recombinant plasmids

[0118] Using 18 synthesized candidate gene fragments (CSP504-9, CSP3788, CSP6959, CSP7503, CSP4850, CSP504-19, CSP2970, CSP2705, CSP3110, CSP3491, CSP34-48, CSP1029, CSP7032, CSP7688-38, CSP7688-48, CSP44, CSP225, and CSP88) as templates, PCR amplification was performed using corresponding primers to obtain the target gene fragments. The linearized vector fragments were then mixed with the 18 target gene fragments at a molar ratio of 1:3, and recombinant plasmids were constructed using an INP fusion expression strategy. In vitro homologous recombination was performed using T5 exonuclease. The reaction system was as follows: DNA fragment 0.12 pM, linearized vector 0.04 pM, 10×Buffer 4 0.5 μL, T5 exonuclease 0.5 μL, and ddH2O added to a final volume of 5 μL. After reacting on ice for 5 min, *E. coli* DH5α competent cells were added and mixed thoroughly. After standing for 30 min, the cells were heat-shocked at 42 °C for 45 s, cooled on ice for 3 min, and then added to LB liquid medium. The cells were incubated at 37 °C and 250 rpm for approximately 1 h to activate the cells. The resulting inoculum was then spread on LB plates supplemented with the appropriate antibiotics and incubated upside down at 37 °C.

[0119] 2.4 Validation of positive clones

[0120] After single colonies grew, PCR verification was performed. A single colony was picked from a plate, added to 10 μL of sterile water, and mixed thoroughly by pipetting. 1 μL of this mixture was then added to the prepared reaction mixture. The PCR tube was centrifuged to remove air bubbles. The reaction mixture consisted of: Primer-F 0.4 μL, Primer-R 0.4 μL, T5 mix 5 μL (Beijing Qingke), Template 1 μL, and H2O 3.2 μL. The PCR amplification program was as follows: 98 ℃ pre-denaturation for 3 min; 98 ℃ denaturation for 10 s, 55 ℃ annealing for 10 s, 72 ℃ extension (set at 10 s / kb according to fragment length), for 30 cycles; 72 ℃ extension for 10 min; and storage at 4 ℃. Band sizes consistent with expectations were verified by sequencing.

[0121] Single clones were selected for colony PCR verification, and positive clones were sent for sequencing confirmation. The 18 recombinant plasmids with correct sequencing results were named pEZ15A-INP-CSP504-9, pEZ15A-INP-CSP3788, pEZ15A-INP-CSP6959, pEZ15A-INP-CSP7503, pEZ15A-INP-CSP4850, pEZ15A-INP-CSP504-19, pEZ15A-INP-CSP2970, pEZ15A-INP-CSP2705, pEZ15A-INP-CSP2705, pEZ15A-INP-CSP2970 ... The plasmids p-CSP3110, pEZ15A-INP-CSP3491, pEZ15A-INP-CSP34-48, pEZ15A-INP-CSP1029, pEZ15A-INP-CSP7032, pEZ15A-INP-CSP7688-38, pEZ15A-INP-CSP7688-48, pEZ15A-INP-CSP44, pEZ15A-INP-CSP225, and pEZ15A-INP-CSP88 were constructed using an INP fusion expression strategy and can express the INP-CSP fusion protein in *Cytomonas motile*.

[0122] 2.5 Preparation of competent cells of *Morphozoa motileis*

[0123] 2.5.1 Preparation Phase:

[0124] (1) Prepare 200 mL of ultrapure water, 200 mL of 10% glycerol, 50 mL of round-bottom centrifuge tubes, 1.5 mL of EP tubes, and 100 mL of RMG5 culture medium; after preparing the above items, sterilize them at 108 °C for 30 min.

[0125] (2) Take out the glycerol bacteria placed in the -80 °C refrigerator and activate them on a plate, then incubate them in a 30 °C constant temperature incubator.

[0126] (3) Pick a single colony and put it into RMG5 medium and incubate at 30 °C overnight.

[0127] (4) When transferring the bacterial culture to 100 mL of RMG5 medium, control the initial OD. 600 The concentration was approximately 0.025-0.03, and the sample was incubated in a shaker at 30°C and 100 rpm.

[0128] (5) After 5 h of incubation, a 1 mL sample can be taken and the OD value measured using a spectrophotometer. If the OD value is... 600 A value of 0.4-0.6 is sufficient for collecting bacteria in 50 mL centrifuge tubes for later use.

