Highly stable cellulolytic enzyme and use thereof

Abstract

The present application relates to the technical field of biology, in particular to a high-stability cellulose-degrading enzyme and application thereof, the amino acid sequence of the cellulose-degrading enzyme is shown as SEQ ID NO.1; or the amino acid sequence obtained by substitution, deletion or addition of one or more amino acids of the amino acid sequence shown as SEQ ID NO.1, and has the same enzyme activity as the amino acid sequence shown as SEQ ID NO.1, the cellulose-degrading enzyme has high alkali resistance, good tolerance to temperature, most metal ions and common chemical reagents.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to highly stable cellulosic degrading enzymes and their applications. Background Technology

[0002] β-glucosidase (EC 3.2.1.21) is an important component of the cellulase system. It can specifically hydrolyze non-reducing terminal β-D-glucosidic bonds, degrading cellobiose and other cellooligosaccharides into glucose. It is a key enzyme for the complete degradation of lignocellulose into fermentable sugars. It has been widely used in biomass conversion, bioenergy, textiles, detergents, food, and medicine.

[0003] In the industrial utilization of lignocellulose, alkaline pretreatment is a common method to disrupt its dense crystalline structure and improve the efficiency of subsequent enzymatic hydrolysis. The pH of the treated system is typically maintained at 8.0–10.0. Furthermore, industrial processes such as textile refining, detergent formulation, and pulp bleaching also involve alkaline operating conditions. These applications all require β-glucosidase to maintain its catalytic activity and stability under high pH conditions.

[0004] Currently, commercially available β-glucosidases are mainly derived from *Trichoderma reesei* or *Aspergillus niger*. Their optimal reaction conditions are predominantly neutral or acidic, and they exhibit poor stability and are easily inactivated in alkaline environments, making them unsuitable for high-pH industrial applications and limiting their industrial application scope. Therefore, it is still necessary to explore novel β-glucosidases with excellent tolerance to alkalis and a wide pH range, as well as to temperature variations, most metal ions, and common chemical reagents, to expand their applications in fields such as biomass conversion.

[0005] In summary, there is an urgent need for a cellulase that exhibits excellent tolerance to a wide pH range, temperature, most metal ions, and common chemical reagents. Summary of the Invention

[0006] This invention addresses the technical problem of how to provide a cellulase that exhibits excellent tolerance to a wide pH range, temperature, most metal ions, and common chemical reagents.

[0007] To achieve the above objectives, a first aspect of the present invention provides a cellulase, wherein the amino acid sequence of the cellulase is as shown in SEQ ID NO.1; or

[0008] An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as shown in SEQ ID NO.1, and having the same enzyme activity as the amino acid sequence shown in SEQ ID NO.1.

[0009] A second aspect of the present invention provides a gene encoding the above-mentioned cellulase, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0010] A third aspect of the present invention provides a recombinant vector comprising the above-described coding gene.

[0011] A fourth aspect of the present invention provides a recombinant strain comprising the above-described recombinant vector.

[0012] The fifth aspect of the present invention provides the application of the above-mentioned cellulase; or the above-mentioned encoding gene; or the above-mentioned recombinant vector; or the above-mentioned recombinant strain in the decomposition of cellulosic oligosaccharides and glycosides.

[0013] The sixth aspect of the present invention provides the use of the above-mentioned cellulase; or the above-mentioned encoding gene; or the above-mentioned recombinant vector; or the above-mentioned recombinant strain in catalyzing β-glucosidic bonds and β-xyloside bonds.

[0014] The seventh aspect of the present invention provides the application of the above-mentioned cellulase; or the above-mentioned encoding gene; or the above-mentioned recombinant vector; or the above-mentioned recombinant strain in the fields of biomass conversion, bioenergy, food, feed, textiles and medicine.

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

[0016] (1) The cellulase provided by the present invention exhibits catalytic activity over a wide pH range of 3.0 to 11.0. Taking the highest enzyme activity (pH 9.0, 45℃) as 100%, it maintains more than 90% relative enzyme activity in the pH range of 4.0 to 9.0, and retains more than 70% relative enzyme activity in the pH range of 9.0 to 11.0. After incubation for 1 h in different pH buffers, the residual enzyme activity of the cellulase provided by the present invention remains above 70% in the pH range of 3.5 to 11.0, and the residual enzyme activity remains above 90% in the pH range of 5.0 to 11.0.

