Endoglucanase as well as coding gene and application thereof

Abstract

The invention provides endoglucanase as well as a coding gene and application thereof. The endoglucanase disclosed by the invention has high endoglucanase activity, excellent thermal stability, pH tolerance and / or various enzyme activities, and has potential application values in a plurality of fields such as food, feed, textile, medical treatment and biological energy sources.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering. More specifically, the present invention relates to endoglucanase, its encoding gene and applications. Background Art

[0002] More than 400 million tons of pulp are converted into paper and paperboard every year, which is one of the largest biorefining processes. In the papermaking process, the refining of pulp is a key process step to improve fiber bonding, tensile strength, uniformity, air resistance and many other quality parameters. Cellulose is composed of D-glucose linked by β-1,4-glycosidic bonds, forming a highly ordered crystal structure, which appears in two different forms. During the pulping process of wood chips, part of the cellulose is separated from lignin and hemicellulose. Refining causes damage to the internal and external structures of cellulose fibers because hydrogen bonds are broken, increasing flexibility and stripping the fibers. The main disadvantage of mechanical refining technology is high energy consumption, usually between 150 kWh and 500 kWh / ton of paper, accounting for 30% to 50% of the total energy consumption in papermaking. Enzymes are becoming an ideal tool for cellulose fiber refining, which can reduce energy consumption by up to 40%, but the source and stability of enzymes have become a major factor restricting the development of this tool.

[0003] Cellulase is a general term for enzymes involved in cellulose degradation in nature, including endoglucanase, cellobiohydrolase and β-glucosidase. These three cellulases work together to convert cellulose into fermentable sugars. Members of the glycoside hydrolase family cleave the glycosidic bond between carbohydrates into non-carbohydrates. Endocellulase mainly acts on β-1,4-glucoside bonds to convert long cellulose chains into short chains, making an important contribution to cellulose hydrolysis. More importantly, this exposes more ends in the cellulose chain, providing more sites for the hydrolysis process of exocellulase. β-Glucosidase hydrolyzes cellobiose and oligo-cellulose into glucose for further fermentation or conversion into other chemicals.

[0004] Hemicellulose is a general term for plant-derived heteropolysaccharides composed of different sugar monomers coupled by different bondings and substitutions. Due to the heterogeneity of hemicellulose polymers, their degradation processes require the cooperative and / or sequential action of multiple enzymes. Studies have found that the digestion of hemicellulose requires a large number of polysaccharidases and disaccharidases, including xylanase, β-xylosidase, α-glucuronidase, α-arabinofuranosidase and acetylxylan esterase; xylanase and β-xylosidase cleave the main chain of xylan to release xylose. Hemicellulase is a major component of pretreated biomass, so hemicellulase plays an indispensable role in the overall function of biomass hydrolases.

[0005] Most of the existing endoglucanases are derived from culturable microorganisms, but they are accompanied by problems such as poor enzyme stability and low sustainability. With the further development of microbial sequencing technology and the development of metagenomic sequencing technology for environmental samples through genetic engineering technology, the research on herbivorous animal feces has gradually deepened, and it has become possible to mine genes with high hemicellulase activity. The application of genes with high hemicellulase activity provides a reference direction for mining endoglucanase genes and provides better candidate enzymes for biological fermentation. Summary of the invention

[0006] In order to solve the problems of narrow access to endoglucanase, poor stability and low sustainability in the prior art, the inventors screened and obtained two genes with high endoglucanase activity from the monogastric microorganisms of Equus przewalskii through metagenomics technology, which opened up a new source for the acquisition of endoglucanase, enriched the genetic resources for obtaining endoglucanase, helped to break through the limitations of traditional acquisition methods, and provided more options for industrial production and scientific research.

[0007] Therefore, the present invention provides a kind of endoglucanase on the one hand, it is selected from:

[0008] (1) a protein with an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0009] (2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acids in the amino acid sequence of the protein of (1); and

[0010] (3) A protein having an amino acid sequence that is at least 80% (e.g., 85%, 90%, 95%, 98% and 99%) homologous to SEQ ID NO: 1 or SEQ ID NO: 2 and having the same function.

[0011] In a specific embodiment, the endoglucanase is selected from the protein whose amino acid sequence is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0012] In a specific embodiment, the endoglucanase further has one or more of the following activities: filter paper enzyme activity, hemicellulase activity, and β-glucosidase activity, preferably filter paper enzyme activity and hemicellulase activity. Among them, the protein with the amino acid sequence shown in SEQ ID NO:1 (the protein encoded by the M1F-1 gene) has an endoglucanase activity of 0.001 U / mg to 0.135 U / mg, and the protein with the amino acid sequence shown in SEQ ID NO:1 (the protein encoded by the M1F-3 gene) has an endoglucanase activity of 0.0193 to 0.1343 U / mg, and at the same time has a filter paper enzyme activity of 0.001 to 0.0673 U / mg and a hemicellulase activity of 0.06 to 0.368 U / mg.