[0129] 2.5.2 Preparation stage:

[0130] (1) Centrifuge the bacterial solution at 4000 rpm, 8 min, 25 °C to collect the bacteria. Then discard the supernatant, add 10 mL of sterile water to gently resuspend the bacteria, and then add sterile water to make up to 40 mL.

[0131] (2) After centrifugation at 4000 rpm, 8 min, and 25 °C, discard the supernatant and add 10 mL of 10% glycerol to gently resuspend the bacterial cells. Then add 10% glycerol to make up to 40 mL.

[0132] (3) Repeat step 2.

[0133] (4) Add 300 μL of 10% glycerol to resuspend the bacterial cells, dispense into 1.5 mL EP tubes, place in liquid nitrogen for quick freezing, and then transfer to a -80 °C freezer.

[0134] (5) Test whether the competent cells are contaminated and the electroporation efficiency.

[0135] 2.6 Construction of recombinant strains of *Morphozoa motilityis*

[0136] The 18 recombinant plasmids constructed above were electroporated into ZMNP competent cells of *Bacillus simulans*.

[0137] (1) The electric transfer cup was pre-cooled on ice in advance. The competent cells of motile fermentation monoclonal bacteria were taken out from the -80 °C freezer and placed on ice to thaw.

[0138] (2) Take 50 μL of competent cells and add them to the electroporation vessel, then add about 500 ng of DNA sample.

[0139] (3) Perform the electric rotation according to the procedure of 1.8 kV, 25 μF, 200 Ω.

[0140] (4) Resuspend the sample in the electroporation cup with 1 mL RMG5, then transfer it to an EP tube and incubate at 30°C for 3-6 h.

[0141] (5) After taking out the sample, spread it on the plate with the corresponding resistance and incubate it in a constant temperature incubator at 30 °C for 2-3 days.

[0142] Next, colony PCR was performed for verification (the system and plasmid construction colony PCR were the same, and aseptic technique was observed). The primers for PCR detection are as follows:

[0143] pEZ15A-F:5'-GGCAAAGCCACCCTATTTTTAG-3' (SEQ ID NO:23);

[0144] pEZ15A-R:5'-CACTTCACTGACACCCTCAT-3' (SEQ ID NO:24).

[0145] Eighteen positive recombinant strains were obtained and named ZMNP / pEZ15A-INP-CSP504-9, ZMNP / pEZ15A-INP-CSP6959, etc. Meanwhile, ZMNP / pEZ15A-INP (expressing only INP and lacking the CSP gene) was used as a negative control.

[0146] 2.7 Panblue plate screening units

[0147] The 18 recombinant strains and negative control strains were inoculated into RMG5 liquid medium and cultured until OD500. 600 Approximately 0.5. Take 2 μL of bacterial suspension and inoculate it onto an RMG2-Spe trypan blue plate containing 1% CMC and spectinomycin. Incubate at 30 ℃ for 2-3 days, observing the formation of a clear hydrolysis zone around the colony. Results are as follows... Figure 1 As shown in Table 2.

[0148] Table 2. Results of trypan blue plate assay for endoglucan leaf beetle-derived endoglucan leaf beetle.

[0149]

[0150] Figure 1 This figure shows the trypan blue plate screening results of 18 endoglucanases derived from *Leymus chinensis* expressing *Fermentomonas motilityis* ZMNP. As shown in the figure, the negative control strain ZMNP / pEZ15A-INP showed no clear hydrolysis zone, while among the recombinant strains expressing the 18 candidate endoglucanases, 8 strains showed obvious hydrolysis zones, and the remaining 10 strains showed no obvious hydrolysis zone or very small hydrolysis zones. These results indicate that these 8 endoglucanases were successfully expressed in *Fermentomonas motilityis* and possess high CMC degradation activity.

[0151] Based on the hydrolysis zone area ratio, eight highly active endoglucanases were screened: CSP2705, CSP44, CSP7688-48, CSP504-9, CSP6959, CSP7503, CSP4850, and CSP3788, for further research. The sources and predicted signal peptides of these eight endoglucanases are shown in Table 3.

[0152] Table 3. Screening results of endocellulase from *Ichthyophthirius multifiliis*.

