Endoglucanase capable of degrading cellulose as well as preparation method and application of endoglucanase

Abstract

The invention discloses endoglucanase capable of degrading cellulose as well as a preparation method and application of the endoglucanase, and belongs to the technical field of biology. The endoglucanase for degrading cellulose, which has an amino acid sequence as shown in SEQ ID NO.1, and the preparation method and application of the endoglucanase, provided by the invention, have the characteristics of heat resistance, alkali resistance and salt resistance, and specifically, the endoglucanase still has catalytic activity under the conditions that the temperature is 100 DEG C, the pH value is 4.5-10.5 and the salt (NaCl) concentration is 1-4 M; under the conditions that the temperature is 100 DEG C, the pH value is 7.5-10.5 and the NaCl concentration is 4 M (about 234 mg / mL), the catalytic activity of the catalyst is basically kept unchanged, even under the optimal treatment condition, the catalyst can adapt to degradation of cellulose in more extreme samples, especially high-salt samples, and the catalyst has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of enzyme engineering, and particularly relates to an endoglucanase capable of degrading cellulose as well as a preparation method and application thereof. BACKGROUND

[0002] Cellulose is one of the most abundant renewable resources on earth. It is of great significance to solve the energy crisis and environmental problems to effectively convert cellulose into useful energy and chemicals. Cellulase is a kind of enzyme capable of degrading cellulose, including endoglucanase, exoglucanase and beta-glucosidase. Among them, endoglucanase (endoenzyme) can randomly cut the internal beta-1, 4-glycosidic bond of cellulose molecules, and is one of the key enzymes for cellulose hydrolysis.

[0003] In the process of enzymatic hydrolysis, temperature has an important influence on the hydrolysis efficiency. With the increase of temperature, on the one hand, the viscosity of the reaction system is reduced, and the mass transfer effect is enhanced; on the other hand, the increase of Brownian motion increases the contact frequency of enzyme and substrate. Therefore, in the process of enzymatic hydrolysis of cellulose, a higher reaction temperature is preferred, and the thermal stability of the enzyme degrading cellulose is of great significance.

[0004] However, most of the known endoglucanases capable of degrading cellulose have poor stability at high temperature, which limits their application in industrial production. For example, in the fields of biomass energy production, paper industry, textile industry and the like, it is necessary to degrade and process cellulose under high temperature conditions to improve the reaction rate and efficiency.

[0005] Therefore, it is of great application value to develop endoglucanase capable of degrading cellulose with high temperature resistance (heat resistance). SUMMARY

[0006] 1. Invention purposes The purpose of the present application is to provide an endoglucanase capable of degrading cellulose as well as a preparation method and application thereof. The endoglucanase capable of degrading cellulose has the characteristics of heat resistance, alkali resistance and salt resistance, and can adapt to more extreme, especially high-salt sample degradation of cellulose. Moreover, it has high stability.

[0007] 2. Technical solutions In order to achieve the above-mentioned purposes of the application, the technical solutions adopted by the present application are as follows: In a first aspect, the present application provides the application of a protein in degrading cellulose, wherein the protein is selected from: (i) a protein with amino acids as shown in SEQ ID NO. 1; or (ii) a protein with more than 95% homology with the amino acid sequence shown in SEQ ID NO. 1 and having hydrolytic activity of cellulose; As a further illustration of the present application, cellulose refers to linear polysaccharide or polysaccharide derivative, such as sodium carboxymethyl cellulose; degrading cellulose refers to linear polysaccharide or polysaccharide derivative capable of acting on β-1, 4-glycosidic bond; degrading cellulose activity refers to degrading activity of polysaccharide or polysaccharide derivative such as sodium carboxymethyl cellulose as a substrate.

[0008] Further, the amino acid sequence of the above protein having more than 95% homology with the amino acid sequence shown in SEQ ID NO. 1 and having degrading cellulose activity is shown in SEQ ID NO. 3. As a further illustration of the present application, although the protein having the amino acid sequence shown in SEQ ID NO. 3 has been described in the prior art, it is only a hypothetical protein, and its function is unknown, and the applicant first discovered that it has endoglucanase activity of degrading cellulose, and has the characteristics of heat resistance, alkali resistance and salt resistance, and can be applied to efficient degradation of cellulose, and has good application prospect.