[0013] Another aspect of the present invention also provides a gene encoding the endoglucanase according to the present invention.

[0014] In a specific embodiment, the gene is selected from:

[0015] (1) DNA with the nucleotide sequence shown in SEQ ID NO:3 (M1F-1 gene) or SEQ ID NO:4 (M1F-3 gene);

[0016] (2) A DNA molecule that hybridizes with the DNA described in (1) under stringent conditions; and

[0017] (3) A DNA molecule having at least 80% or more (such as 85%, 90%, 95%, 98%, and 99%) homology with the DNA described in (1).

[0018] Another aspect of the present invention also provides a recombinant expression vector, expression cassette, transgenic cell line, or recombinant bacterium containing the gene according to the present invention.

[0019] In a specific embodiment, the cell line is a eukaryotic or prokaryotic cell line, and / or the recombinant bacterium is a bacterium or a fungus.

[0020] In a specific embodiment, the recombinant bacterium is Escherichia coli, Bacillus subtilis, or yeast.

[0021] Another aspect of the present invention also provides the application of the gene according to the present invention or the recombinant expression vector, expression cassette, transgenic cell line, or recombinant bacterium according to the present invention in the production of endoglucanase. In a specific embodiment, the application is a non-disease treatment and non-disease diagnosis application.

[0022] In a specific embodiment, the endoglucanase is used in the fields of food, feed, textile, medical treatment, or bioenergy (such as cellulose ethanol preparation).

[0023] Another aspect of the present invention also provides the use of the endoglucanase according to the present invention, the gene according to the present invention, or the recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium according to the present invention in degrading cellulose or hemicellulose. In a specific embodiment, the use is for non-disease treatment and non-disease diagnosis applications.

[0024] The endoglucanase of the present invention has cleavage activity against lignocellulose, has significant advantages in the degradation of cellulose and hemicellulose, has excellent enzyme activity, thermal stability, pH tolerance and / or multiple enzyme activities, and has potential application value in multiple fields such as food, feed, textile, medical treatment, and bioenergy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] From the following detailed description in conjunction with the accompanying drawings, the above features and advantages of the present invention will become more apparent, wherein:

[0026] Figure 1 is a schematic diagram of the structure of the recombinant plasmid of the present invention;

[0027] Figure 2 is the SDS-PAGE electrophoresis diagram of the recombinant protein expression in the present invention;

[0028] Figure 3 shows the thermal stability of M1F-1 endoglucanase;

[0029] Figure 4 shows the optimal reaction temperature of M1F-1 endoglucanase;

[0030] Figure 5 shows the optimal pH of M1F-1 endoglucanase;

[0031] Figure 6 shows the effect of different metal ions on the activity of M1F-1 endoglucanase;

[0032] Figure 7 shows the thermal stability of M1F-3 endoglucanase;

[0033] Figure 8 shows the optimal reaction temperature of M1F-3 endoglucanase;

[0034] Figure 9 shows the optimal pH of M1F-3 endoglucanase;

[0035] Figure 10 shows the effect of different metal ions on the activity of M1F-3 endoglucanase;

[0036] Figure 11 shows the thermal stability of M1F-3 filter paper enzyme;

[0037] Figure 12Show the optimal reaction temperature of M1F-3 filter paper enzyme;

[0038] Figure 13 Show the optimal pH of M1F-3 filter paper enzyme;

[0039] Figure 14 Show the effects of different metal ions on the activity of M1F-3 filter paper enzyme;

[0040] Figure 15 Show the thermal stability of M1F-3 hemicellulase;

[0041] Figure 16 Show the optimal reaction temperature of M1F-3 hemicellulase;

[0042] Figure 17 Show the optimal pH of M1F-3 hemicellulase;

[0043] Figure 18 Show the effects of different metal ions on the activity of M1F-3 hemicellulase. Detailed implementation manners

[0044] The following will describe the technical solutions in the embodiments of the present invention clearly and completely in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0045] The experimental methods used in the examples are all conventional methods unless otherwise specified.

[0046] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be obtained through commercial channels.

[0047] Unless otherwise indicated, the terms used herein have the general technical meanings understood by those skilled in the art. For the definitions and terms in this field, it is particularly recommended that those skilled in the art refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0048] As used herein, the term "comprising" or "including" is an open-ended description that includes all specified components or steps described, as well as other specified components or steps that do not materially affect; when used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention.

[0049] As used herein, the term "and / or" includes all combinations of the items connected by this term, and each combination should be considered as having been separately listed herein. For example, "A and / or B" includes "A", "A and B", and "B". Another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0050] As used herein, the term "substitution of amino acids" means that one amino acid is replaced by another amino acid with similar or dissimilar properties, including conservative substitutions (i.e., having a minor impact on the structure or function of the protein) or non-conservative substitutions (i.e., having a major impact on the structure or function of the protein). In the present invention, it is preferred that the amino acid substitution is a conservative substitution.