[0153]

[0154] 2.8 Information on the gene sequence of endoglucanase obtained through screening

[0155] The amino acid sequences of the eight endoglucanases obtained through the above screening and the nucleotide sequences of their encoding genes are shown in Table 1 above. The PCR amplification primer sequences for these genes are shown in Table 4.

[0156] Table 4. Primer sequences for PCR amplification of 8 endoglucanase genes

[0157]

[0158] Example 3: Heterologous expression and purification of endoglucanase in Escherichia coli and Cytogenes motiles

[0159] 3.1 Expression plasmid construction

[0160] To further verify the enzymatic properties of the eight endoglucanases obtained in Example 2, they were cloned into the pTZ28a vector (CN114774453B) to construct expression plasmids suitable for *Escherichia coli* BL21(DE3) and *Fermentomonas motilityis* ZM4-T7. Additionally, a vector was constructed using the *Fermentomonas motilityis* gene CelA, an endogenous endoglucanase gene, as a control. The nucleotide sequence of the CelA gene is shown in SEQ ID NO:41, and its encoded amino acid sequence is shown in SEQ ID NO:42.

[0161] Using the synthesized CSP genes and their own CelA fragment (SEQ ID NO:41) as templates, PCR amplification was performed using the corresponding primers described in Table 5 below to obtain the target gene fragments.

[0162] Table 5. Primer sequences for PCR amplification used to construct the pTZ28a-CSP expression plasmid.

[0163]

[0164] Simultaneously, using pTZ28a plasmid as a template, reverse PCR amplification was performed using primers pTZ28a reverse amplification vector-F and pTZ28a reverse amplification vector-R to obtain linearized vector fragments.

[0165] pTZ28a back-amplifier-F: 5'-TGAGATCCGGCTGCTAACAAAGC-3' (SEQ ID NO: 61);

[0166] pTZ28a reverse amplification vector-R: 5'-GTGATGATGATGATGATGGCTGC-3' (SEQ ID NO:62).

[0167] Simultaneously, the target fragment was amplified using the aforementioned primers, and the pTZ28a vector fragment was reverse-amplified and transformed into DH5α via T5 ligation. The operation procedure was the same as in Example 2. The PCR reaction system consisted of: 1 μL Template DNA, 2 μL Primer-F, 2 μL Primer-R, 25 μL 2×Phanta Master Mix, and ddH2O to a final volume of 50 μL; the PCR reaction program was: 95 ℃ for 3 min; 95 ℃ for 10 s, 58 ℃ for 5 s, 72 ℃ for 10 s (adjusted according to fragment length), 72 ℃ for 10 min; <12 ℃ for 5 min; the T5 ligation system consisted of: 0.04 pM linearized vector, 0.12 pM DNA fragment, 0.5 μL T5 exonuclease, 40.5 μL 10×Buffer, and ddH2O to a final volume of 5 μL.

[0168] The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing kanamycin (0.03 mg / mL), and incubated overnight at 37°C. Single clones were picked for colony PCR verification, and positive clones were sent for sequencing confirmation. The correctly sequenced recombinant plasmids were named pTZ28A-CSP504-9, pTZ28A-CSP6959, pTZ28A-CSP7503, pTZ28A-CSP2705, pTZ28A-CSP7688-48, pTZ28A-CSP3788, pTZ28A-CSP4850, pTZ28A-CSP44, and pTZ28A-CSPCelA, respectively.

[0169] 3.2 Construction and Induction Expression of Recombinant Strains

[0170] The recombinant plasmids constructed above were transformed into Escherichia coli BL21(DE3) and ZM4-T7 competent cells, respectively.

[0171] (1) Recombinant strain of *Escherichia coli* BL21(DE3): The plasmid was transformed into BL21(DE3) competent cells and plated on LB agar plates containing kanamycin (0.03 mg / mL), and cultured overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing kanamycin (0.03 mg / mL), and cultured overnight at 37°C with shaking. The cells were then transferred to fresh LB medium (containing 0.03 mg / mL kanamycin) at a 1:100 inoculation rate and cultured at 37°C until OD500. 600 The concentration is approximately 0.7-0.9. Add IPTG to a final concentration of 0.2-0.5 mM and induce expression at 18 ℃ for 16-20 h.