[0017] (2) The cellulose-degrading enzyme provided by this invention has a certain effect on Na+. + K + Mg 2+ Ca 2+ Co 2+ Mn 2+ Ni 2+ Metal ions are resistant to methanol, ethanol, and EDTA.

[0018] (3) The cellulose degrading enzyme provided by the present invention has good tolerance to pH, temperature, metal ions and chemical reagents, and has broad application prospects in papermaking, textiles, detergents, biomass conversion and other fields. Attached Figure Description

[0019] Figure 1 The SDSPAGE electrophoresis results of β-glucosidase MbBgl3 are shown in the figure. Lane M is the protein marker and lane 1 is the purified β-glucosidase MbBgl3.

[0020] Figure 2 A 3D structural simulation of β-glucosidase MbBgl3;

[0021] Figure 3 Figure showing the results of the optimal temperature analysis for β-glucosidase MbBgl3;

[0022] Figure 4 Figure showing the optimal pH analysis results for β-glucosidase MbBgl3;

[0023] Figure 5 The figure shows the results of pH stability analysis of β-glucosidase MbBgl3.

[0024] Figure 6 Figure showing the results of temperature stability analysis of β-glucosidase MbBgl3;

[0025] Figure 7 Figure showing the glucose tolerance results of β-glucosidase MbBgl3;

[0026] Figure 8 The figure shows the metal ion tolerance results of β-glucosidase MbBgl3. Detailed Implementation

[0027] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0028] In this invention, high stability refers to good tolerance to a wide pH range, temperature, most metal ions, and common chemical reagents.

[0029] In this invention, the cellulase is a β-glucosidase, named β-glucosidase MbBgl3.

[0030] A first aspect of the present invention provides a cellulase, wherein the amino acid sequence of the cellulase is as shown in SEQ ID NO.1; or

[0031] An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as shown in SEQ ID NO.1, and having the same enzyme activity as the amino acid sequence shown in SEQ ID NO.1.

[0032] In this invention, the β-glucosidase gene was obtained from the genome of microorganisms in the root sediments of the mangrove forest in Futian, Shenzhen. After removing the signal peptide sequence, the cellulase described in this invention was obtained through artificial synthesis.

[0033] According to the present invention, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.1 includes:

[0034] The amino acid sequence of the cellulase is shown in SEQ ID NO.3; or

[0035] The amino acid sequence of the cellulase is shown in SEQ ID NO.4; or

[0036] The amino acid sequence of the cellulase is shown in SEQ ID NO.5.

[0037] In this invention, the cellulase also includes an enzyme derived from the amino acid sequence shown in SEQ ID NO.1.

[0038] According to the present invention, the pH range for the cellulose-degrading enzyme to perform its enzymatic hydrolysis function is 3.0 to 11.0.

[0039] According to the present invention, the temperature range in which the cellulose-degrading enzyme performs its enzymatic hydrolysis function is 20~100℃.

[0040] A second aspect of the present invention provides a gene encoding the above-mentioned cellulase, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0041] A third aspect of the present invention provides a recombinant vector comprising the above-described coding gene.

[0042] In this invention, the recombinant vector comprises the coding gene and the initial vector.

[0043] According to a preferred embodiment of the present invention, the initial vector is pET-28a, and the coding gene is cloned between the NdeI and BamHI restriction sites of the initial vector pET-28a.

[0044] The present invention does not impose any particular limitation on the preparation method of the recombinant vector; conventional recombinant vector preparation methods in the art can be used.

[0045] A fourth aspect of the present invention provides a recombinant strain comprising the above-described recombinant vector.

[0046] In this invention, the recombinant strain is obtained by transferring the recombinant vector into an empty vector.

[0047] According to a preferred embodiment of the present invention, the empty carrier bacteria is Escherichia coli.

[0048] More preferably, the empty carrier bacteria is Escherichia coli BL21(DE3) strain.

[0049] The present invention does not impose any particular limitation on the preparation method of the recombinant strain; conventional methods for preparing recombinant strains in the art can be used.

[0050] The fifth aspect of the present invention provides the application of the above-mentioned cellulase; or the above-mentioned encoding gene; or the above-mentioned recombinant vector; or the above-mentioned recombinant strain in the decomposition of cellulosic oligosaccharides and glycosides.