[0051] As used herein, the term "protein with the same function" refers to a protein having the same endoglucanase activity, β-glucosidase activity, filter paper enzyme activity, or hemicellulase activity.

[0052] As used herein, the term "hybridize with... under stringent conditions" means hybridizing and washing the membrane at 65 °C in a DNA or RNA hybridization experiment using a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

[0053] An object of the present invention is to provide two genes with high endoglucanase activity.

[0054] Further defined, the starting vector of the recombinant vector containing the gene is a plasmid vector applicable to Escherichia coli, Bacillus subtilis, and yeast fungi.

[0055] The present invention also provides a recombinant microbial cell, which is a recombinant microbial cell carrying the above-mentioned gene or the above-mentioned recombinant vector.

[0056] The present invention also provides the application of the above-mentioned gene, the above-mentioned recombinant vector, and the above-mentioned recombinant microbial cell in the preparation of endoglucanase.

[0057] The present invention provides two methods for producing endoglucanase, and the method is to obtain endoglucanase by fermenting the above-mentioned recombinant microbial cell.

[0058] The application of the two genes with high endoglucanase activity in bacterial or fungal expression is achieved in the following manner:

[0059] 1) Express a gene with high endoglucanase activity to obtain the corresponding recombinant engineering bacteria;

[0060] 2) Use the DNS method to measure the filter paper enzyme activity, endoglucanase activity, and hemicellulase activity with filter paper, CMC-Na, and corncob xylan as substrates respectively; use p-nitrophenyl-β-D-galactopyranoside (PNPG) as the substrate, and terminate the reaction with 1 mol / L Na2CO3 to measure the β-glucosidase activity;

[0061] 3) Use the supernatant of the recombinant bacterial solution as the crude enzyme solution to measure the substrate specificity of the recombinant enzyme.

[0062] Furthermore, in the step 2), the protein encoded by the M1F-1 gene has an endoglucanase activity of 0.001 U / mg - 0.135 U / mg, and the protein encoded by the M1F-3 gene has an endoglucanase activity of 0.0193 - 0.1343 U / mg, and at the same time has a filter paper enzyme activity of 0.001 - 0.0673 U / mg and a hemicellulase activity of 0.06 - 0.368 U / mg.

[0063] Furthermore, in the step 3), the order of substrate specificity is CMC-Na > xylan > filter paper.

[0064] The present invention provides the application of two genes with high endoglucanase activity. The obtained genes can be expressed in Escherichia coli and have multiple enzyme functions at the same time. The recombinant microbial cells containing the M1F-1 gene have an endoglucanase activity of 0.001 U / mg - 0.135 U / mg. The corresponding optimal temperature of the endoglucanase is 43 °C, and in the temperature range of 37 °C - 80 °C, the enzyme activity is stably maintained above 80%; the optimal pH is 7.0, and in the pH range of 5.0 - 9.0, the enzyme activity is maintained above 60%, showing excellent temperature and pH stability. The M1F-3 gene has multiple enzyme functions, and the protein encoded by it has an endoglucanase activity of 0.0193 - 0.1343 U / mg, and at the same time has a filter paper enzyme activity of 0.001 - 0.0673 U / mg and a hemicellulase activity of 0.06 - 0.368 U / mg. The excellent performance of these genes and their expression products in terms of enzyme activity, functional diversity, and stability provides high-quality gene resources and enzyme preparation options for bioengineering fields such as cellulose degradation and biomass conversion, helps to improve the production efficiency of related industries, and promotes the application and development of bioengineering technologies in fields such as food, feed, and energy.

[0065] All patents and publications mentioned in this application are incorporated herein by reference in their entirety. Those skilled in the art should recognize that certain changes can be made to the present invention without departing from the spirit or scope of the present invention. The following examples further illustrate the present invention and should not be construed as limiting the present invention or the scope of the specific methods described herein.

[0066] Example 1 Obtaining the Endoglucanase Gene

[0067] 1. Experimental Materials

[0068] Luria - Bertani medium (LB medium): 10 g of tryptone, 10 g of NaCl, 5 g of yeast extract, add distilled water to 1000 mL.

[0069] LB solid medium: Add 1.8 - 2 g of agar to 100 mL of LB liquid medium, autoclave at 121 °C for 20 min.

[0070] Whatman No. 1 filter paper, purchased from whatman company, product number: 1001 - 047.

[0071] Sodium carboxymethyl cellulose (CMC - Na), purchased from Solarbio company, product number: C8621.

[0072] Corncob xylan, purchased from Solarbio company, product number: X8163.

[0073] pNPG (p - nitrophenyl - β - D - glucopyranoside), purchased from Solarbio company, product number: N8700.