[0009] Further, the above application includes adding the above protein to a sample containing linear polysaccharide or polysaccharide derivative, such as cellulose.

[0010] Further, the above application includes adding the above protein to a sample containing linear polysaccharide or polysaccharide derivative, such as cellulose, under the condition that the temperature is at least 50°C, the pH is at least 4.5, and / or the salt concentration is at least 1 M.

[0011] Further, in the above application, the temperature of degradation is at least 50°C.

[0012] Further, in the above application, the temperature of degradation is 50-100°C, 60-100°C, 70-100°C, 80-100°C or 90-100°C.

[0013] Further, in the above application, the temperature of degradation is 90-100°C. As a further illustration of the present application, the research results of the present application show that the above protein has the characteristics of thermophilic, and especially the protein having the amino acid sequence shown in SEQ ID NO: 1, the optimum reaction temperature is 100°C.

[0014] Further, in the above application, the temperature of degradation is 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100°C.

[0015] Further, in the above application, the pH of degradation is at least 4.5.

[0016] Further, in the above application, the pH for degradation is 4.5-10.5, 5.5-10.5, 6.5-10.5, 7.5-10.5, 8.5-10.5, or 9.5-10.5.

[0017] Further, in the above application, the pH for degradation is 7.5-10.5, 8.5-10.5, or 9.5-10.5. As a further illustration of the present application, the research results of the present application show that the above protein has good alkali resistance.

[0018] Further, in the above application, the pH for degradation is 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, or 10.5.

[0019] Further, in the above application, the salt concentration for degradation is at least 1 M, and the salt concentration is calculated based on NaCl.

[0020] Further, in the above application, the salt concentration for degradation is 1-4 M, 2-4 M, or 3-4 M.

[0021] Further, in the above application, the salt concentration for degradation is 3-4 M. As a further illustration of the present application, the research results of the present application show that the above protein has good salt resistance and halophilic characteristics.

[0022] Further, in the above application, the salt concentration for degradation is 1, 2, 3, or 4 M.

[0023] In a second aspect, the present application provides an endocellulase for degrading cellulose, and the amino acid sequence of the endocellulase for degrading cellulose is shown in SEQ ID NO. 1. As a further illustration of the present application, the above endocellulase for degrading cellulose is a protein first discovered and screened from nature by the applicant, and it has been verified that it has endocellulase activity for degrading cellulose. Moreover, the endocellulase for degrading cellulose has the characteristics of heat resistance (the optimum temperature is 100°C), alkali resistance (the pH breakthrough is 8), and salt resistance (the breakthrough is 4 M based on NaCl).

[0024] In a third aspect, the present application further provides a polynucleotide encoding the above endocellulase for degrading cellulose.

[0025] Further, the nucleotide sequence of the above polynucleotide is shown in SEQ ID NO. 2, and it encodes the amino acid sequence shown in SEQ ID NO. 1.

[0026] In a fourth aspect, the present application further provides a recombinant expression vector comprising the above polynucleotide encoding the endocellulase for degrading cellulose.

[0027] Further, the polynucleotide is located in a proper position of the recombinant expression vector, so that the polynucleotide can be correctly and smoothly replicated, transcribed or expressed; in order to meet the requirements of the recombination operation, a restriction enzyme cutting site can be added at both ends of the polynucleotide sequence in the recombinant expression vector, or a start codon, a stop codon and the like can be additionally added; the recombinant expression vector can be a prokaryotic expression vector or a eukaryotic expression vector.

[0028] Further, the recombinant expression vector is selected from any one or more of pET-28a, pET-dute1 or pRSF-dute1.

[0029] Further, the recombinant expression vector is selected from pET-28a.

[0030] In a fifth aspect, the present application further provides a genetically engineered bacterium containing the polynucleotide encoding the cellulose-degrading endoglucanase or the recombinant expression vector.

[0031] Further, the genetically engineered bacterium is selected from any one or more of Escherichia coli MG1655, Escherichia coli BL21 (DE3) or Escherichia coli BL21 (DE3) pLysS.

[0032] Further, the genetically engineered bacterium is selected from Escherichia coli BL21 (DE3).

[0033] In a sixth aspect, the present application further provides the use of the polynucleotide, the recombinant expression vector and / or the genetically engineered bacterium in the preparation of the cellulose-degrading endoglucanase.

