Endoglucanase mutant eg06, its coding gene and product and application thereof

Abstract

The application discloses an endoglucanase mutant EG06, a coding gene thereof, a product of the endoglucanase mutant EG06 and application thereof, and belongs to the field of genetic engineering and enzyme engineering. The technical problem to be solved is that the endoglucanase activity is low, and the heat resistance and acid resistance are poor in the prior art. The endoglucanase mutant EG06 has an amino acid sequence as shown in SEQ ID NO. 2. The sequence of EG03 as shown in SEQ ID NO. 1 is subjected to S153M, G189W, N193P and S209W mutations to obtain the endoglucanase mutant EG06. The endoglucanase mutant EG06 has outstanding performance at high temperature and in acidic pH, has the optimal pH 6.0, the optimal temperature 40 DEG C, and the high-efficiency catalytic characteristics of maintaining 30% enzyme activity at 20 DEG C, and has significant application potential in the industrial field.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering and enzyme engineering, in particular, the present application relates to an endoglucanase mutant EG06, its coding gene and product and application. BACKGROUND

[0002] Technical content for understanding the present application:

[0003] In the field of feed nutrition and food processing, endoglucanase is a key enzyme preparation that can efficiently decompose cellulose and beta-glucan in plant raw materials. It can degrade large molecular polysaccharides into short-chain oligosaccharides by specific hydrolysis of beta-1, 4-glycosidic bonds, thereby destroying the structure of plant cell walls and releasing the wrapped nutrients. In feed applications, this enzyme can significantly improve feed digestion and utilization, reduce chyme viscosity, reduce animal intestinal problems, and improve energy and protein absorption efficiency; in food processing, it can be used to improve the quality of grain products and improve fruit juice clarity. Compared with traditional phytase acting on phytic acid, endoglucanase mainly acts on cellulose substances, and the two are often used together in feed additives to improve the nutritional value. With the progress of enzyme engineering technology, endoglucanase products with high temperature resistance, acid resistance and other properties are providing more efficient solutions for green breeding and sustainable food industry.

[0004] Retrieved relevant patent documents:

[0005] The document discloses an endoglucanase mutant and its application. The endoglucanase mutant includes: in the amino acid sequence of the wild-type endoglucanase, at least one of the following sites: D194, T232 or G247, the amino acid sequence is mutated, and the protein has endoglucanase activity. Compared with the wild type, the specific activity of the endoglucanase mutant provided in the document is higher, which helps the wide application of beta-endoglucanase in industry.

[0006] Retrieved relevant non-patent documents:

[0007] Journal name: Journal of Food and Biotechnology, document name: Site-directed mutagenesis technology to improve endoglucanase gene F-10 enzyme activity, publication date: 2014.08.15, which discloses an endoglucanase mutant and its application. The document uses high-enzyme-activity F-10 mutant endoglucanase gene for site-directed mutagenesis, mutates 91 (K91E) and 369 (K369R) amino acids, constructs mutant plasmids (K91E), (K369R) and (K91E / K369R), transforms E. coli BL21, and obtains mutant endoglucanase gene engineering strains K91E, K369R and K91E / K369R through screening, and obtains endoglucanase strains with improved enzyme activity.

[0008] The prior art represented by the foregoing documents at least has the following unsolved technical problems or defects:

[0009] The pH stability of the endoglucanase mutant of patent document CN120424912A is not significantly improved compared with wild-type endoglucanase. The relative enzyme activity of the endoglucanase mutant of non-patent document “Site-directed mutagenesis technology to improve endoglucanase gene F-10 enzyme activity” is less than 20% at pH 10. SUMMARY

[0010] The purpose of the present application is to provide:

[0011] An endoglucanase mutant EG06, its encoding gene and its product and application, to solve the problems of low endoglucanase activity, poor heat resistance and poor acid resistance in the prior art.

[0012] Term explanation:

[0013] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the claims belongs. Unless otherwise indicated, all patents, patent applications, publications, and other documents cited herein are hereby incorporated by reference in their entirety. If there is a plurality of definitions for a term herein, the definition in this section prevails.

