Alkali-resistant cellulase StrEgl and application thereof

Abstract

The invention provides alkali-resistant cellulase StrEgl and application thereof, and belongs to the technical field of bioengineering, the amino acid sequence of the cellulase StrEgl is as shown in SEQ ID NO.1, and the nucleotide sequence for coding the cellulase StrEgl gene is as shown in SEQ ID NO.2. The invention further provides a preparation method of the alkali-resistant cellulase StrEgl. The alkali-resistant cellulase StrEgl provided by the invention has alkali resistance and can degrade cellulose under an alkaline condition; in an environment with a pH value of 9-10, the enzymatic hydrolysate still has good enzymatic hydrolysis activity; meanwhile, the cellulase StrEgl has good tolerance to temperature, metal ions and chemical reagents, is very suitable for industrial application, and has wide application prospects in the fields of papermaking, spinning, detergents and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to an alkali-tolerant cellulase StrEgl and its application. Background Art

[0002] Cellulase, as a highly active biocatalyst, has wide application value in the conversion and utilization of cellulose. According to the substrate, action site and released products of cellulase, cellulase can be divided into three main categories: endo-1,4-β-D-glucanase, exo-1,4-β-D-glucannase and β-1,4-glucosidase. Endo-1,4-β-D-glucanase can randomly cut the β-1,4-glycosidic bond of cellulose molecules from the inside, generating shorter sugar chains or cellooligosaccharides, providing new sugar chain ends for the next step of degradation. Exo-1,4-β-D-glucannase acts on the reducing end or non-reducing end of the cellulose polysaccharide chain, while β-1,4-glucosidase can degrade cellobiose into glucose.

[0003] Cellulase has wide applications and plays an important role in industrial fields such as pulp and paper, food brewing, feed production, and washing. Most of the cellulases used in the market currently are derived from Trichoderma reesei or Aspergillus niger, which have low thermal stability and act in an acidic environment, and it is difficult to meet the requirements of alkaline conditions such as papermaking (pH value between 7.5 and 9.5) and washing (pH value should be around 7 to 10). Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an alkali-tolerant cellulase StrEgl and its application; the cellulase StrEgl has alkali tolerance and thermal stability, can efficiently exert the enzyme activity of cellulase in high temperature and alkaline environments, and can be used for the development and application of industrial products.

[0005] The present invention provides an alkali-tolerant cellulase StrEgl, and the amino acid sequence of the cellulase StrEgl is as shown in SEQ ID NO.1.

[0006] Preferably, the pH value range for the cellulase StrEgl to exert its enzymatic hydrolysis function is 4 to 10.

[0007] Preferably, the temperature range for the cellulase StrEgl to exert its enzymatic hydrolysis function is 40 to 100 °C.

[0008] The present invention provides a gene encoding the cellulase StrEgl, and the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

[0009] The present invention provides a recombinant vector expressing the said gene, which comprises the said gene and an initial vector.

[0010] Preferably, the initial vector is pET-28a, and the gene is cloned between the Nco I and Xho I restriction sites of the initial vector pET-28a.

[0011] The present invention provides a recombinant strain expressing the said gene, which is obtained by transferring the said recombinant vector into an empty vector bacterium.

[0012] Preferably, the empty vector bacterium is Escherichia coli.

[0013] The present invention provides the application of the cellulase StrEgl, the said gene, the said recombinant vector, and the said recombinant strain in decomposing cellulose under alkaline conditions.

[0014] The present invention provides the application of the cellulase StrEgl, the said gene, the said recombinant vector, and the said recombinant strain in the fields of papermaking, textile, or detergent.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The alkali-tolerant cellulase StrEgl provided by the present invention has alkali-tolerant properties and can decompose cellulose under alkaline conditions; in an environment with a pH value of 4 to 8, it has a relatively high enzyme activity, all above 80%; in an environment with a pH value of 9 to 10, it still has good enzymatic hydrolysis activity, and its relative enzyme activities are 60% and 38% respectively.

[0017] The cellulase StrEgl has good enzyme activity stability in the pH range of 5 to 9, and the residual enzyme activity after treatment for 1 h basically does not decrease. When the environmental pH value is 10, 62.54% of the enzyme activity remains after treatment.

[0018] At the same time, the cellulase StrEgl has good thermal stability and can decompose cellulose under medium and high temperature conditions; when the temperature is 40 to 60 °C, its relative enzyme activity is higher than 90%, and when the temperature is 80 to 100 °C, it still has a relative enzyme activity of more than 40%.