[0034] Further, the use comprises: culturing the genetically engineered bacterium; collecting the supernatant after cell disruption to obtain the cellulose-degrading endoglucanase.

[0035] Further, the use further comprises: purifying the supernatant to obtain the purified cellulose-degrading endoglucanase.

[0036] In a seventh aspect, the present application further provides a preparation method of the cellulose-degrading endoglucanase, which comprises: culturing the genetically engineered bacterium; collecting the supernatant after cell disruption to obtain the cellulose-degrading endoglucanase.

[0037] Further, the preparation method further comprises: purifying the supernatant to obtain the purified cellulose-degrading endoglucanase.

[0038] 3. Beneficial effects Compared with the prior art, the present application has the beneficial effects that: (1) The endoglucanase provided by the application has the amino acid sequence shown in SEQ ID NO. 1, has the characteristics of heat resistance, alkali resistance and salt resistance, and specifically has catalytic activity under the conditions of 100 DEG C, pH 4.5-10.5 and salt (NaCl) concentration of 1-4 M; and the catalytic activity is basically unchanged under the conditions of 100 DEG C, pH 7.5-10.5 and NaCl concentration of 4 M (about 234 mg / mL), and even under the optimal treatment conditions, it can adapt to more extreme, especially the degradation of cellulose in high-salt samples. And it has high stability, the activity is basically not reduced under the condition of 100 DEG C for 2 h, and the half-life is more than 1 month at room temperature.

[0039] (2) The application of the protein with the amino acid sequence shown in SEQ ID NO. 1 or the amino acid sequence shown in SEQ ID NO. 3 in hydrolyzing cellulose is first discovered by the applicant to have endoglucanase activity of degrading cellulose. And it has the characteristics of heat resistance, alkali resistance and salt resistance, and can be applied to efficient degradation of cellulose, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 The protein electrophoretogram of the target protein EG1, the sample order from left to right is Marker, crude enzyme liquid, purified enzyme liquid 1 (5 μL is loaded), and purified enzyme liquid 2 (10 μL is loaded).

[0041] Figure 2 The protein electrophoretogram of the target protein EG2, the sample order from left to right is Marker, purified enzyme liquid 1 (5 μL is loaded), purified enzyme liquid 2 (10 μL is loaded), and crude enzyme liquid. The band size of Marker is as follows: the red target band is 70 KD, and the bands below are 55 KD, 40 KD, 35 KD and 25 KD in turn.

[0042] Figure 3 The glucose standard curve diagram.

[0043] Figure 4 The relative enzyme activity of the target protein EG1 under different temperatures.

[0044] Figure 5 The relative enzyme activity of the target protein EG2 under different temperatures.

[0045] Figure 6 The relative enzyme activity of the target protein EG1 under different pH values.

[0046] Figure 7 The relative enzyme activity of the target protein EG2 under different pH values.

[0047] Figure 8 is the relative activity of the target protein EG1 under different concentrations of NaCl.

[0048] Figure 9 is the relative activity of the target protein EG2 under different concentrations of NaCl.

[0049] Figure 10 is the relative activity of the target protein EG1 after different temperature incubation.

[0050] Figure 11 is the relative activity of the target protein EG2 after different temperature incubation. DETAILED DESCRIPTION

[0051] The present application is further described in the following non-limiting examples.

[0052] 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 this application belongs; the term "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0053] Unless otherwise indicated, conventional methods and techniques of molecular biology, microbiology, and recombinant DNA, which are within the skill of the art, can be used for the practice of the present application. Such methods and techniques are explained fully in the literature. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Techniques, 4th Ed., John Wiley & Sons, Inc., Hoboken, N.J. (1999); and the series Methods in Enzymology, Academic Press, San Diego, Calif.