[0014] It should be understood that the above brief summary and the following detailed description are exemplary and explanatory only and are not restrictive of the subject matter of the present application. In the present application, the use of singular includes plural unless specifically stated otherwise. It should also be noted that the use of “or” or “and” means “and / or” unless otherwise indicated. In addition, the use of the term “include” and other forms, such as “includes,” “including,” and “contain,” are not limiting.

[0015] Definitions of standard chemical terms are found in the reference “Molecular Cloning: A Laboratory Manual” (3rd Ed.), J. Sambrook.

[0016] Unless otherwise indicated, conventional methods of the art are employed, such as methods of culturing strains, passaging strains, preserving strains, assaying enzyme activity, and the like.

[0017] Unless specifically defined, the use of each term herein is understood to be in accordance with its standard usage in the art. For example, the use of a vendor's instructions for a kit, or the use of a method according to a known procedure or the description of the application, is contemplated. The foregoing techniques and methods can generally be performed according to conventional methods well known in the art, as described in various general and more specific references that are cited and discussed throughout the present specification.

[0018] The term "optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, that the description includes instances where the event or circumstance occurs, and instances where it does not. For example, the definition "the host cell is any one or more of a eukaryotic cell, a prokaryotic cell" according to the following means that the host cell can be a eukaryotic cell, or the host cell can be a prokaryotic cell, or the host cell can be a eukaryotic cell and a prokaryotic cell.

[0019] The term "mutation" as used herein refers to a heritable change in the genetic material (nucleotide sequence) of an organism, including base substitutions, insertions, deletions, inversions, or translocations, as a result of DNA replication, environmental factors (e.g., radiation, chemical mutagens), or human manipulation (e.g., site-directed mutagenesis, error-prone PCR). If the mutation occurs in a coding region, it can result in a change in the amino acid sequence encoded by the region, which in turn can affect the structure and function of the protein.

[0020] The term "enzyme activity" as used herein refers to the ability of an enzyme to catalyze a specific chemical reaction, and is a key indicator of the functional efficiency of an enzyme. It is typically quantified as the amount of substrate consumed or product produced per unit time (e.g., U / mL, U / mg protein). The term "enzyme activity" as used herein with respect to endoglucanase refers to the rate at which the enzyme catalyzes the hydrolysis of cellulose to produce reducing sugars (e.g., glucose) under specific reaction conditions (e.g., temperature, pH).

[0021] The terms "gene", "polynucleotide", "nucleic acid sequence", "nucleotide sequence", or "nucleic acid molecule" as used herein include both double- and single-stranded DNA and / or RNA. They also include known types of modifications, for example, methylation, "capping", substitution of one or more of the naturally occurring nucleotides with an analog.

[0022] The term "nucleotide" as used herein refers to a small molecule consisting of a five-carbon sugar, a phosphate group, and a nitrogenous base, which is the basic unit of nucleic acids (DNA and RNA) and plays a key role in genetic information storage, energy metabolism, and cell signal transmission; in nucleotides: the five-carbon sugar provides the backbone structure for RNA or DNA, DNA contains deoxyribose, and RNA contains ribose; the phosphate group is used to connect sugar molecules (five-carbon sugars) to form phosphodiester bonds of nucleic acid chains; the nitrogenous base: purine (adenine, guanine) and pyrimidine (cytosine, thymine, uracil), which is paired by hydrogen bond (such as A-T, C-G); different nucleotides are sometimes represented by the nitrogenous base contained thereon.

[0023] The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid. The vector can be a plasmid, cosmid, virus, or bacteriophage. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells.

[0024] The term "recombinant strain" as used herein refers to an engineered strain that can stably carry and express an exogenous gene after the recombinant vector carrying the exogenous gene of interest is introduced into a host strain (such as E. coli, Pichia) by transformation, transfection, conjugation, etc.; the strain can synthesize the protein encoded by the gene of interest (such as recombinant endoglucanase) and can be identified and cultured by screening markers (such as resistance screening).

[0025] In a first aspect, the present application provides an endoglucanase mutant EG06.