[0172] (2) Recombinant strain of *M. motile fermentomonas* ZM4-T7: The plasmid was electroporated into ZM4-T7 competent cells and plated on RMG5 plates containing kanamycin (0.03 mg / mL). The cells were incubated at 30°C for 2-3 days. Single colonies were picked and inoculated into RMG5 liquid medium containing kanamycin (0.03 mg / mL). The cells were then incubated statically at 30°C overnight. The initial OD was determined... 600 0.1 mg / mL was transferred to fresh RMG5 medium (containing 0.03 mg / mL kanamycin) and incubated at 30°C until OD500. 600 The concentration was approximately 0.6-0.8. Add arabinose to a final concentration of 3 mg / mL and induce expression at 30°C for 24 h.

[0173] 3.3 Protein purification

[0174] (1) Collect the induced bacterial culture, centrifuge at 8000 rpm for 5-10 min at 4 ℃, and discard the supernatant; resuspend the bacterial cells in 10 mL of 50 mM Tris-HCl buffer (pH 7.5, 200 mM NaCl), and homogenize the cells using a high-pressure homogenizer. Place the lysis buffer in a refrigerated centrifuge, centrifuge at 8000 rpm for 10 min, and collect the supernatant.

[0175] (2) Pretreatment of the Ni-NTA column: Ethanol was flushed out from the Ni-NTA gravity column, and 5 volumes of filtered water were slowly added for rinsing. The Ni column was then equilibrated with 5 volumes of Tris-HCl buffer. The collected supernatant was filtered through a 0.45 μm filter to remove cell debris and other insoluble impurities. The supernatant was then slowly added to the Ni column and incubated at 4°C for 1-2 h to allow the histidine tag protein to fully bind to the Ni column.

[0176] (3) Elute contaminating proteins with 20 mM, 40 mM, and 80 mM imidazole, and elute the target protein with 300 mM imidazole. During the process, use Coomassie Brilliant Blue G250 to detect color changes in the eluent to monitor protein elution. Take 20 μL of each gradient eluted protein solution and add 5 μL of 5×SDS-PAGE loading buffer, then heat in a 100 ℃ metal bath for 10 min. After brief centrifugation, use SDS-PAGE electrophoresis to detect the purity of the target protein under each imidazole gradient, loading 5-10 μL. Results are as follows: Figure 2-17 As shown.

[0177] (4) Wash and equilibrate the ultrafiltration cup (10 kDa molecular weight cutoff) with filtered water and buffer, and add the eluted target protein to the ultrafiltration cup. Centrifuge at 4 °C and 3800 rpm to concentrate the protein to the required volume. Add the concentrated protein to a desalting column (PD-10 Columns) that has been washed and equilibrated with filtered water and buffer to remove residual imidazole. After desalting, further concentrate to the required concentration, add 10%-20% glycerol, aliquot, and flash freeze in liquid nitrogen. Store at -80 °C.

[0178] Figure 2-18 SDS-PAGE analysis of the purified endoglucanase expressed in *Escherichia coli* BL21(DE3) and *Fermentomonas motilityis* ZM4-T7 was performed. In the above figures, lane M represents the protein molecular weight marker; lane 1 is the lysed supernatant; lane 2 is the lysed precipitate; lane 3 is the flow-through; and lanes 4-7 are the elution fractions of 20 mM, 40 mM, 80 mM, and 300 mM imidazole, respectively. The results showed that the target protein bands, consistent with the expected molecular weight, were visible in the 300 mM imidazole elution fraction, and the purity was high, suitable for subsequent enzyme activity assays.

[0179] 3.4 Protein Concentration Determination

[0180] A standard curve was plotted using the Bradford assay kit. The test protein was diluted to a certain concentration, and 200 μL of G250 staining solution was added to each well of a 96-well plate. 5 μL of sample was used for the reaction. Three parallel experiments were conducted. The absorbance (OD) values ​​of different concentrations of the standard were measured using a microplate reader. 595 ), calculate protein concentration.

[0181] Example 4: Determination of Endoglucanase Activity and Comparison of Enzyme Activity

[0182] 4.1 Method for Assay of Endoglucanase Activity

[0183] Using 10 mg / mL sodium carboxymethyl cellulose (CMC) solution as the substrate, the reaction system contained 20 mM sodium citrate buffer (pH 4.5). Specific steps: Add 100 μL CMC, 75 μL buffer, and 25 μL enzyme solution to a 2 mL EP tube, mix well, and incubate at 50°C for 30 min. Then add 150 μL of 3,5-dinitrosalicylic acid (DNS) reagent, heat at 100°C for 5 min to terminate the reaction, cool to room temperature, and bring the volume to 1 mL with sterile water. Mix well, and then take 200 μL of the sample to measure the absorbance at 540 nm using a microplate reader.