[0019] The cellulase StrEgl is also tolerant to metal ions and chemical reagents; it is found that metal ions Mn 2+ and Co 2+ have a promoting effect on the cellulase StrEgl, and chemical reagents 1% Tween20 and 1% Tween80 have a promoting effect on the cellulase StrEgl. While EDTA, PMSF, Triton X-100, and Urea have no effect on the activity of the cellulase StrEgl.

[0020] In summary, the alkaline-resistant cellulase StrEgl provided by the present invention has good tolerance to pH, temperature, metal ions and chemical reagents, is very suitable for industrial applications, and has broad application prospects in the fields of papermaking, textiles, detergents, etc. Description of the Drawings

[0021] Figure 1 It is the SDS-PAGE electrophoresis result of cellulase StrEgl, where lane M is the protein Marker, lane 1 is the lysate of BL21-StrEgl bacteria solution without IPTG induction, lane 2 is the total protein of the engineered strain BL21-StrEgl induced by IPTG, lane 3 is the flow-through solution of the crude enzyme solution of cellulase StrEgl passing through the column, and lane 4 is the NTA-60 elution flow-through solution;

[0022] Figure 2 It is the 3D structure simulation diagram of cellulase StrEgl;

[0023] Figure 3 It is the analysis result of the enzymatic properties of cellulase StrEgl, where A is the relative enzyme activity of cellulase StrEgl at different pH values, and B is the relative enzyme activity of cellulase StrEgl at different temperatures;

[0024] Figure 4 It is the analysis result of the pH stability of cellulase StrEgl;

[0025] Figure 5 It is the result of the metal ion tolerance of cellulase StrEgl;

[0026] Figure 6 It is the result of the chemical reagent tolerance of cellulase StrEgl. Detailed Embodiments

[0027] The present invention provides an alkaline-resistant cellulase StrEgl, and the amino acid sequence of the cellulase StrEgl is shown in SEQ ID NO.1, specifically as follows:

[0028] MDTTVCEQYGSTVIQGRYVVQNNRWGTGATQCVTATDTGFRVTRADGSVPTNGAPKSYPSVFNGCHYTNCSPGTSLPARLSTISGAPSSISYGYVSGAVYNASYDIWLDPTPRTDGVNRTEIMIWFNRVGPVQPIGSPVGTATVGGRSWEVWTGSNGSNDVISFVAPSAISSWSFDVMDFVDQAVARGLAQSNWYLTSIQAGFEPWQNGAGLAVHSFSSTVDLGGGNPGDPGDPVTACRVTYATNVWQGGFTADVTVENTGSSAVDNWRLGFTLPSGQLVTNSWNTTLSGSSGAVTASAVAHNAQIAPGGSQTFGFQGTHSGTFAQPTGFSLNGTACTSA。

[0029] Source of the alkaline-resistant cellulase StrEgl described in the present invention: It is obtained by encoding the cellulase gene from Streptomyces isolated and screened from the metagenomic DNA of the Taklimakan Desert soil sample in Yuli County, Xinjiang, after removing the signal peptide sequence.

[0030] In the present invention, the pH value range for the cellulase StrEgl to exert its enzymatic hydrolysis function is 4 - 10, and the temperature range is 40 - 100 °C; the cellulase StrEgl is also tolerant to metal ions and chemical reagents.

[0031] The present invention provides a gene encoding the cellulase StrEgl, and the nucleotide sequence of the gene is as shown in SEQ ID NO.2, specifically as follows:

[0032] ATGGACACCACGGTCTGCGAGCAGTACGGCTCGACCGTCATCCAGGGGCGCTACGTCGTCCAGAACAACCGCTGGGGCACCGGCGCCACCCAGTGCGTCACCGCCACCGACACCGGCTTCCGGGTCACCCGGGCCGACGGCTCGGTACCCACCAACGGCGCCCCGAAGTCGTACCCGTCCGTCTTCAACGGCTGCCACTACACGAACTGTTCGCCGGGCACCAGCCTTCCGGCCCGGCTCAGCACCATCTCCGGTGCGCCCAGCAGCATCTCCTACGGCTATGTCTCGGGCGCCGTGTACAACGCCTCGTACGACATCTGGCTGGACCCGACGCCCCGCACCGACGGCGTCAACCGGACCGAGATCATGATCTGGTTCAACCGGGTGGGCCCGGTCCAGCCGATCGGCTCGCCGGTGGGCACCGCCACCGTCGGTGGCCGCAGCTGGGAGGTGTGGACCGGCAGCAACGGCTCCAACGACGTGATCTCCTTCGTCGCCCCCTCGGCGATCAGCAGCTGGAGCTTCGACGTCATGGACTTCGTCGACCAGGCCGTCGCCCGGGGCCTGGCGCAGAGCAACTGGTATCTGACGAGTATCCAGGCCGGGTTCGAGCCGTGGCAGAACGGCGCCGGGCTCGCGGTGCACTCCTTCTCCTCCACCGTCGATCTCGGCGGCGGCAACCCCGGTGACCCCGGCGACCCGGTGACGGCCTGCCGGGTGACCTACGCGACCAACGTCTGGCAGGGCGGCTTCACCGCCGACGTCACGGTCGAGAACACCGGTTCCAGCGCCGTCGACAACTGGCGGCTCGGCTTCACCCTGCCCTCCGGGCAGCTCGTCACCAACTCCTGGAACACCACCCTGTCCGGCTCCTCGGGCGCGGTCACGGCGAGCGCCGTGGCGCACAACGCGCAGATCGCCCCCGGCGGCAGCCAGACCTTCGGCTTCCAGGGCACCCACAGCGGCACGTTCGCGCAGCCGACCGGGTTCAGCCTGAACGGAACGGCCTGCACCAGCGCGTGA。

[0033] The cellulase gene from Streptomyces is separated and screened from the metagenomic DNA of soil samples from the Taklimakan Desert in Yuli County, Xinjiang, and after removing the signal peptide sequence, it is commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis.

[0034] The present invention also provides a recombinant vector for expressing the gene, comprising the gene and an initial vector. The initial vector is preferably pET-28a, and the gene is preferably cloned between the Nco I and Xho I restriction sites of the initial vector pET-28a. The present invention has no particular limitation on the preparation method of the recombinant vector, and the preparation method of the recombinant vector known in the art can be used.

[0035] The present invention provides a recombinant strain expressing the gene, wherein the recombinant strain is obtained by transferring the recombinant vector into an empty vector bacterium. In the present invention, the empty vector bacterium is preferably Escherichia coli, and more preferably Escherichia coli BL21 (DE3) strain. The present invention does not specifically limit the preparation method of the recombinant strain, and a preparation method known in the art can be used.

[0036] The present invention provides application of the cellulase StrEgl, the gene, the recombinant vector and the recombinant strain in decomposing cellulose under alkaline conditions.

[0037] The present invention also provides application of the cellulase StrEgl, the gene, the recombinant vector and the recombinant strain in the fields of papermaking, textile or detergent.

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] Example 1

[0040] (1) After removing the signal peptide sequence of the cellulase gene from Streptomyces in the metagenomic DNA of the Taklimakan Desert soil sample in Yuli County, Xinjiang, Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the StrEgl gene sequence, and the sequence information is shown in SEQ ID NO.2.

[0041] Primer design: Using the nucleotide sequence of the synthetic cellulase gene StrEgl as a DNA template, primers StrEgl-F / R for amplifying the cellulase gene StrEgl were designed and commissioned to Suzhou Genewise Biotechnology Co., Ltd. for synthesis. The nucleotide sequence of primers StrEgl-F / R is shown in Table 1.

[0042] Table 1 Nucleotide sequence of primer StrEgl-F / R

[0043] Name Sequence (5’~3’) StrEgl-F GAAGGAGATATACCATGGGCATGGACACCACGGTCTG(SEQ ID NO.3) StrEgl-R TGGTGGTGGTGGTGCTCGAGCGCGCTGGTGCAG(SEQ ID NO.4)

[0044] (2) PCR amplification: The PCR amplification system is calculated as 20 μL and includes 10 μL of 2×PhantaMax Master Mix, 1 μL of DNA template (synthesized cellulase gene StrEgl), 1 μL of upstream primer StrEgl-F with an initial concentration of 10 μmol / L, 1 μL of downstream primer StrEgl-R with an initial concentration of 10 μmol / L, and 7 μL of ddH 2 O. The PCR amplification program is denaturation at 95°C for 3 min → (denaturation at 95°C for 30 s → annealing at 60°C for 30 s → extension at 72°C for 1 min) × 35 cycles → extension at 72°C for 5 min → storage at 4°C to obtain the cellulase gene StrEgl, and the sequence is as shown in SEQ ID NO.2.

[0045] The amino acid sequence of the cellulase StrEgl encoded by the cellulase gene StrEgl is as shown in SEQ ID NO.1.