[0026] Among them, the technical feature includes: endoglucanase mutant EG06.

[0027] Among them, the technical feature endoglucanase mutant EG06 has an amino acid sequence as shown in SEQ ID NO. 2.

[0028] SEQ ID NO. 2:

[0029] EFQQTVWGQCGGIGWSGPTNCAPGSACSTLNPYYAQCIPGATTITTSTRPPSGPTTTTRATSTSSSTPPTSSGVRFAGVNIAGFDFGCTTDGTCVTSKVYPPLKNFTGSNNYPDGIGQMQHFVNEDGMTIFRLPVGWQYLVNNNLGGNLDSTSISKYDQLVQGCLSLGAYCIVDIHNYARWNGGIIGQGGPTNAQFTSLWSQLASKYASQSRVWFGIMNEPHDVNINTWAATVQEVVTAIRNAGATSQFISLPGNDWQSAGAFISDGSAAALSQVTNPDGSTTNLIFDVHKYLDSDNSGTHAECTTNNIDGAFSPLATWLRQNNRQAILTETGGGNVQSCIQDMCQQIQYLNQNSDVYLGYVGWGAGSFDSTYVLTETPTSSGNSWTDTSLVSSCLARK.

[0030] The technical feature of the endoglucanase mutant EG06 is obtained by mutating the amino acid sequence shown in SEQ ID NO. 1.

[0031] SEQ ID NO. 1:

[0032] EFQQTVWGQCGGIGWSGPTNCAPGSACSTLNPYYAQCIPGATTITTSTRPPSGPTTTTRATSTSSSTPPTSSGVRFAGVNIAGFDFGCTTDGTCVTSKVYPPLKNFTGSNNYPDGIGQMQHFVNEDGMTIFRLPVGWQYLVNNNLGGNLDSTSISKYDQLVQGCLSLGAYCIVDIHNYARWNGGIIGQGGPTNAQFTSLWSQLASKYASQSRVWFGIMNEPHDVNINTWAATVQEVVTAIRNAGATSQFISLPGNDWQSAGAFISDGSAAALSQVTNPDGSTTNLIFDVHKYLDSDNSGTHAECTTNNIDGAFSPLATWLRQNNRQAILTETGGGNVQSCIQDMCQQIQYLNQNSDVYLGYVGWGAGSFDSTYVLTETPTSSGNSWTDTSLVSSCLARK.

[0033] Preferably, the mutation comprises mutating the serine at position 153 to methionine, the glycine at position 189 to tryptophan, the asparagine at position 193 to proline and the serine at position 209 to tryptophan of SEQ ID NO. 1.

[0034] Based on further solving or simultaneously solving multiple technical problems of the technical problem of the present application, in the technical solution provided by the first aspect of the present application, the preferred solution comprises:

[0035] The mutation comprises mutating the serine at position 153 to methionine, the glycine at position 189 to tryptophan, the asparagine at position 193 to proline and the serine at position 209 to tryptophan of SEQ ID NO. 1. This technical solution, on the basis of solving the technical problem of "low endoglucanase activity, poor heat resistance and poor acid resistance in the prior art", provides a new endoglucanase mutant EG06 which performs outstandingly at high temperature and acidic pH, and simultaneously has high efficient catalytic properties of optimal pH 6.0, optimal temperature 40℃, and 30% enzyme activity at 20℃.

[0036] In a second aspect, the present application provides a gene eg06 .

[0037] Among them, the technical features include: the gene eg06 .

[0038] Among them, the technical features include: the gene eg06 Encoding the above-mentioned endoglucanase mutant EG06.

[0039] Preferably, it has a nucleotide sequence as shown in SEQ ID NO. 3.

[0040] SEQ ID NO. 3:

[0041]

[0042] Based on further solving or simultaneously solving multiple technical problems of the technical problem of the present application, in the technical scheme provided by the second aspect of the present application, the preferred scheme includes:

[0043] Coding gene eg06 The nucleotide sequence is shown as SEQ ID NO. 3. The technical scheme solves the technical problem of "low endoglucanase activity, poor heat resistance and poor acid resistance in the prior art", and provides a coding gene of endoglucanase mutant EG06.