[0051] The sixth aspect of the present invention provides the use of the above-mentioned cellulase; or the above-mentioned encoding gene; or the above-mentioned recombinant vector; or the above-mentioned recombinant strain in catalyzing β-glucosidic bonds and β-xyloside bonds.

[0052] The seventh aspect of the present invention provides the application of the above-mentioned cellulase; or the above-mentioned encoding gene; or the above-mentioned recombinant vector; or the above-mentioned recombinant strain in the fields of biomass conversion, bioenergy, food, feed, textiles and medicine.

[0053] The present invention will be described in detail below through embodiments.

[0054] Example 1: Preparation of β-glucosidase MbBgl3

[0055] (1) Synthesis of coding genes

[0056] The β-glucosidase gene was isolated and screened from the root sediment microbial genome of Kandelia candel in the Futian mangrove forest of Shenzhen. After removing the signal peptide sequence, the MbBgl3 gene sequence was synthesized by Changzhou Kana Gene Technology Co., Ltd. The nucleotide sequence is shown in SEQ ID NO.2.

[0057] (2) Preparation of recombinant vector

[0058] The pET-28a plasmid and the obtained MbBgl3 gene sequence were simultaneously digested with NdeI and BamHI. After digestion at 37°C for 2 h, nucleic acid electrophoresis was performed for verification, and the target gene and plasmid were recovered by gel extraction. The recovered products were ligated using DNA ligase at 16°C for 1 h. The ligation product was transformed into DH5α competent cells and cultured overnight on kanamycin-resistant solid LB medium. Multiple transformants were picked and cultured in kanamycin-containing LB liquid medium at 37°C and 220 rpm for 12 h. The plasmid was extracted and verified by PCR. The recombinant plasmid was sent to a sequencing company for sequencing, and the recombinant plasmid with the correct sequence was named pET-28a-MbBgl3.

[0059] (3) Preparation of recombinant strains

[0060] The correctly sequenced recombinant plasmid pET-28a-MbBgl3 was transformed into BL21(DE3) competent cells to form a recombinant strain. Multiple transformants were picked and cultured in kanamycin-resistant LB medium at 37°C and 220 rpm for 10 h. Plasmids were extracted and verified by PCR. The recombinant plasmids were then sent to a sequencing company for sequencing. The strain from which the correctly sequenced recombinant plasmid originated was the desired target strain, i.e., the recombinant strain.

[0061] (4) Protein induction expression and purification

[0062] 5 mL of seed culture (OD) of the recombinant strain 600 =5) Inoculate into 500 mL LB medium (30 ng / μL kanamycin) and incubate at 37°C until OD. 600 =0.8, to obtain MbBgl3 bacterial culture; add isopropyl thiogalactoside to the culture to a final concentration of 60 ng / μL, induce protein expression overnight at 16℃, collect the bacterial cells by centrifugation at 6000 rpm for 10 min, and resuspend in sodium dihydrogen phosphate (PBS) buffer. Sonicate the cells (400 W, 2 s operation followed by a 6 s interval, for a total of 99 cycles), then centrifuge at 12000 rpm for 10 min to collect the supernatant, obtaining crude enzyme solution; use HisTrap TM The crude enzyme solution was purified by affinity chromatography using an HP column. Equilibration was performed with a mobile phase containing 0.3 mol / L NaCl and 20 mmol / L PBS (pH 8.0). A gradient elution with imidazole at concentrations of 0–0.5 mol / L was then used. Tubes with high UV signals were collected for enzyme activity assay. The tubes showing enzyme activity were combined and purified using HiPrep. TMDesalting was performed using a 26 / 10 Desalting column to obtain purified β-glucosidase MbBgl3 enzyme solution. SDS-PAGE results showed that the purified recombinant protein appeared as a single protein band at approximately 95 kDa, slightly deviating from the theoretical molecular weight (88.1 kDa) calculated based on the amino acid sequence. This deviation may be related to the influence of the His tag on the protein's migration rate in SDS-PAGE, but the single master band indicates that the protein has reached a high electrophoretic purity, which meets the requirements for subsequent enzymatic characterization.