[0046] (3) Vector construction and transformation: The cellulase gene StrEgl was ligated onto the pET-28a vector containing the T7 promoter obtained after double digestion with Nco I and Xho I using C115-01 recombinase to construct the expression vector pET28a-StrEgl. For the specific steps, refer to the pET-28a vector instruction manual; then transform the Escherichia coli BL21(DE3) strain. For the specific steps, refer to the Escherichia coli BL21(DE3) strain instruction manual to obtain the engineered strain BL21-StrEgl.

[0047] (4) Protein induction expression and purification: Inoculate 5 mL of the engineered strain BL21-StrEgl seed solution (OD 600 = 5) into 500 mL of LB culture medium (50 μg / mL Kan) and culture at 37°C until OD 600= 0.8 to obtain the BL21-StrEgl bacterial solution; IPTG was added to it to a final concentration of 0.5 mmol / L, and after protein induction expression at 16 °C for 20 h, the bacteria were collected by centrifugation at 5000 rpm for 10 min, resuspended with NTA-0 buffer and ultrasonically disrupted for 10 min. The ultrasonic power was set at 400 W, and after every 3 s of ultrasonic treatment, there was an intermittent period of 5 s; after the ultrasonic treatment ended, the supernatant was collected by centrifugation at 5000 rpm for 30 min to obtain the crude enzyme solution; the crude enzyme solution was used to wash the nickel column of type 70666-3, and it was washed twice in total. The washing flow rate was 1 mL / min, and then gradient elution was carried out using NTA-10 buffer, NTA-20 buffer, NTA-40 buffer, NTA-60 buffer, NTA-80 buffer, and NTA-100 buffer. The elution flow rate was 1 mL / min. At the same time, the E112-01 protein detection solution was used for detection and the elution peak was collected. After ultrafiltration centrifugation to remove imidazole, the purified StrEgl enzyme solution was obtained; SDS-PAGE electrophoresis was carried out on the StrEgl enzyme solution, and the results were as Figure 1 shown. The NTA buffer was purchased from Sangon Biotech (Shanghai) Co., Ltd., and the product number was C600304-0500.

[0048] (5) Protein structure analysis: The structure of the cellulase StrEgl was analyzed using Swiss-Model (https: / / swissmodel.expasy.org / ), and the results were as Figure 2 shown.

[0049] The PCR amplification results showed that the full length of the cellulase gene StrEgl was 1020 bp (after removing the stop codon), and the encoded cellulase StrEgl had a total of 340 amino acids. The results of protein induction expression and purification showed that the cellulase StrEgl with His-Tag was successfully purified using the Escherichia coli expression system, and the SDS-PAGE electrophoresis results showed that a single protein band appeared at about 35 kDa, which was consistent with the predicted result. The protein purity of the StrEgl enzyme solution was relatively high and the expression level was relatively large.

[0050] Example 2

[0051] The Optimal pH of Cellulase StrEgl

[0052] Prepare 0.1 mol / L citric acid - sodium citrate buffer solutions with pH values of 3, 4, 5, and 6 respectively, 0.2 mol / L disodium hydrogen phosphate - sodium dihydrogen phosphate buffer solutions with pH values of 7 and 8 respectively, and 0.2 mol / L glycine - sodium hydroxide buffer solutions with pH values of 9, 10, 11, and 12 respectively; use 1% sodium carboxymethyl cellulose solution (CMC - Na) as the substrate. Mix 100 μL of 1% CMC - Na solution, 80 μL of buffer solutions with different pH values, and 20 μL of StrEgl enzyme solution with a final concentration of 400 mg / L evenly to obtain a reaction system. React in a water bath at 50 °C for 30 min. Use the DNS method to determine the specific enzyme activity of cellulase StrEgl at different pH values. Specifically: Add 300 μL of DNS reagent to each reaction system to terminate the reaction, then perform a color reaction in a boiling water bath for 5 min. After cooling to room temperature, make up to 1 mL with distilled water. Take 200 μL each and place them in a 96 - well microplate reader, and use a microplate reader to measure the OD 540 value. Calculate the reducing sugar content in each reaction system according to the glucose standard curve formula, and calculate the specific enzyme activity of cellulase StrEgl at different pH values according to the specific enzyme activity formula. Taking the highest enzyme activity as 100%, determine the optimal pH of cellulase StrEgl. The results are shown in Table 2 and Figure 3 as follows.