[0044] In a third aspect, the present application provides a recombinant vector.

[0045] Among them, the technical features include: a recombinant vector.

[0046] Among them, the technical features of the recombinant vector include the above-mentioned coding gene eg06 .

[0047] Specifically, the recombinant vector is constructed by: a coding gene eg06 Fused with an expression vector to construct a recombinant vector.

[0048] Preferably, the expression vector is selected from any one or more of pPIC9K, pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pHIL-S1, pPICZα, pYAM75P, PNZ8149-usp45.

[0049] Further preferably, the expression vector is pPIC9K.

[0050] Based on further solving or simultaneously solving multiple technical problems of the technical problem of the present application, in the technical scheme provided by the third aspect of the present application, the preferred scheme includes:

[0051] The recombinant vector is constructed by: a coding gene eg06 Fused with an expression vector to construct a recombinant vector, and the expression vector is pPIC9K. The technical scheme solves the technical problem of "low endoglucanase activity, poor heat resistance and poor acid resistance in the prior art", and provides a recombinant vector for expressing the coding gene eg06 .

[0052] In a fourth aspect, the present application provides a recombinant strain.

[0053] Among them, the technical features include: a recombinant strain.

[0054] The technical feature recombinant strain comprises the recombinant vector.

[0055] Specifically, the recombinant strain is obtained by transforming a host cell with the recombinant vector.

[0056] Preferably, the host cell is any one or more of eukaryotic cells and prokaryotic cells.

[0057] Further preferably, the host cell is Pichia pastoris.

[0058] Still further preferably, the host cell is Pichia pastoris GS115.

[0059] Based on further solving or simultaneously solving multiple technical problems of the technical problem of the present application, in the technical scheme provided in the fourth aspect of the present application, the preferred scheme comprises:

[0060] The recombinant strain is obtained by transforming a host cell with the recombinant vector, and the host cell is Pichia pastoris GS115. eg06 The technical scheme solves the technical problem of low endoglucanase activity, poor heat resistance and poor acid resistance in the prior art, and provides a recombinant strain comprising a recombinant vector expressing a coding gene.

[0061] In the fifth aspect, the present application provides a preparation method of the endoglucanase mutant EG06.

[0062] The technical features comprise the endoglucanase mutant EG06 and the preparation method.

[0063] The endoglucanase mutant EG06 has an amino acid sequence as shown in SEQ ID NO. 2.

[0064] The preparation method comprises the following steps:

[0065] S1, the coding gene is fused with an expression vector to construct a recombinant vector, and the recombinant vector is transformed into a host cell to obtain a recombinant strain. eg06

[0066] S2, the recombinant strain is cultured to induce expression of the endoglucanase mutant EG06.

[0067] S3, the endoglucanase mutant EG06 is recovered and purified.

[0068] ​In particular, the expression vector in step S1 is selected from any one or more of pPIC9K, pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pHIL-S1, pPICZ alpha, pYAM75P, PNZ8149-usp45.

[0069] Preferably, the expression vector in step S1 is pPIC9K.

[0070] In particular, the host cell in step S1 is any one or more of a eukaryotic cell, a prokaryotic cell.

[0071] Preferably, the host cell in step S1 is Pichia pastoris.

[0072] Further preferably, the host cell is Pichia pastoris GS115.

[0073] Based on further solving or simultaneously solving multiple technical problems of the technical problem of the present application, in the technical scheme provided in the fifth aspect of the present application, the preferred scheme includes:

[0074] The preparation method of the endoglucanase mutant EG06 includes fusing the above-mentioned coding gene eg06 with an expression vector to construct a recombinant vector, transforming the host cell with the recombinant vector to obtain a recombinant strain; culturing the recombinant strain to induce the expression of the endoglucanase mutant EG06; and recovering and purifying to obtain the endoglucanase mutant EG06. This technical scheme solves the technical problem of "low activity, poor heat resistance and poor acid resistance of endoglucanase in the prior art" and provides a method for preparing the endoglucanase mutant EG06.