[0184] Enzyme activity (U / mL) is defined as the amount of enzyme required to catalyze the production of 1 μg of glucose per minute.

[0185] Enzyme activity (U / mg) is defined as the number of enzyme activity units contained in one milligram of protein.

[0186] 4.2 Enzyme activity assay results

[0187] The activities of each purified endoglucanase in Example 3 were determined using the method described above. Elution buffer without the target protein was used as a negative control. The enzyme activity (U / mL) and protein concentration of each enzyme sample were measured, and the specific enzyme activity (U / mg) was calculated. The results are shown in Table 6 and... Figure 18-19 As shown.

[0188] Table 6 Results of endoglucanase activity assay from *Ichthyophthirius multifiliis*

[0189]

[0190] Figure 19 This study compared the enzyme activities (U / mL) of eight recombinant endoglucanases in *Zygomorpha motilityis* ZM4-T7 and *Escherichia coli* BL21. The results showed that, under the same conditions, the endogenous endoglucanase CelA had an activity of 28.15 U / mL in ZM4-T7. In both ZM4-T7 and BL21, CSP6959 exhibited the highest enzyme activity (ZM4-T7: 43.19 U / mL; BL21: 48.79 U / mL); in ZM4-T7, CSP4850 had the lowest activity (20.59 U / mL); and in BL21, CSP44 had the lowest activity (19.48 U / mL).

[0191] Figure 20This study compared the specific enzyme activities (U / mg) of eight recombinant endoglucanases from *Fermentomonas motilityis* ZM4-T7 and *Escherichia coli* BL21. The results showed that, under the same conditions, the endogenous endoglucanase CelA had an activity of 10.82 U / mg in ZM4-T7. In ZM4-T7, CSP2705 exhibited the highest specific enzyme activity at 22.10 U / mg; in BL21, CSP504-9 showed the highest specific enzyme activity at 15.70 U / mg. Notably, except for CSP44, the specific enzyme activities of the other seven enzymes expressed in ZM4-T7 were higher than those in BL21, indicating that these endoglucanases derived from the intestinal flora of *Leymus chinensis* have better adaptability and higher catalytic efficiency in the *Fermentomonas motilityis* chassis cells.

[0192] Based on the plasmid construction method of the above embodiments, those skilled in the art can easily obtain recombinant vectors containing nucleotide sequences (nucleotide sequences shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16) encoding the endoglucanase of this application (amino acid sequences shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15), such as the pEZ15A-INP-CSP series of recombinant plasmids constructed in Example 2, and the pTZ28A-CSP series of recombinant plasmids constructed in Example 3.

[0193] Transforming the above-mentioned recombinant vector into suitable host cells yields corresponding recombinant host cells. These host cells include, but are not limited to, *M. motile fermentation monoclonal bacteria* (such as ZMNP and ZM4-T7) and *Escherichia coli* (such as BL21(DE3) and DH5α). For example, the ZMNP / pEZ15A-INP-CSP series of recombinant strains obtained in Example 2, and the BL21(DE3) / pTZ28A-CSP and ZM4-T7 / pTZ28A-CSP series of recombinant strains obtained in Example 3, are all recombinant host cells of this application.

[0194] Based on the aforementioned protein expression and purification methods, high-purity endoglucanases of this application can be prepared. The specific steps are as follows: (1) Culturing the recombinant host cells described in Example 5 (such as BL21(DE3) / pTZ28A-CSP series or ZM4-T7 / pTZ28A-CSP series recombinant strains), and inducing them to express endoglucanase under suitable conditions; (2) Collecting the bacterial cells, and collecting the supernatant after disruption and centrifugation; (3) Purifying the target protein by Ni-NTA affinity chromatography to obtain high-purity target protein. Using this method, the endoglucanases shown in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, and 15 of this application can be prepared stably and efficiently.

[0195] By mixing one or more of the endoglucanases prepared by the methods of the above embodiments with a pharmaceutically or industrially acceptable carrier, excipient, or excipient, and formulating according to conventional methods in the art, a cellulase preparation can be obtained. The preparation can be in liquid or solid form (such as lyophilized powder) and can be used for subsequent cellulose degradation applications.