[0054] As used herein, the term "about" is used to provide flexibility to a numerical range endpoint by providing support for a stated numerical value as being "close" to the actual value within a reasonable measurement accuracy. The degree of flexibility of such endpoints will depend on the context within which they are used. As used herein, the term "at least one of" is intended to mean one or more of the listed items can be present and / or can be used, alone or in any combination of one or more of the listed items. For example, the phrase "at least one of A, B, and C" includes A alone, B alone, C alone, as well as combinations such as AB, AC, BC, and ABC. Concentrations, amounts, and other numerical data can be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be construed as having been followed to following purpose. It is to be further understood that such a range format is used merely for convenience and brevity and should be construed as having been followed to following purpose. It is to be further understood that such a range format is used merely for convenience and in the interest of providing a clear and concise specification. Accordingly, the endpoints of the ranges should be construed as being included in the ranges themselves. It is to be further understood that, although the use of ranges are expressly recited in the specification, the endpoints of the ranges should be construed as being included in the ranges themselves. It must be noted that all numerical values are mutationally adaptable to constitute more precise ranges. It is therefore contemplated to be within the scope of the claimed application to use alternative minimum and maximum values which are different from the specified ones. It is intended, therefore, that within the scope of this application, changes can be made in the precise amount of compounds or their derivatives, and other conditions of the application without departing from the scope of the application, which is further defined by the following claims.

[0055] The reagents and preparation methods used in the present application are as follows: 0.1 M sodium citrate (pH 6.5): 21.0 g of citric acid monohydrate was dissolved in 950 mL of deionized water, 1.0 M NaOH was added to adjust the pH to 6.5, and then diluted to 1000 mL.

[0056] 1.0% (w / v) sodium carboxymethyl cellulose (CMC-Na): 1.0 g of CMC-Na was added to 100 mL of 0.1 M sodium citrate buffer (pH 6.5), heated to dissolve, and used after standing at room temperature for 2 days.

[0057] Example 1 The present example provides screening of target proteins with potential endoglucanase activity for degrading cellulose.

[0058] Atlantic Ocean sediment samples were taken, DNA was extracted and subjected to metagenomic sequencing (contracted to Beijing Nuoweziyuan Technology Co., Ltd. to complete); After sequencing, the data obtained by sequencing was annotated, and according to the annotation results, genes with cellulose endoglucanase-related functional annotations were screened as potential coding genes for endoglucanase degrading cellulose; The amino acid sequence encoded by the potential endoglucanase coding gene for degrading cellulose was subjected to similarity comparison with the existing cellulose endoglucanase sequence database, and the protein with high similarity to the known cellulose endoglucanase sequence was selected as the target protein with potential endoglucanase activity for degrading cellulose; The target protein EG1 with potential endoglucanase activity for degrading cellulose was finally obtained, and the amino acid sequence thereof is shown as SEQ ID NO. 1: MKLAVTLSMLATTAMGQTACSQYDSASSPPYSVNQNLWGEYQGTGSQCAYVDKLSSSGASWHTKWTWSGGEGTVKSYSNSGKTFDKKLVSDVSSIPTSVKWSQDDTNVQADVSYDLFTAANADHATSSGDYELAIWLARYGTVQPIGKQIYTATVGGKSWSVWYGTSVQAGASQKTYSFVAGSPITSYSGDIKDFANYLTQNQGTPASSQHLITLQKGTEPFTGGPETFTVDEWTASVN (SEQ ID NO. 1).

[0059] At the same time, through bioinformatics analysis, a hypothetical protein ASPTUDRAFT_49330 [GenBank: OJI89655.1;Aspergillus tubingensis CBS134.48] (target protein EG2), the amino acid sequence of which is shown as SEQ ID NO. 3: MKLAVTLSMLATTAMGQTMCSQYDSASSPPYSVNQNLWGEYQGTGSQCVYVDKLSSSGASWHTKWTWSGGEGTVKSYSNSGLTFDKKLVSDVSSIPTSVKWSQDDTNVQADVSYDLFTAANADHATSSGDYELMIWLARYGTVQPIGKQIATATVGGKSWEVWYGTSVQAGAEQKTYSFVAGSPINSYSGDIKDFFNYLTQNQGFPASSQHLITLQFGTEPFTGGPATFTVDNWTASVN (SEQ ID NO. 3).

[0060] Example 2 This example provides the preparation of a target protein (an endoglucanase potentially degrading cellulose).

[0061] In order to further determine whether the target protein EG1 and the target protein EG2 have the function of degrading cellulose endoglucanase, the target protein is prepared by the method of heterologous expression.