[0075] In the sixth aspect, the present application provides the application of the above-mentioned endoglucanase mutant EG06, coding gene eg06 , recombinant vector or recombinant strain in the textile industry, food industry, pulp and paper industry or feed industry.

[0076] Among them, the technical features include: application in the textile industry, food industry, pulp and paper industry, feed industry.

[0077] Among them, the technical feature of the textile industry: In recent years, there have been more and more environmental problems and environmental constraints in the textile industry, so treating cotton fibers with cellulase (such as endoglucanase) has become an important environmentally friendly way. Washing denim with cellulase not only improves the color brightness of the fabric, but also significantly improves the appearance of the clothes.

[0078] In the food industry, cellulase is mixed with other enzymes (xylanase and pectinase) to extract and purify fruit and vegetable juice, produce fruit juice with fruit particles and olive oil, the addition of enzymes reduces the viscosity of the juice, improves the stability, increases the aroma of the juice, and also reduces the risk of filtering the juice in the filter membrane. In beer and liquor production, the addition of glucanase improves the germination rate of barley, and can also be used for the extraction of artificial pigments such as carotenoids.

[0079] In the pulp and paper industry, mechanical processing of pulp such as refining and polishing of wood raw materials can cause high fine particle content and high hardness of the pulp, and biological processing of pulp with cellulase can not only save 20%-40% of energy in the refining process, but also improve the strength of the pulp. The mixture of cellulase and hemicellulase can change the properties of cellulose, remove ink on the surface of cellulose, and degrade pollutants such as colloids in the wastewater of paper mills, thereby reducing environmental pollution. It is found that endoglucanase is essential for improving the drainage performance of secondary fibers.

[0080] In the feed industry, the pretreatment of grain feed with cellulase and xylanase improves the nutritional value. Cellulase can convert anti-nutritional factors and cellulose in feed into components that are easily absorbed by animals, improving the health and physical fitness of animals, so the application potential of cellulase in animal feed is huge.

[0081] Based on the further solution or simultaneous solution of multiple technical problems of the technical problem of the application, in the technical scheme provided in the sixth aspect of the application, the preferred scheme comprises:

[0082] Endoglucanase mutant EG06, coding gene eg06 The application of the recombinant vector or the recombinant strain in the textile industry, the food industry, the pulp and paper industry or the feed industry, which solves the technical problem of "low activity, poor heat resistance and poor acid resistance of endoglucanase in the prior art", provides the application scene of the endoglucanase mutant EG06, the coding gene eg06

[0083] The beneficial effects of the application are:

[0084] The endoglucanase mutant EG06 performs outstandingly at high temperature and acidic pH, and has high efficient catalytic properties of optimal pH 6.0, optimal temperature 40℃, and 30% enzyme activity at 20℃, which has significant application potential in the industrial field. BRIEF DESCRIPTION OF DRAWINGS

[0085] Figure 1 The optimal pH of the recombinant endoglucanase mutant.​

[0086] Figure 2 pH stability of the recombinant endoglucanase mutant.

[0087] Figure 3 Optimum temperature of the recombinant endoglucanase mutant.

[0088] Figure 4 Thermal stability of the recombinant endoglucanase mutant. DETAILED DESCRIPTION

[0089] The following non-limiting examples can provide a more complete understanding of the application to one of ordinary skill in the art, but are not intended to limit the application in any way. The following merely illustrates the scope of the application and the manner of practicing it. Various changes and modifications can be made to the application as disclosed, and such changes and modifications are intended to fall within the scope of the application as claimed.

[0090] The application is further described in the following specific examples. The various instruments, devices, equipment, reagents, products, etc. used in the examples of the application are obtained through conventional commercial channels, unless otherwise stated.

[0091] Test materials and reagents

[0092] 1. Strains and vectors: endoglucanase gene of the application eg06 synthesized by Beijing Ruibo Xingke Biotechnology Co., Ltd., Pichia pastoris expression vector pPIC9K and strain GS115 purchased from Invitrogen Co.