[0196] The endoglucanase activity assay method described in the foregoing embodiments has confirmed that all eight endoglucans of this application possess the activity to degrade sodium carboxymethyl cellulose (CMC), and can catalyze the hydrolysis of cellulose substrates to generate reducing sugars (calculated as glucose). Therefore, the endoglucans of this application can be used to degrade cellulose substrates to produce glucose and other oligosaccharides. In specific applications, the endoglucanase of this application (or the recombinant host cell expressing the enzyme) can be reacted with cellulose-containing substrates (such as CMC, microcrystalline cellulose, filter paper, natural lignocellulose biomass, etc.) under suitable conditions (such as temperature 30-60 ℃, pH 4.0-6.0) to achieve cellulose degradation and glucose production.

[0197] *Fermentomonas motilityis* is a naturally occurring and highly efficient ethanol-producing strain. The endoglucanase obtained in this application exhibits excellent expression activity and specific enzyme activity in *Fermentomonas motilityis*. The nucleotide sequence encoding the endoglucanase of this application is constructed into a suitable expression vector, which is then transformed into *Fermentomonas motilityis*. The resulting recombinant strain possesses both the ability to degrade cellulose and produce ethanol. Taking the ZM4-T7 / pTZ28A-CSP series of recombinant strains constructed in the aforementioned examples as an example, this strain, when cultured in a medium containing cellulose substrate, can express and secrete endoglucanase, degrading cellulose into fermentable sugars, and subsequently fermenting to produce ethanol. Through optimization of fermentation conditions, efficient production of cellulosic ethanol can be achieved. Therefore, the endoglucanase and its recombinant host cell of this application can be used in the production process of fuel ethanol.

[0198] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. An endoglucanase selected from at least one of the following (a) to (h): (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:1; (b) A protein consisting of the amino acid sequence shown in SEQ ID NO:3; (c) A protein consisting of the amino acid sequence shown in SEQ ID NO:5; (d) A protein consisting of the amino acid sequence shown in SEQ ID NO:7; (e) A protein consisting of the amino acid sequence shown in SEQ ID NO:9; (f) A protein consisting of the amino acid sequence shown in SEQ ID NO:11; (g) A protein consisting of the amino acid sequence shown in SEQ ID NO:13; (h) A protein consisting of the amino acid sequence shown in SEQ ID NO:

15.

2. A nucleic acid molecule encoding the endoglucanase of claim 1.

3. The nucleic acid molecule according to claim 2, wherein it is selected from at least one of the following (1)-(8): (1) The nucleotide sequence is shown in SEQ ID NO:2, which encodes the amino acid sequence shown in SEQ ID NO:1; (2) The nucleotide sequence is shown in SEQ ID NO:4, which encodes the amino acid sequence shown in SEQ ID NO:3; (3) The nucleotide sequence is shown in SEQ ID NO:6, which encodes the amino acid sequence shown in SEQ ID NO:5; (4) The nucleotide sequence is shown in SEQ ID NO:8, which encodes the amino acid sequence shown in SEQ ID NO:7; (5) The nucleotide sequence is shown in SEQ ID NO:10, which encodes the amino acid sequence shown in SEQ ID NO:9; (6) The nucleotide sequence is shown in SEQ ID NO:12, which encodes the amino acid sequence shown in SEQ ID NO:11; (7) The nucleotide sequence is shown in SEQ ID NO:14, which encodes the amino acid sequence shown in SEQ ID NO:13; (8) The nucleotide sequence is shown in SEQ ID NO:16, which encodes the amino acid sequence shown in SEQ ID NO:

15.

4. A recombinant vector containing the nucleic acid molecule as described in claim 2 or 3.

5. A recombinant host cell containing the nucleic acid molecule of claim 2 or 3 or the recombinant vector of claim 4.

6. The recombinant host cell according to claim 5, wherein the host cell is *Mammotrophic Fermentatosporium* or *Escherichia coli*.

7. Fifth aspect, a method for preparing the endoglucanase of claim 1, comprising the following steps: (1) Culturing the recombinant host cells described in the fourth aspect to express the endoglucanase; and (2) The endoglucanase was isolated and purified from the culture.

8. A cellulase preparation comprising the endoglucanase of claim 1.

9. The use of the dextranase of claim 1 or the recombinant host cell of claim 5 in the degradation of cellulose or the production of glucose.

10. The use of the dextranase of claim 1 or the recombinant host cell of claim 5 in the production of fuel ethanol.

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