[0063] (5) Protein structure analysis

[0064] The three-dimensional structure of β-glucosidase MbBgl3 was predicted using AlphaFold3.

[0065] The results are as follows Figure 2 As shown, this enzyme consists of an N-terminal TIM barrel domain, an α / β sandwich domain, and a C-terminal β-sandwich domain, exhibiting typical multi-domain characteristics of the glycoside hydrolase family 3.

[0066] Example 2: Optimal temperature of β-glucosidase MbBgl3

[0067] Using a 2 mg / mL p-nitrophenyl-β-D-glucopyranoside solution as a substrate, a reaction system was prepared by mixing 50 μL of pH 7.0 citrate-disodium hydrogen phosphate buffer, 40 μL of 2 mg / mL p-nitrophenyl-β-D-glucopyranoside solution, and 10 μL of enzyme solution. 10 μL of β-glucosidase MbBgl3 enzyme solution was added at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃, respectively, and reacted for 10 min. The reaction was terminated by adding 100 μL of 2% sodium carbonate, and the absorbance at 405 nm was measured to determine the optimal reaction temperature of the enzyme.

[0068] Enzyme activity was characterized by measuring the absorbance of the reaction system at 405 nm using p-nitrophenyl-β-D-glucopyranoside as a substrate. Figure 3 The results showed that β-glucosidase MbBgl3 exhibited the highest absorbance at 45℃, thus determining the optimal reaction temperature for β-glucosidase MbBgl3 as 45℃, with the absorbance at 45℃ representing 100%. The relative enzyme activity remained above 90% within the 20–55℃ range, and retained over 26% relative enzyme activity within the 55–70℃ range. This indicates that β-glucosidase MbBgl3 possesses excellent activity retention over a wide temperature range, making it a β-glucosidase with both high catalytic efficiency and temperature adaptability.

[0069] Example 3: Optimal pH of β-glucosidase MbBgl3

[0070] Prepare 20 mmol / L disodium citrate-phosphate buffer solutions (pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5), 20 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate (PBS) buffer solutions (pH 7.0, 7.6, 8.0, 8.6, 9.0), and 20 mmol / L sodium carbonate-sodium bicarbonate buffer solutions (pH 9.0, 9.5, 10.0, 10.5, 11.0). Using 2 mg / mL p-nitrophenyl-β-D-glucopyranoside solution as the substrate, mix 50 μL of each pH gradient buffer solution, 40 μL of 2 mg / mL pNPG solution, and 10 μL of β-glucosidase MbBgl3 enzyme solution to obtain the reaction system. Incubate at 45 °C for 10 min, then add 100 μL of 2% sodium carbonate to terminate the reaction. Measure 405. The absorbance at nm is used to determine the optimal pH for enzyme reaction.

[0071] Enzyme activity was characterized by measuring the absorbance of the reaction system at 405 nm using p-nitrophenyl-β-D-glucopyranoside as a substrate. Figure 4 The results show that β-glucosidase MbBgl3 exhibits catalytic activity across a wide pH range of 3.0–11.0. The absorbance of β-glucosidase MbBgl3 is highest at pH 9.0, therefore, the optimal reaction pH for β-glucosidase MbBgl3 is determined to be 9.0. With the absorbance at pH 9.0 as 100%, it maintains over 90% relative enzyme activity within the pH range of 4.0–9.0, and retains over 70% relative enzyme activity within the pH range of 9.0–11.0. This indicates that β-glucosidase MbBgl3 possesses excellent acid and alkali resistance and can effectively hydrolyze β-glucosidic bonds over a wide pH range.

[0072] Example 4 pH stability of β-glucosidase MbBgl3

[0073] 3 μL of enzyme solution was mixed with 15 μL of different pH buffers prepared in Example 3 and incubated at 4°C for 1 h. Then, 32 μL of citrate-disodium hydrogen phosphate buffer (pH 7.0) and 40 μL of substrate were added, and the reaction was carried out at 45°C for 10 min. The reaction was terminated by adding 100 μL of 2% sodium carbonate, and the absorbance at 405 nm was measured. The relative enzyme activity was calculated with the highest enzyme activity (pH 9.0, 45°C) as 100%.