[0093] 2. Enzymes and other biochemical reagents: endonuclease purchased from TaKaRa Co., ligase purchased from Invitrogen Co. Mannan purchased from Sigma Co., and others are domestic reagents (all can be purchased from ordinary biochemical reagent companies).

[0094] 3. Culture medium

[0095] (1) YPD medium for yeast culture: 1% peptone, 0.5% yeast extract, 1% glucose, 2% agar, pH 7.0.

[0096] (2) LB medium for E. coli culture: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0.

[0097] (3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 1% glycerol (V / V).

[0098] (4) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 0.5% methanol.

[0099] Example 1

[0100] 1. Trichoderma reesei (Trichoderma reesei) Trichoderma reesei synthesis of endoglucanase encoding gene eg06

[0101] The present application carries out site-directed mutagenesis on the endoglucanase gene from Trichoderma reesei (Trichoderma reesei) Trichoderma reesei eg03 The amino acid sequence of the endoglucanase EG03 obtained from Trichoderma reesei is SEQ ID NO. 1.

[0102] The theoretical molecular weight of the mature endoglucanase EG03 is 42.19 kDa. The present application provides a site-directed mutagenesis endoglucanase EG06, which is a stable endoglucanase EG03 mutant with improved stability produced by replacing multiple amino acids in the endoglucanase EG03 with the amino acid sequence of SEQ ID NO. 1. The amino acid substitutions are: the 153rd serine is mutated to methionine (S153M), the 189th glycine is mutated to tryptophan (G189W), the 193rd asparagine is mutated to proline (N193P), and the 209th serine is mutated to tryptophan (S209W). EcoRI and NotI restriction enzyme cutting sites are added to the 5' and 3' ends of the mutated sequence, respectively. The sequence is sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for artificial synthesis of the gene. The amino acid sequence of the artificially synthesized endoglucanase mutant EG06 is shown in SEQ ID NO. 2, and the nucleotide sequence is shown in SEQ ID NO. 3.

[0103] 2. Cloning of endoglucanase gene eg06

[0104] The synthesized gene vector is stored in the form of a puncture fungus. The puncture fungus is picked up with a sterile toothpick in a clean bench and placed in an LB shaking tube containing Amp (working concentration: 100 μg / mL) antibiotic. It is cultured overnight at 37°C, 220 rpm. The next day, the vector containing the gene is extracted according to the steps of the Kangwei Plasmid Extraction Kit PurePlasmid Mini Kit (CW0500) instruction manual.

[0105] According to the sequence of the endoglucanase gene, the following primers are designed and synthesized:

[0106] P1: 5'- GAATTCCAGCAGACAGTCTGGG-3' (SEQ ID NO. 4); ​​

[0107] P2: 5'-GCGGCCGCAAATTTATTTCCTAGCCAG-3' (SEQ ID NO. 5).

[0108] PCR amplification was performed using the extracted vector as template. The PCR reaction parameters were as follows: denaturation at 95°C for 5 min, then 30 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1.5 min, and finally incubation at 72°C for 10 min. An about 1212 bp fragment was obtained, which was recovered and ligated to pMD19 vector, and then sent to Beijing Riboebio Biotechnology Co., Ltd. for sequencing. The predicted protein molecular weight was 42.45 kDa.

[0109] According to the obtained nucleotide sequence, the obtained nucleotide sequence was compared with eg06 the sequence by DNA Man software, and no error was confirmed.

[0110] 3. Preparation of recombinant endoglucanase EG06

[0111] The expression vector pPIC9K was double-digested with EcoR I + Not I , and the gene encoding endoglucanase was also double-digested with eg06 + EcoR I + Not I The gene fragment encoding the mature endoglucanase was digested, and the expression vector pPIC9K was ligated to obtain the recombinant plasmid pPIC9K-EG06 containing the endoglucanase gene eg06, which was transformed into Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115 / EG06.

[0112] The GS115 strain containing the recombinant plasmid and the control strain (i.e. the unmutated strain GS115 / EG03) were inoculated into 300 mL of BMGY culture solution, and cultured at 30°C with 200 rpm shaking for 48 h. The bacterial cells were collected by centrifugation. Then, the bacterial cells were resuspended in 150 mL of BMMY medium, and cultured at 30°C with 200 rpm shaking. After 72 h of induction, the supernatant was collected by centrifugation, and the activity of endoglucanase was determined.