[0062] Taking the preparation of the target protein EG1 as an example, it specifically includes: (1) Construction of an engineering bacterium expressing the target protein EG1 According to the wild type gene sequence coding the target protein EG1 obtained by screening, the codon optimization is performed, the coding gene fragment with the nucleotide sequence shown as SEQ ID NO. 2 is obtained, and the Beijing Qikexin Biotechnology Co., Ltd. is entrusted to synthesize the gene fragment: ATGAAACTGGCGGTGACCCTGAGCATGCTGGCGACCACCGCGATGGGTCAGACCGCGTGCAGTCAGTATGATAGCGCGAGCAGCCCGCCGTATAGCGTGAATCAGAACCTGTGGGGCGAATATCAAGGCACCGGCAGTCAGTGCGCGTATGTGGATAAACTGAGCAGTAGCGGCGCGAGCTGGCATACCAAATGGACCTGGAGCGGCGGCGAAGGCACCGTGAAAAGCTATAGCAACAGCGGCAAAACCTTTGATAAAAAACTGGTGAGCGATGTGAGCAGCATTCCGACGAGCGTGAAATGGAGCCAAGATGATACCAACGTGCAAGCGGATGTGAGCTATGATCTGTTTACCGCGGCGAACGCGGATCATGCGACGAGCAGCGGCGATTATGAACTGGCGATTTGGCTGGCGCGCTATGGCACCGTGCAGCCGATTGGCAAACAGATTTATACCGCGACCGTGGGCGGCAAAAGCTGGAGCGTGTGGTATGGCACGAGCGTGCAAGCGGGCGCGAGTCAGAAAACCTATAGCTTTGTGGCGGGCAGCCCGATTACGAGCTATAGCGGCGATATTAAAGATTTTGCGAACTATCTGACGCAGAACCAAGGCACCCCGGCGAGCAGTCAGCATCTGATTACCCTGCAGAAAGGCACCGAACCGTTTACCGGCGGCCCGGAAACCTTTACCGTGGATGAATGGACCGCGAGCGTGAAC (SEQ ID NO. 2); The synthetic nucleotide sequence as shown in SEQ ID NO. 2 was inserted into the pET-28a plasmid by N co I and X ho The endonuclease site I was inserted into the pET-28a plasmid, and the obtained recombinant plasmid was named pE1. The above recombinant plasmid pE1 was transformed into Escherichia coli BL21 (DE3) to obtain an engineered bacterium expressing the target protein EG1, which was named BL (pE1). The engineered bacterium expressing the target protein EG1 was successfully constructed by extracting the plasmid of BL (pE1) and confirming by sequencing.

[0063] Similarly, the coding gene fragment (nucleotide sequence as shown in SEQ ID NO. 4) encoding the target protein EG2 is inserted into the pET-28a plasmid to obtain a recombinant plasmid, named pE2; the above-mentioned recombinant plasmid pE2 is transformed into E. coli BL21 (DE3) to obtain an engineering bacterium expressing the target protein EG2. The nucleotide sequence of the coding gene fragment is: ATGAAACTGGCGGTTACTCTGTCCATGCTGGCGACCACCGCGATGGGTCAGACCATGTGCAGCCAGTACGACTCTGCGTCTAGTCCACCGTACTCTGTGAACCAGAACCTGTGGGGTGAATACCAGGGCACCGGTTCTCAGTGCGTTTACGTTGATAAACTGAGCTCTTCCGGCGCAAGCTGGCACACCAAATGGACTTGGTCTGGTGGTGAAGGCACCGTTAAGAGCTATAGCAACTCCGGTCTGACCTTCGACAAGAAACTGGTTTCTGATGTGTCCAGCATTCCGACTAGCGTTAAATGGAGCCAAGATGACACCAACGTTCAGGCAGATGTTTCTTACGACCTGTTCACCGCGGCGAACGCTGACCACGCGACCTCTTCCGGTGACTACGAACTGATGATCTGGCTGGCACGTTACGGTACTGTTCAGCCGATCGGTAAACAGATCGCTACTGCCACTGTAGGCGGTAAGAGCTGGGAAGTTTGGTACGGTACTTCTGTGCAGGCAGGCGCAGAACAAAAAACCTACTCTTTCGTTGCTGGTAGTCCAATCAACAGCTACAGCGGCGATATCAAAGACTTCTTCAACTACCTGACTCAGAACCAGGGCTTTCCGGCGTCTAGCCAGCACCTGATCACTCTGCAGTTCGGTACTGAACCGTTTACCGGTGGTCCAGCTACCTTCACCGTTGACAACTGGACCGCGTCCGTAAAC (SEQ ID NO. 4).