[0074] DNS (Ghose method) reagent, purchased from Shanghai Yuanye Bio - technology Co., Ltd., product number: R30591.

[0075] 0.1 moL / L pH 4.8 citrate - sodium citrate buffer: Mix 460 mL of 0.1 moL / L citric acid solution with 540 mL of 0.1 moL / L sodium citrate solution evenly, filter sterilize, and store in a 4 °C refrigerator.

[0076] 0.1 mol / L pH 5.5 acetic acid - sodium acetate buffer: Dissolve 46.76 g of sodium acetate and 5.7 mL of acetic acid to a volume of 1 L, filter sterilize, and store in a 4 °C refrigerator.

[0077] pH 5.5 citrate - disodium hydrogen phosphate buffer: Mix 425 mL of 0.1 moL / L citric acid solution with 575 mL of 0.2 moL / L disodium hydrogen phosphate solution evenly, filter sterilize, and store in a 4 °C refrigerator.

[0078] 1 mol / L Na2CO3 solution: 106 g of Na2CO3 was made up to 1 L.

[0079] 1% (W / V) carboxymethyl cellulose suspension: 1 g of carboxymethyl cellulose was dissolved in 100 mL of acetic acid - sodium acetate buffer solution with pH 5.5, mixed well, and stored in a refrigerator at 4°C for later use.

[0080] 1% (W / V) corncob xylan solution: 1 g of corncob xylan was dissolved in 100 mL of acetic acid - sodium acetate buffer solution with pH 5.5, mixed well, and stored in a refrigerator at 4°C for later use.

[0081] 5 mmol / L pNPG solution: 0.1506 g of pNPG was dissolved in 100 mL of citric acid - disodium hydrogen phosphate buffer solution with pH 5.5, mixed well, and stored in a refrigerator at 4°C for later use.

[0082] 2. Extraction of DNA and gene synthesis

[0083] Using the monogastric microorganisms of Przewalski's horses as experimental samples, the genomic DNA was extracted using a soil DNA extraction kit (brand: Yeasen, product number: 18815ES08), and the operation was carried out according to the operation manual provided by the merchant. Then it was sent to a sequencing company for metagenomic sequencing and annotation. The functional genes of endoglucanase were screened through data analysis and experimental verification, and the functional genes with endoglucanase were obtained and named M1F-1 (SEQ ID NO: 3) and M1F-3 (SEQ ID NO: 4), and the encoded amino acid sequences are shown as SEQ ID NO: 1 and SEQ ID NO: 2 respectively.

[0084] Amino acid sequence encoded by M1F-1 gene - SEQ ID NO: 1

[0085] MTNIQLNQIGYRPSDTKIATFRIGDTKDVSDSINIEGKISDGFRIINVANGEIVYDGKVSETISSTINGEINAIADFSELKAEGVYVIDSDANGRSCEFAIKEKIYDDVFMASMKMFYLQRCGCEIPKIYAGKYAHPACHNTKARIYGTDRYIDVTGGWHDAGDYGRYIVAAAKAVTDLMMAYEFYPEVMERAFNDVEKENDVPEILKEIKYELDWMIKMQDPETGGVYHKVTCASFPGFVMPEEETEELIVCPISDTATCDFAASMAYAYRVYSKLEEKYGEKAAFYSDAADEYLMAAKKAMNFLEYHTAGEFLNPEGIVTGEYGDDRTIDEFFWAYAEMFKTTGEKKYENALKELDLNDVPGEFGWKEVGDYGFYAYITAEVEKDEELYKAVYKRFTDENEDKMKNYKADPYRSTIYGHYYWGCLMEVADNAKRGIIIDRLKGTDDYREMASAQLHYILGNNPNAKCFVTGFGTNQVENPHHRPSAAVKKAMPGMLVGGMEPLLLDDVAKQMLNGLPAVKCYLDHLESYSTNEITIYWNSPLVFLLAYMRS

[0086] Amino acid sequence encoded by the M1F-3 gene - SEQ ID NO:2

[0087] MSELKKKDAIDVEISGNVQINQVGYRPNDRKTATFRIGDTMSVSDFCNSNGKISDEFKVVKADTGEVVYTGKMSDITSSDFNGEVNAVADFTSVKEPGKYRVVSDDNGSSYEFTIEDDVYNDLFVSSMRMFYLQRCGVELPEDLAGKYAHKACHNSLARVYGTDKMIDVSGAWHDAGDYGRYIVAAAKAVVDLLMAYEYFPETMETSFNIPESGNGIPDIIDEVKFELDWMLKMQDEESGGVYHKVTCKTFPGFVMPEYETDELVVSPISDTATCDFAATMAYAYRVFSKLPDKYGNDAKLYADAADKYLAAANKAMGFLENNRAGEFKNPSDIVTGEYGDSRTIDEFFWAYAEMYKTTGDKKYEDALKKINIDDVPGEFGWKEVGDYGFYAYITSPWDKDDALYNKCYERLEEEAIEKLEKYNTDPYGGSIYGYYYWGCNMEVANNAIRGIILDKLNGTNKYEEMAKAQLSYILGNNPSAKCFVTGFGSNSPKNPHHRPSVAKKEAMPGMLVGGPEPLLLDDVAKKQLKGYPPAKCYLDDTDSYSTNEITIYWNSPLVFLIAYCMQ