[0113] Example 2

[0114] The site-directed mutagenesis modified endoglucanase EG07 is an endoglucanase EG03 mutant with improved stability, which is generated by replacing multiple amino acids in the endoglucanase EG03 with the amino acid sequence of SEQ ID NO. 1, and the amino acid substitutions are respectively the mutation of serine at position 153 to methionine (S153M) and the mutation of glycine at position 189 to tryptophan (G189W). EcoR I and Not I restriction enzyme cutting sites are added at the 5' end and the 3' end of the sequence after mutation respectively, and the sequence is sent to Beijing Ruibo Xingke Biotechnology Company for artificial synthesis of gene. The amino acid sequence of the artificially synthesized endoglucanase mutant EG07 is shown in SEQ ID NO. 6. The specific preparation method of the recombinant endoglucanase EG07 is as described in the above examples.

[0115] SEQ ID NO. 6:

[0116] EFQQTVWGQCGGIGWSGPTNCAPGSACSTLNPYYAQCIPGATTITTSTRPPSGPTTTTRATSTSSSTPPTSSGVRFAGVNIAGFDFGCTTDGTCVTSKVYPPLKNFTGSNNYPDGIGQMQHFVNEDGMTIFRLPVGWQYLVNNNLGGNLDSTMISKYDQLVQGCLSLGAYCIVDIHNYARWNGGIIGQWGPTNAQFTSLWSQLASKYASQSRVWFGIMNEPHDVNINTWAATVQEVVTAIRNAGATSQFISLPGNDWQSAGAFISDGSAAALSQVTNPDGSTTNLIFDVHKYLDSDNSGTHAECTTNNIDGAFSPLATWLRQNNRQAILTETGGGNVQSCIQDMCQQIQYLNQNSDVYLGYVGWGAGSFDSTYVLTETPTSSGNSWTDTSLVSSCLARK.

[0117] Example 3

[0118] The amino acid sequence of the endoglucanase EG03 obtained in Trichoderma reesei is shown in SEQ ID NO. 1. The specific preparation method of the recombinant endoglucanase EG07 is as described in the above examples.

[0119] Example 4 Activity analysis of recombinant endoglucanase EG06

[0120] The reaction system of 5 mL includes 400 μL of the appropriate diluted enzyme solution, 400 μL of the substrate, reaction for 30 min, addition of 1 mL of DNS solution, boiling water bath for 5 min, recovery to room temperature, addition of 3.2 mL of ddH2O, and determination of OD value at 540 nm. The enzyme amount required for releasing 1 μmol of reducing sugar per minute from a β-glucan solution with a concentration of 4 mg / mL under the given conditions is 1 endoglucanase activity unit, which is expressed as U.

[0121] Example 6 Determination of properties of recombinant endoglucanase EG06

[0122] The recombinant endoglucanases EG06 and EG07 were subjected to enzymatic property determination and comparison with the unmutated endoglucanase EG03.

[0123] 1. Determination of optimum pH and pH stability

[0124] (1) Determination of optimum pH

[0125] The purified unmutated endoglucanase EG03, recombinant endoglucanases EG06 and EG07 were subjected to enzymatic reaction at different pH for 30 min to determine the optimum pH. The substrate β-glucan was prepared by using 0.1 mol / L citric acid-sodium phosphate buffer with different pH, and the endoglucanase activity was determined at 37°C.

[0126] The determination results are shown in Table 1. Figure 1 As shown in Table 1, the optimum pH of EG03, EG06 and EG07 is 6.0, but the recombinant endoglucanase EG06 can maintain more than 45% of the relative enzyme activity in a wider pH range (4.0-8.0). The enzyme activity of EG03 and EG07 is less than 20% at pH 4.0, indicating that the recombinant EG06 has superior pH tolerance, and can have a wider applicable pH range in practical application.