[0064] (2) Preparation of target protein Taking the preparation of the target protein EG1 as an example, it specifically includes: The obtained engineering bacteria BL (pE1) expressing target protein EG1 was cultured in LB liquid medium (containing 100 μg / mL kanamycin) at 37℃ and 220 rpm overnight; the culture was transferred into 50 mL fresh LB medium (250 mL flask) at a ratio of 1:100 and cultured at 37℃ and 220 rpm; When the optical density (OD) at 600 nm reached about 0.6, isopropyl thiogalactoside (IPTG) was added to a final concentration of 1 mM, and the engineering bacteria were cultured at 25℃ for 16 hours; 600 12000 rpm, 4℃ centrifugation for 10 min, discard the supernatant, resuspend the cell pellet with pre-cooled 20 mM Tris-HCl buffer (pH 8.5) at 100 g / L, and then ultrasonic disruption; then 12000 rpm, 4℃ centrifugation for 30 min, collect the supernatant, i.e. crude enzyme solution, and store at -20℃.

[0065] The above crude enzyme solution can also be further purified to obtain purified enzyme solution using ion exchange method. Ion exchange method purification includes: purifying the crude enzyme solution with a pre-packed column containing 5 mL of HiTrap Q FF ion exchange resin to adsorb the target protein. The specific purification process is as follows: First, wash with buffer B for 3 column volumes (CV) at a flow rate of 2.5 mL / min; then equilibrate with buffer A for 10 CV at a flow rate of 2.5 mL / min; then load the sample ring at a flow rate of 2.5 mL / min; then wash the impurities with buffer A for 8 CV at a flow rate of 2.5 mL / min; finally, perform linear gradient elution with buffer B, 0~40%B, 10 CV, at a flow rate of 2.5 mL / min; wherein, the preparation of buffer A includes: weighing 2.42 g of Tris (tris-hydroxymethyl aminomethane) and dissolving it in 1 L of deionized water, adjusting the pH to 8.0 with hydrochloric acid to obtain a 20 mM Tris-HCl buffer at pH 8.5; the preparation of buffer B includes: weighing 2.42 g of Tris and dissolving it in 1 L of deionized water, then adding 58.44 g of NaCl (sodium chloride), adjusting the pH to 8.5 with hydrochloric acid to obtain a 20 mM Tris-HCl, 1M NaCl buffer.

[0066] The crude enzyme solution and the purified enzyme solution were subjected to polyacrylamide gel electrophoresis, and the results are shown in Figure 1 , the molecular weight of the target protein is about 26 KD, and the purified target protein is obtained.

[0067] Similarly, the polyacrylamide gel electrophoresis result of the target protein EG2 is shown in Figure 2 , the molecular weight of the target protein is about 26 KD, and the purified target protein is obtained.​

[0068] Example 3 This example provides target protein EG1 and target protein EG2 as the activity determination of endocellulase degrading cellulose.

[0069] The activity (enzyme activity) determination of endocellulase degrading cellulose is obtained by calculating the amount of reducing sugar generated during the reaction process.

[0070] (1) Preparation of glucose standard curve 3,5-dinitrosalicylic acid (DNS) has an oxidation-reduction reaction with reducing sugar under alkaline conditions to generate 3-amino-5-nitrosalicylic acid, which shows a brown-red color under boiling conditions, has maximum absorption at a wavelength of 540 nm, and the color depth is proportional to the reducing sugar content within a certain concentration range. The reducing sugar content is determined by colorimetry.

[0071] Based on the principle of DNS method, the standard curve of reducing sugar was established, the concentration gradient of glucose was set according to Table 1, the final glucose solution was mixed and reacted with DNS for 30 min, then boiled in boiling water for 10 min, and then the temperature was reduced to room temperature with cold water. The absorbance of the reaction solution at 540 nm was determined by ultraviolet spectrophotometer, and the glucose standard curve was drawn, as shown in Figure 3 .