[0088] Nucleotide sequence of the M1F-1 gene - SEQ ID NO:3

[0089]

[0090] Nucleotide sequence of the M1F-3 gene - SEQ ID NO:4

[0091]

[0092] Amplification of functional genes and construction of recombinant expression vectors. Using the synthetic gene as a template, primers (5’→3’) are as follows:

[0093] F: CTGATTGGTCAGCAAGTGAACAGC (SEQ ID NO:5)

[0094] R: GACCGAGATAGGGTTGAGTGTTGTTC (SEQ ID NO:6)

[0095] Perform PCR amplification on its functional genes. The high-fidelity enzyme GoldMix (Beijing Tsingke Biotechnology Co., Ltd.) is used for amplification. The amplification conditions are: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 90 s. After 32 cycles, finally extend at 72°C for 10 min. The PCR products are identified by 1.0% agarose gel electrophoresis at 100 V for 90 min, observed under ultraviolet light, and the correct fragments are purified using the Sangon gel extraction and purification kit.

[0096] Use restriction endonucleases (NcoI, XhoI) to perform double digestion on the above-mentioned PCR gel-extracted products and vector pET28a(+) according to the instructions, recover the digested products, ligate the digested PCR products and the vector through T4 ligase, introduce the ligation products into Escherichia coli strain BL21 competent cells by heat shock method, screen positive clones by colony PCR, and finally further verify by sequencing to obtain the recombinant vector (see plasmid map in Figure 1 ).

[0097] Example 2 Study on the properties of recombinant protein

[0098] 1. Preparation of crude enzyme solution

[0099] Cultivate the recombinant strain overnight, and then expand the culture at an inoculation amount of 1% for 3 to 4 h until OD 600 is 0.6 - 0.8. Add 0.2 mmol / L IPTG (isopropyl-β-D-thiogalactoside) to induce for 6 h, and centrifuge the bacterial solution at 4500 rpm. Add 1 mg / mL lysozyme solution to the precipitate, freeze-thaw 3 times repeatedly, ultrasonically disrupt for 30 min, and centrifuge at 4500 rpm. The obtained supernatant is the crude enzyme solution.

[0100] 2. Study on the properties of recombinant protein

[0101] (1) Study on the properties of multifunctional enzyme activity

[0102] Measure the enzyme activity of the recombinant protein on four model substrates: filter paper, CMC-Na, corncob xylan, and pNPG.

[0103] The detection method is as follows:

[0104] Adopt the internationally common filter paper enzyme activity assay method, mainly referring to the standard method NREL / TP-510-42628 formulated by the National Renewable Energy Laboratory of the United States. Put about 25 mg of Whatman No. 1 filter paper (1 cm × 3 cm) into a test tube, add 0.45 mL of citric acid-sodium citrate buffer solution with pH 4.8 and 50 μL of crude enzyme solution, shake well to mix evenly, incubate at 50 °C for 60 min (for the control group, 50 μL of crude enzyme solution is first boiled for 10 min to inactivate, and the inactivated enzyme solution is used as the control group). Then immediately add 1.5 mL of DNS solution to terminate the reaction, heat the test tube in a boiling water bath for 5 min for color development, then quickly cool it, make the volume up to 5 ml, and measure the absorbance value at 540 nm. Calculate the content of glucose in the system according to the glucose standard curve.

[0105] Use the DNS method to determine the activity of endoglucanase. Prepare 1% CMC-Na as the substrate with sodium acetate buffer solution (pH 5.5) as the solvent. Incubate 50 μL of crude enzyme solution with 450 μL of substrate at 50 °C for 30 min (for the control group, 50 μL of crude enzyme solution is first boiled for 10 min to inactivate), add 1.5 mL of DNS solution to terminate the enzymatic reaction, boil the mixture at 100 °C for 5 min, add distilled water to make the volume up to 5 mL, and measure the OD 540 value, and measure the amount of reducing sugar formed during the incubation process. Calculate the content of glucose in the system according to the glucose standard curve.

[0106] Use 5 mmol / L pNPG as the substrate (pH 5.5). Incubate 50 μL of crude enzyme solution with 450 μL of substrate in a water bath at 45 °C for 20 min (for the control group, 50 μL of crude enzyme solution is first boiled for 10 min to inactivate), add 1 mL of 1 mol / L Na2CO3 to terminate the reaction, add distilled water to make the volume up to 5 mL, pipette 200 μL of the supernatant into an enzyme-labeled plate, and measure the absorbance at 410 nm. Define the amount of enzyme required to produce 1 μmol of p-nitrophenol per minute as one unit of endoglucanase activity.