[0127] (2) Determination of pH stability

[0128] The purified unmutated endoglucanase EG03, recombinant endoglucanases EG06 and EG07 were treated in the above buffer with different pH at 37°C for 120 min, and then the enzyme activity was determined in the buffer with pH 6.0 at 37°C to study the pH tolerance of the enzymes.

[0129] The results are shown in Table 2. Figure 2As shown, it is indicated that the recombinant endoglucanase EG06 is very stable between pH 4.0-9.0, and the residual enzyme activity is above 40% after 120 min treatment in this pH range, and the relative enzyme activity is significantly improved compared with EG03 and EG07, which indicates that the recombinant enzyme has good pH stability in a wider pH range.

[0130] 2. Optimum temperature and thermal stability determination

[0131] The optimum temperature determination of the unmutated endoglucanase EG03, the recombinant endoglucanase EG06 and the recombinant endoglucanase EG07 is carried out by enzyme reaction in a citric acid-sodium phosphate buffer (pH 6.0) buffer system and at different temperatures, and then enzyme activity determination is carried out at 37℃.

[0132] The temperature resistance determination of the unmutated endoglucanase EG03, the recombinant endoglucanase EG06 and the recombinant endoglucanase EG07 is carried out by treatment at different temperatures for different time, and then enzyme activity determination is carried out at 37℃.

[0133] The optimum temperature determination result of the recombinant endoglucanase EG06 reaction Figure 3 ) shows that the optimum temperature thereof is 40℃, and it maintains a high enzyme activity at 30-60℃.

[0134] The thermal stability test of the enzyme shows that Figure 4 EG06 has good thermal stability, and can maintain above 62% of enzyme activity after incubation at 85℃ for 3 min. The residual enzyme activity of EG03 is only 35% after treatment at 85℃, and the residual enzyme activity of EG07 is only 41%. Compared with the two, the thermal stability of EG06 is significantly improved, and it has good heat resistance.

[0135] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. An endoglucanase mutant EG06, characterized in that, The amino acid sequence of the endoglucanase mutant EG06 is shown in SEQ ID NO.

2.

2. The endoglucanase mutant EG06 according to claim 1, characterized in that, The endoglucanase mutant EG06 was obtained by mutating the amino acid sequence shown in SEQ ID NO.

1.

3. The endoglucanase mutant EG06 according to claim 2, characterized in that, The mutations include changing serine at position 153 of SEQ ID NO.1 to methionine, glycine at position 189 to tryptophan, asparagine at position 193 to proline, and serine at position 209 to tryptophan.

4. A gene encoding eg06 Its characteristics are, The aforementioned coding gene eg06 Encodes the endoglucanase mutant EG06 according to any one of claims 1-3.

5. The encoding gene according to claim 4 eg06 Its characteristics are, The aforementioned coding gene eg06 The nucleotide sequence is shown in SEQ ID NO.

3.

6. A recombinant vector, characterized in that, The recombinant vector expresses the coding gene according to any one of claims 4-5. eg06 .

7. A recombinant bacterial strain, characterized in that, The recombinant strain described herein comprises the recombinant vector of claim 6.

8. The method for preparing the endoglucanase mutant EG06 according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: S1. The encoding gene according to any one of claims 4-5 eg06 The recombinant vector is fused with an expression vector to construct a recombinant vector. The recombinant vector is then transformed into host cells to obtain recombinant bacterial strains. S2. Culture the recombinant strain and induce the expression of the endoglucanase mutant EG06; S3. After recovery and purification, the endoglucanase mutant EG06 was obtained.

9. The preparation method according to claim 8, characterized in that, The expression vector mentioned in step S1 is selected from any one or more of pPIC9K, pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pHIL-S1, pPICZα, pYAM75P, and PNZ8149-usp45; the host cell is any one or more of eukaryotic cells and prokaryotic cells.

10. The endoglucanase mutant EG06 according to any one of claims 1-3, and the encoding gene according to any one of claims 4-5. eg06 The application of the recombinant vector of claim 6 or the recombinant strain of claim 7 in the textile industry, food industry, pulp and paper industry or feed industry.

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