[0072] Table 1

[0073] (2) Activity (enzyme activity) determination of endocellulase degrading cellulose Carboxymethyl cellulose sodium (CMC-Na) was used as the reaction substrate to determine whether it had corresponding activity.

[0074] The endo-β-1,4-glucosidase activity determination method is as follows: The final reaction system is 500 μL, that is, 50 μL of 0.1 M pH 6.0 citric acid buffer, 250 μL of carboxymethyl cellulose sodium (1% w / v), 200 μL of enzyme solution (crude enzyme solution or purified enzyme solution, purified enzyme solution refers to the purified recombinant protein). When the enzyme concentration is high, the enzyme is diluted accordingly and added to the reaction system.

[0075] Zero system setting: replace 200 μL of enzyme solution in the above reaction system with 200 μL of citric acid buffer, and other components remain unchanged.

[0076] Enzyme blank (enzyme blank) setting: no enzyme solution is added in the reaction system for water bath reaction, and enzyme solution is added before measuring the 540 nm absorbance value, which is used as a reference for enzyme and reaction system components.

[0077] Reaction condition: water bath reaction for 30 min under different temperature gradient (4°C, 23-100°C), immediately add 200 μL of DNS, reaction for 10 min at 100°C in metal bath, put into 4°C refrigerator for 5 min, measure absorbance at 540 nm. According to the standard curve shown in FIG. 4, calculate the release amount of reducing sugar (the release of reducing sugar indicates the activity of endoglucanase which degrades cellulose). Figure 3

[0078] Take 50 μL of the recombinant protein purified according to the above method, and measure the enzyme activity according to the above method. At the same time, the effects of temperature, pH value, and salt concentration on enzyme activity and thermal stability are measured.

[0079] (a) Study on the effect of temperature on enzyme activity.

[0080] Referring to the above reaction system, use citric acid buffer with pH 6.5, add the purified enzyme solution into the above system, and then configure the mixed solution for reaction. Place the sample at different temperatures for reaction for 30 min, and measure the enzyme activity. Take the enzyme activity measured at the optimum reaction temperature as 100% relative enzyme activity. Take the inactivated enzyme as a control, and calculate the relative enzyme activity at different temperatures.

[0081] The results are shown in FIGS. 5 and 6. Figure 4 Figure 5 As shown in FIGS. 5 and 6, the optimum reaction temperature of the target protein EG1 under the current conditions is 100°C, which indicates that the target protein EG1 has a high-temperature preference; the optimum reaction temperature of the target protein EG2 is 30-40°, and the activity decreases to 44% as the temperature increases to 80°, and the activity increases again to 70% of the maximum activity at 90°, which indicates that the target protein EG2 has a dual property of low-temperature and high-temperature preference.

[0082] (b) Study on the effect of pH on enzyme activity.

[0083] Referring to the above reaction system, replace the 0.1 M citric acid buffer with pH 6.0 in the reaction system with any one of the buffers with pH 2.5-10.5 (citric acid buffer with pH 2.5, pH 3.5, pH 4.5, pH 5.5, pH 6.0, pH 6.5, Tris-HcL buffer with pH 6.5, pH 7.5, pH 8.5, pH 9.5, pH 10.5), configure 20 mM citrate buffer with pH interval of 1 from pH 2.5 to pH 10.5, and configure 1% CMC-Na with different pH as the reaction substrate.

[0084] ​​The enzyme activity at different pH was determined according to the DNS method. The sample was reacted at 50 °C for 30 min, and the enzyme activity was determined. The enzyme activity at the optimum pH was taken as 100% relative enzyme activity. The relative enzyme activity under different pH conditions was calculated by taking the inactivated enzyme as a control.

[0085] The results are shown in Figure 6 and Figure 7 The optimum pH of the target proteins EG1 and EG2 is between 6.5 and 10.5, with high and stable enzyme activity. When the pH is lower than 4.5, the activity decreases, indicating that the two proteins (target proteins EG1 and EG2) have good alkali resistance.

[0086] (c) Effect of different concentrations of NaCl on enzyme activity

[0087] Referring to the above reaction system, the target proteins were placed in 0 M, 1 M, 2 M, 3 M, and 4 M NaCl solutions, and an equal amount of CMCNa substrate was added to each solution. The reaction was carried out at 50 °C and pH 5.5 in a citric acid buffer for 30 min, and the enzyme activity was determined. The relative activity was calculated by dividing the reducing sugar produced under various NaCl concentrations by the reducing sugar produced without NaCl (0 M NaCl concentration).