[0053] The glucose standard curve formula is: y = 1.67x - 0.0349 (R 2 = 0.998); Formula I;

[0054] In Formula I, x is the glucose concentration, with the unit of mg / mL, and y is the OD 540 value

[0055] The specific enzyme activity formula is:

[0056] In Formula II: The specific enzyme activity (U / mg) is the amount of enzyme required for the StrEgl enzyme solution to decompose the substrate and release 1 μg of glucose in 1 min. G is the reducing sugar content (mg / mL), M is the dilution factor of the StrEgl enzyme solution, V is the volume of the StrEgl enzyme solution (mL), T is the reaction time (min), and N is the cellulase concentration (mg / mL).

[0057] Table 2 Specific enzyme activity of cellulase StrEgl at different pH values

[0058] pH Specific enzyme activity (U / mg) pH Specific enzyme activity (U / mg) 3 885.39±43.46 8 3876.67±58.75 4 4438.39±53.57 9 3017.74±50.43 5 4861.04±43.92 10 1850.67±7.71 6 4552.91±61.17 11 495.46±13.90 7 4326.59±34.71 12 451.83±26.99

[0059] The results showed that the optimal pH for the cellulase StrEgl to exert its enzymatic hydrolysis function was 5; taking the highest enzyme activity as 100%, it had relatively high relative enzyme activities at pH values from 4 to 8, all above 80%; it still had good enzymatic hydrolysis activity at pH values from 9 to 10, and its relative enzyme activities were 62% and 38% respectively. This indicates that the cellulase StrEgl is a highly active cellulase with alkali tolerance and can degrade cellulose under alkaline conditions.

[0060] Example 3

[0061] Optimal temperature of cellulase StrEgl

[0062] Using 1% CMC-Na solution as the substrate, 100 μL of 1% CMC-Na solution, 80 μL of 0.1 mol / L citric acid-sodium citrate buffer solution (pH = 5), and 20 μL of BvEgl enzyme solution with a final concentration of 400 mg / L were mixed evenly to obtain a reaction system, which was subjected to a water bath reaction at 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, and 100 °C for 30 min. According to the DNS method described in Example 2, the specific enzyme activity of cellulase StrEgl at different temperatures was measured and calculated. Taking the highest enzyme activity as 100%, the optimal temperature of cellulase StrEgl was determined. The results are shown in Table 3 and Figure 3 as follows.

[0063] Table 3 Specific enzyme activity of cellulase BvEgl at different temperatures

[0064] Temperature (℃) Specific enzyme activity (U / mg) Temperature (℃) Specific enzyme activity (U / mg) 20 2837.76±11.57 70 3312.23±87.01 30 36.31.26±58.23 80 2537.82±23.36 40 4454.75±60.23 90 2134.26±54.67 50 4855.59±59.72 100 1965.19±17.72 60 4801.05±58.23

[0065] The results showed that the optimal temperature for the cellulase StrEgl to exert its enzymatic hydrolysis function was 50 °C; its relative enzyme activities were higher than 90% at 40 - 60 °C, and still had relative enzyme activities above 40% at 80 - 100 °C. This indicates that the cellulase StrEgl is a highly active cellulase with high temperature tolerance and can degrade cellulose under medium and high temperature conditions.

[0066] Example 4

[0067] pH stability of cellulase StrEgl

[0068] In the prepared buffer solutions with different pH values (4 - 10) (citric acid-sodium citrate buffer solution), the cellulase StrEgl solution was diluted to 400 mg / L; the specific reaction system was as follows: 100 μL of 1% CMC-Na solution, 80 μL of buffer solution, and 20 μL of BvEgl enzyme with a final concentration of 400 mg / L. After being placed at 50 °C for 1 h, according to the method described in Example 2, the specific enzyme activity of cellulase StrEgl treated with different pH buffer solutions was measured and calculated. The results are as Figure 4 follows.

[0069] The results showed that the cellulase StrEgl had good enzyme activity stability in the pH range of 5 - 9, and the residual enzyme activity after 1 hour of treatment basically did not decrease. When the environment was at pH 10, 62.54% of the enzyme activity remained after treatment. This indicated that the cellulase StrEgl was a highly active cellulase with alkali tolerance.

[0070] Example 5

[0071] Substrate specificity of cellulase StrEgl

[0072] Prepare CMC - Na solution, crystalline cellulose solution (Avicel), beechwood xylan solution, barley glucan solution, β - mannan solution, sucrose solution, laminarin solution, and amylopectin solution with a final concentration of 1% independently. Take 100 μL of each as the substrate, and mix them evenly with 80 μL of 0.1 mol / L citric acid - sodium citrate buffer solution (pH = 5) and 20 μL of StrEgl enzyme solution with a final concentration of 400 mg / L to obtain a reaction system. React in a water bath at 40 °C for 30 min, and measure and calculate the specific enzyme activity of cellulase StrEgl in each reaction system according to the DNS method described in Example 2. The results are shown in Table 4.