[0107] The hemicellulose-degrading enzyme is mainly represented by xylanase. The reaction substrate is 1% corncob xylan, prepared with 0.1 mol / L acetic acid-sodium acetate buffer solution with pH 5.5. Incubate 50 μL of crude enzyme solution with 450 μL of substrate at 45 °C for 30 min (for the control group, 50 μL of crude enzyme solution is first boiled for 10 min to inactivate), then add 1.5 mL of DNS color-developing solution, mix well and heat in boiling water for 5 min for color development, quickly cool and then add distilled water to make the volume up to 5 mL, and measure the absorbance at 540 nm. Calculate the content of xylose in the system according to the xylose standard curve.

[0108] According to the above method, the endoglucanase activity of the M1F-1 gene detected was 0.001 U / mg - 0.135 U / mg, the endoglucanase activity of the M1F-3 gene was 0.0193 - 0.1343 U / mg, and it also had a filter paper enzyme activity of 0.001 - 0.0673 U / mg and a hemicellulase activity of 0.06 - 0.368 U / mg.

[0109] (2) Determination of the optimal temperature and temperature stability of the recombinant protein M1F-1 endoglucanase

[0110] The His-tag gravity column protein purification steps for the crude enzyme are as follows: First, perform column pretreatment. Rinse the column with deionized water to remove the preservation solution ethanol. Equilibrate the column with the equilibration buffer (containing 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) for 15 - 20 min, and slowly add the protein supernatant after cell disruption (dissolved in the binding buffer, 2 - 4 mM imidazole) to the column and bind for 2 h. Elute the unbound miscellaneous proteins with the washing buffer (containing 20 - 50 mM imidazole), elute the target protein with the elution buffer (containing 50 - 500 mM imidazole), collect the eluate (collect in multiple tubes to avoid dilution), and verify the protein purity by SDS-PAGE.

[0111] Under the condition of pH 5.5, the enzyme activity of the purified endoglucanase (see the SDS-PAGE electrophoresis map of the purified protein in Figure 2 ) was measured at different temperatures (37 - 80 °C). The experimental results ( Figure 3 ) showed that its optimal temperature range was 40 - 45 °C, and the enzyme activity presented an obvious peak in this interval. Further narrowing the interval study found that the enzyme activity was at the highest level at 43 °C, which was 0.121 U / mg ( Figure 4 ).

[0112] The enzyme and commercial cellulase (Novozymes cellulase 1.5 L) were simultaneously assayed for enzyme activity in the same temperature range, and the specific enzyme activities of the two enzymes were measured respectively. The results are as Figure 3 shown. In the same temperature range, the specific enzyme activity of the commercial cellulase was much lower than that of this enzyme.

[0113] (3) Determination of the optimal pH and pH stability of the recombinant protein M1F-1 endoglucanase

[0114] To determine the optimal pH of the purified and expressed endoglucanase, enzymatic reactions were carried out in buffer systems with different pH values. The pH suitability results measured at 43 °C ( Figure 5) It was shown that the optimal pH of this endoglucanase was 7.0, at which the specific activity was in a relatively high range, being 0.133 U / mg. The enzyme activity of this endoglucanase remained above 60% within the range of pH 5.0 - 9.0.

[0115] The enzyme and commercial cellulase (Novozymes cellulase 1.5 L) were simultaneously assayed for enzyme activity within the same pH range, and the specific enzyme activities of the two enzymes were measured separately. The results are as Figure 5 shown. In the range of pH 6.0 - 9.0, the specific enzyme activity of the commercial cellulase was far lower than that of this enzyme.

[0116] (4) Determination of the effects of different metal ions on the activity of recombinant protein M1F - 1 endoglucanase

[0117] To determine the effects of metal ions and organic reagents on the endoglucanase, different metal ions Zn 2+ , Fe 3+ , Ni 2+ , Mg 2+ , Cu 2+ , Ca 2+ , K 2+ (each 20 mM) were added to the enzyme solution at 43 °C and pH 7.0, and the specific enzyme activity was measured after 30 min of treatment. The measurement results are as Figure 6 shown. This enzyme had certain enzyme activity in the environments of metal ions Zn 2+ , Fe 3+ , Ni 2+ , Mg 2+ , Ca 2+ , K 2+ ; only in Cu 2+ did it show a relatively low activity range.