[0088] The experimental results are shown in Figure 8 and Figure 9 The vertical axis represents the relative activity (%), and the horizontal axis represents the concentration of NaCl (unit: M). In the target protein EG1 system, the amount of reducing sugar increased (relative activity reached 176%) as the concentration of NaCl increased, indicating that the enzyme has salt tolerance. At a concentration of 3 M NaCl, the amount of reducing sugar decreased slightly, but remained at a high level. At a concentration of 4 M NaCl, the relative activity reached 195%. The activity of target protein EG1 varied from 0 M to 4 M NaCl concentration, and the activity showed a trend of first increasing, then slightly decreasing, and then increasing. At a concentration of 4 M NaCl, the enzyme activity of target protein EG1 was the highest, indicating that it has good salt tolerance and salt-loving characteristics. Target protein EG2 also showed similar salt activation benefits, and the enzyme activity gradually increased with increasing salt concentration, reaching the highest activity at 4 M NaCl. This indicates that the two enzymes (target proteins EG1 and EG2) have good salt tolerance and salt-loving characteristics.

[0089] (d) Stability study of target proteins Enzymes with thermal stability under high temperature conditions can maintain activity in industrial applications and have high application value.

[0090] After the purified recombinant protein (0 h, 0.5 h, 1 h, 1.5 h, 2 h) is bathed at different temperature gradients (50°C, 70°C, 100°C), the bathed recombinant protein is added into the reaction system, and then reacted at 50°C for 30 min. Then 200 μL of DNS is immediately added, and reacted at 100°C for 10 min in a metal bath. The reaction is terminated by placing in a 4°C refrigerator, and the absorbance at 540 nm is measured.

[0091] The enzyme activity measured at 0 h of bathing is taken as 100% relative enzyme activity. The target protein EG1 can still maintain 70% activity after being bathed at 50°C, 70°C and 100°C for 2 h, Figure 10 which indicates that the target protein (enzyme) has good thermal stability, providing a basis for industrial application. The target protein EG2 has good thermal stability at 50°C and 70°C, and the activity decreases to 40% of the initial activity after being bathed for 2 h at 100°C, Figure 11 which indicates that the thermal stability of the enzyme decreases, but still has activity.

[0092] The above only describes the preferred embodiments of the present application, and it should be noted that those of ordinary skill in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. The application of protein in the degradation of cellulose, characterized in that, The protein is selected from: (i) Proteins with amino acids as shown in SEQ ID NO.1; or (ii) A protein that has more than 95% homology with the amino acid sequence shown in SEQ ID NO.1 and has cellulose-degrading activity.

2. The application according to claim 1, characterized in that, The amino acid sequence of the protein that has more than 95% homology with the amino acid sequence shown in SEQ ID NO.1 and has cellulose-degrading activity is shown in SEQ ID NO.

3.

3. The application according to claim 1 or 2, characterized in that, The application includes adding protein to a cellulose-containing sample under conditions of a degradation temperature of at least 50°C, and / or a degradation pH of at least 4.5, and / or a degradation salt concentration of at least 1 M.

4. An endoglucanase for degrading cellulose, characterized in that, The amino acid sequence of the cellulose-degrading endoglucanase is shown in SEQ ID NO.

1.

5. A polynucleotide encoding the cellulose-degrading endoglucanase of claim 4.

6. The polynucleotide according to claim 5, characterized in that, The nucleotide sequence of the polynucleotide is shown in SEQ ID NO.

2.

7. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide as described in claim 5 or 6.

8. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria contain the polynucleotide as described in claim 5 or 6 or the recombinant expression vector as described in claim 7.

9. The use of the polynucleotide of claim 5 or 6, the recombinant expression vector of claim 7, or the genetically engineered bacteria of claim 8 in the preparation of cellulose-degrading endoglucanase.

10. A method for preparing an endoglucanase for degrading cellulose, characterized in that, The method includes culturing the genetically engineered bacteria of claim 8; collecting the supernatant after cell disruption to obtain an endoglucanase that degrades cellulose.

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