[0073] Table 4 Specific enzyme activity of cellulase StrEgl towards different substrates

[0074] Substrate Specific enzyme activity (U / mg) CMC-Na solution 4861.04±43.92 Microcrystalline cellulose solution 1077.79±54.41 Beechwood xylan solution 892.91±4.67 Barley glucan solution —— β-Mannan solution —— Sucrose solution —— Laminarin solution —— Amylopectin solution ——

[0075] The results showed that the cellulase StrEgl had a super strong degradation ability towards cellulose substrates containing β - 1,4 glycosidic bonds, and the specific enzyme activity could reach 4861.04 ± 43.92 U / mg. This indicated that the cellulase StrEgl was a highly active cellulase.

[0076] Example 5

[0077] Tolerance of cellulase StrEgl to metal ions and chemical reagents

[0078] Use 80 μL of K diluted by 0.1 M Tris - HCl pH 5 buffer solution with concentrations of 1 mM and 10 mM 1+ 、Mg 2+ 、Cu 2+ 、Mn 2+ 、Ni 2+ 、Ca 2+ 、Co 2+ 、Zn2+ , Fe 3+ Nine metal ion solutions, 80 μL of SDS, EDTA, PMSF, β-ME, Urea diluted from 5 mM with 0.1 M Tris-HCl buffer at pH 5, or 80 μL of Tween20, Tween80, and TritonX-100 diluted from 1% with 0.1 M Tris-HCl buffer at pH 5 were respectively mixed with 20 μL of StrEgl enzyme solution with a final concentration of 400 mg / L to obtain cellulase StrEgl dilution solutions with a concentration of 100 mg / L diluted with different metal ions and chemical reagents. The specific enzyme activity of cellulase StrEgl in each reaction system was measured and calculated according to the DNS method described in Example 2, and the results are as Figure 5 and Figure 6 shown.

[0079] According to Figure 5 and Figure 6 , it can be seen that metal ions Mn 2+ and Co 2+ have a promoting effect on cellulase StrEgl, and chemical reagents 1% Tween20 and 1% Tween80 have a promoting effect on cellulase StrEgl. EDTA, PMSF, Triton X-100, and Urea have no effect on the activity of cellulase StrEgl, indicating that cellulase StrEgl has great potential for commercial development.

[0080] It can be seen from the above examples that the alkaline-resistant cellulase StrEgl provided by the present invention has good tolerance to pH, temperature, metal ions, and chemical reagents, is very suitable for industrial applications, and has broad application prospects in the fields of papermaking, textiles, detergents, etc.

[0081] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An alkali-resistant cellulase StrEgl, characterized in that The amino acid sequence of the cellulase StrEgl is shown in SEQ ID NO.

1.

2. The cellulase StrEgl according to claim 1, characterized in that The pH value range of the cellulase StrEgl for performing enzymatic hydrolysis is 4 to 10.

3. The cellulase StrEgl according to claim 1 or 2, characterized in that The temperature range in which the cellulase StrEgl exerts its enzymatic function is 40 to 100°C.

4. A gene encoding the cellulase StrEgl according to any one of claims 1 to 3, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

5. A recombinant vector expressing the gene according to claim 4, characterized in that: Comprising the gene as claimed in claim 4 and an initial vector.

6. The recombinant vector according to claim 5, characterized in that The initial vector is pET-28a, and the gene is cloned between the Nco I and Xho I restriction sites of the initial vector pET-28a.

7. A recombinant strain expressing the gene according to claim 4, characterized in that: The recombinant strain is obtained by transferring the recombinant vector according to claim 5 or 6 into an empty vector bacterium.

8. The recombinant strain according to claim 7, characterized in that The empty-load bacteria are Escherichia coli.

9. Use of the cellulase StrEgl according to any one of claims 1 to 3, the gene according to claim 4, the recombinant vector according to claim 5 or 6, or the recombinant strain according to claim 7 or 8 in decomposing cellulose under alkaline conditions.

10. Use of the cellulase StrEgl according to any one of claims 1 to 3, the gene according to claim 4, the recombinant vector according to claim 5 or 6, or the recombinant strain according to claim 7 or 8 in the fields of papermaking, textiles or detergents.

Images