[0118] (5) Determination of the optimal temperature and temperature stability of recombinant protein M1F - 3 endoglucanase

[0119] Under the condition of pH 5.5, the purified endoglucanase (for the SDS - PAGE electrophoresis pattern of the purified protein, see Figure 2 ) was assayed for enzyme activity at different temperatures (37 - 80 °C). The experimental results ( Figure 7 ) showed that its optimal temperature range was 40 - 45 °C, and there was an obvious peak of enzyme activity within this range. After further narrowing the range, the enzyme activity was at the highest level at 43 °C, being 0.113 U / mg ( Figure 8 ).

[0120] This enzyme and commercial cellulase 1.5L The enzyme activity was measured simultaneously within the same temperature range, and the specific enzyme activities of the two enzymes were measured respectively. The results are as Figure 7 shown. Within the same temperature range, the specific enzyme activity of the commercial cellulase is far less than that of this enzyme.

[0121] (6) Determination of the optimal pH and pH stability of the recombinant protein M1F-3 endoglucanase

[0122] To determine the optimal pH of the purified and expressed endoglucanase, an enzymatic reaction was carried out in buffer systems with different pH values. The pH suitability results measured at 43 °C ( Figure 9 ) showed that the optimal pH of this endoglucanase is 6.0. At this time, the specific activity is in a relatively high range, which is 0.134 U / mg. The enzyme activity of this endoglucanase remains above 60% within the range of pH 5.0 - 9.0.

[0123] This enzyme and the commercial cellulase 1.5L The enzyme activity was measured simultaneously within the same pH range, and the specific enzyme activities of the two enzymes were measured respectively. The results are as Figure 9 shown. Within the pH range of 6.0 - 10.0, the specific enzyme activity of the commercial cellulase is far less than that of this enzyme.

[0124] (7) Determination of the effect of different metal ions on the activity of the recombinant protein M1F-3 endoglucanase

[0125] To determine the effects of metal ions and organic reagents on the endoglucanase, different metal ions (Zn 2+ , Fe 3+ , Ni 2+ , Mg 2+ , Cu 2+ , Ca 2+ , K 2+ (20 mM)) were added to the enzyme solution at 43 °C and pH 6.0, and the specific enzyme activity was measured after 30 min of treatment. The measurement results are as Figure 10 shown. This enzyme shows good enzyme activity performance in the environment of metal ions Zn 2+ , Mg 2+ , Cu 2+ , Ca 2+ , K 2+ , far higher than that of the commercial enzyme 1.5L, and has an obvious inhibitory effect on the commercial enzyme; while in the environment of Fe 3+ , Ni 2+ , the specific enzyme activity of this cellulase is in the range less than that of the commercial enzyme.

[0126] (8) Determination of the optimal temperature and temperature stability of the recombinant protein M1F-3 filter paper enzyme

[0127] Under the condition of pH 4.8, the enzyme activity of the purified filter paper enzyme was measured at different temperatures (37 - 80 °C). The experimental results ( Figure 11 ) showed that its optimal temperature range was 40 - 45 °C, and there was an obvious peak in enzyme activity within this range. After further narrowing the range, the enzyme activity was at the highest level at 42 °C, reaching 0.039 U / mg ( Figure 12 ).

[0128] This enzyme and commercial cellulase 1.5 L were simultaneously assayed for enzyme activity within the same temperature range, and the specific enzyme activities of the two enzymes were measured respectively. The results are as Figure 11 shown. Within the same temperature range, the specific enzyme activity of this enzyme was in the same range as that of commercial cellulase.

[0129] (9) Determination of the optimal pH and pH stability of the recombinant protein M1F - 3 filter paper enzyme

[0130] To determine the optimal pH of the purified and expressed filter paper enzyme, the enzyme - catalyzed reaction was carried out in buffer systems with different pH values. The results of the pH suitability measured at 42 °C ( Figure 13 ) showed that the optimal pH of this filter paper enzyme was 6.0, and at this time the specific activity was in a relatively high range, reaching 0.067 U / mg. The enzyme activity of this filter paper enzyme remained above 60% within the range of pH 5.0 - 10.0.

[0131] This enzyme and commercial cellulase 1.5 L were simultaneously assayed for enzyme activity within the same pH range, and the specific enzyme activities of the two enzymes were measured respectively. The results are as Figure 13 shown. Within the pH range of 5.0 - 7.0, the specific enzyme activity of commercial cellulase was much lower than that of this enzyme.

[0132] (10) Determination of the effect of different metal ions on the activity of the recombinant protein M1F - 3 filter paper enzyme

[0133] To determine the effects of metal ions and organic reagents on the filter paper enzyme, different metal ions Zn 2+ , Fe 3+ , Ni 2+ , Mg 2+ , Cu 2+ , Ca 2+ , K 2+ (20 mM) were added to the enzyme solution at 43 °C and pH 6.0 respectively. After treatment for 30 min, the specific enzyme activity was measured. The measurement results are as Figure 14 shown. The adaptability of this enzyme to Zn 2+ , Fe 3+ was not as good as that of the commercial enzyme, but it also showed a certain enzyme activity.

[0134] (11) Determination of the optimal temperature and temperature stability of recombinant protein M1F-3 hemicellulase

[0135] Under pH 5.5, the enzyme activity of purified hemicellulase was measured at different temperatures (37-80°C). Figure 15 ) showed that its optimum temperature range is 50-60℃, and the enzyme activity has a significant peak in this range. After further narrowing the range, the enzyme activity is at its highest range at 57℃, which is 0.136U / mg ( Figure 16 ).

[0136] The enzyme was mixed with commercial cellulase (Novozymes Cellulase 1.5L (Novozymes liquid on the picture), Novozymes xylanase Pentopan Mono (Novozymes solid on the picture), Yuanye xylanase S23566, Aladdin xylanase X1952724) were tested for enzyme activity at the same temperature range, and the specific enzyme activities of the two enzymes were tested respectively. The results are shown in Figure 15 As shown, within the same temperature range, the specific enzyme activity of commercial cellulase is in a much smaller range than that of this enzyme.

[0137] (12) Determination of the optimal pH and pH stability of recombinant protein M1F-3 hemicellulase

[0138] To determine the optimal pH of the purified expressed hemicellulase, the enzyme reaction was carried out in buffer systems with different pH values. The pH adaptability results measured at 43°C ( Figure 17 ) showed that the optimum pH of the hemicellulase was 7.0, at which time the specific activity was in a relatively high range of 0.177 U / mg. The enzyme activity of the hemicellulase was maintained in the range of more than 80% in the pH range of 3.0-11.0.

[0139] The enzyme activity of the enzyme and commercial cellulase were tested simultaneously in the same pH range, and the specific enzyme activities of the two enzymes were measured respectively. Figure 17 As shown, in the pH range of 3.0-11.0, the specific enzyme activity of commercial cellulase is much smaller than that of this enzyme.

[0140] (13) Determination of the effects of different metal ions on hemicellulase activity of recombinant protein M1F-3

[0141] To determine the effects of metal ions and organic reagents on endoglucanase, different metal ions Zn, 2+ , Fe 3+ , Ni 2+ ,Mg 2+ , Cu 2+, Ca 2+ , K 2+ (20 mM), after treatment for 30 min, the specific enzyme activity was measured. The measurement results are as Figure 18 shown. This enzyme showed a relatively high enzyme activity range in the metal ion Ni 2 + environment; it showed a certain enzyme activity in other metal ion environments.

[0142] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept scope of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0143] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0144] Furthermore, any combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. Endoglucanase, which is selected from: (1) a protein with an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2; (2) a protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acids in the amino acid sequence of the protein in (1); and (3) a protein with an amino acid sequence having at least 80% homology with SEQ ID NO:1 or SEQ ID NO:2 and having the same function.

2. The endoglucanase according to claim 1, which is selected from a protein with an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:

2.

3. The endoglucanase according to claim 1 or 2, which further has one or more of the following activities: filter paper enzyme activity, hemicellulase activity, and β-glucosidase activity, wherein the protein with an amino acid sequence as shown in SEQ ID NO:1 (the protein encoded by the M1F-1 gene) has an endoglucanase activity of 0.001 U / mg to 0.135 U / mg, and the protein with an amino acid sequence as shown in SEQ ID NO:1 (the protein encoded by the M1F-3 gene) has an endoglucanase activity of 0.0193 to 0.1343 U / mg, and at the same time has a filter paper enzyme activity of 0.001 to 0.0673 U / mg and a hemicellulase activity of 0.06 to 0.368 U / mg.

4. A gene encoding the endoglucanase according to any one of claims 1 to 3.

5. The gene according to claim 4, which is selected from: (1) DNA with a nucleotide sequence as shown in SEQ ID NO:3 or SEQ ID NO:4; (2) a DNA molecule that hybridizes with the DNA in (1) under stringent conditions; and (3) a DNA molecule having at least 80% homology with the DNA in (1).

6. A recombinant expression vector, expression cassette, transgenic cell line, or recombinant bacterium containing the gene according to claim 4 or 5.

7. The recombinant expression vector, expression cassette, transgenic cell line, or recombinant bacterium according to claim 6, wherein the cell line is a eukaryotic cell line or a prokaryotic cell line, and / or the recombinant bacterium is a bacterium or a fungus.

8. Use of the gene according to claim 4 or 5 or the recombinant expression vector, expression cassette, transgenic cell line, or recombinant bacterium according to claim 6 or 7 in the production of endoglucanase.

9. The use according to claim 8, wherein the endoglucanase is used in the fields of food, feed, textile, medicine, or bioenergy.

10. Use of the endoglucanase according to any one of claims 1 to 3, the gene according to claim 4 or 5, or the recombinant expression vector, expression cassette, transgenic cell line, or recombinant bacterium according to claim 6 or 7 in the degradation of cellulose or hemicellulose.

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