[0074] The results showed that after incubation with different buffers within the pH range of 3.0–11.0 for 1 h, the residual enzyme activity remained above 70%, as indicated in the instruction manual. Figure 5 As shown, the residual enzyme activity remained above 90% within the pH range of 5.0 to 11.0. This indicates that MbBgl3 exhibits good pH stability and strong alkali tolerance.

[0075] Example 5 Temperature stability of β-glucosidase MbBgl3

[0076] The enzyme solution was incubated at 4°C, 25°C, 30°C, 35°C, and 45°C for 24 h, and samples were taken at 2 h, 4 h, 8 h, 18 h, and 24 h to determine residual enzyme activity. For the assay, 10 μL of enzyme solution, 50 μL of citrate-disodium hydrogen phosphate buffer (pH 7.0), and 40 μL of substrate were mixed thoroughly to form the reaction system. The reaction was carried out at 45°C for 10 min, and 100 μL of 2% sodium carbonate was added to terminate the reaction. The absorbance at 405 nm was then measured. The enzyme solution at 0 h was used as a blank control, and its activity was defined as 100%. The relative enzyme activity at each time point was calculated.

[0077] The results showed that the enzyme exhibited excellent stability under medium and low temperature conditions. After incubation at 4℃, 25℃, and 30℃ for 24 h, the residual enzyme activity remained above 80%; after incubation at 35℃ for 24 h, the residual enzyme activity remained above 50%. (See attached instructions). Figure 6 As shown, at 45℃, enzyme activity gradually decreased with prolonged incubation time. This indicates that β-glucosidase MbBgl3 is a β-glucosidase with good stability at medium and low temperatures.

[0078] Example 6: Substrate specificity of β-glucosidase MbBgl3 and inhibition of its products

[0079] 40 μL of each of the following 2 mg / mL p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-α-D-glucopyranoside, p-nitrophenyl-β-D-galactopyranoside, p-nitrophenyl-β-D-xylopyranoside, and p-nitrophenyl-β-D-cellobiose were used as substrates. These were mixed with 50 μL of citrate-disodium hydrogen phosphate solution (pH 7.0) and 10 μL of enzyme solution to obtain a homogeneous reaction system. The reaction was carried out in a water bath at 45 °C for 10 min, and the reaction was terminated by adding 100 μL of 2% sodium carbonate. The absorbance at 405 nm was measured. The p-nitrophenol content in each reaction was calculated by constructing a p-nitrophenol standard curve, and the enzyme activity of each substrate was calculated using the enzyme activity formula. A 10 mg / mL glucose standard stock solution was prepared and serially diluted to obtain glucose standard solutions with concentrations of 10 mg / mL, 5 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.2 mg / mL. Liquid chromatography was performed using an Aminex HPX-87H column with 5 mmol / L dilute sulfuric acid as the mobile phase, a flow rate of 0.6 mL / min, a column temperature of 60°C, and a detection time of 40 min. A glucose standard curve was plotted based on the detection results. 50 μL of 100 mmol / L cellobiose solution, 100 μL of enzyme solution, and 350 μL of pH 9.0 citrate-disodium hydrogen phosphate buffer were mixed thoroughly, reacted at 45°C for 5 min, and then inactivated at 100°C for 5 min. The reacted samples were analyzed by liquid chromatography, and enzyme activity was calculated based on the glucose standard curve. The results are shown in Table 1.

[0080] A 4 mol / L glucose stock solution was prepared and dissolved in pH 9.0 citrate-disodium hydrogen phosphate buffer. 10 μL of enzyme solution, 50 μL of glucose-containing citrate-disodium hydrogen phosphate buffer, and 40 μL of substrate were mixed thoroughly to obtain the reaction system. The final glucose concentrations in the reaction system were 0.2, 0.5, 1, and 2 mol / L, with a glucose-free buffer serving as a control, indicating 100% activity. The reaction system was incubated at 45°C for 10 min, and the reaction was terminated by adding 100 μL of 2% sodium carbonate. The absorbance at 405 nm was measured. The results are as follows: Figure 7 As shown.

[0081] The formula for the pNP standard curve is: y = 0.0017x + 0.0456 (R 2 =0.9999) Formula I;

[0082] In Formula I, x is the concentration of p-nitrophenol (pNP) in μmol / L, and y is the OD value. 405 value;

[0083] The specific enzyme activity formula is: Formula II;

[0084] In Formula II: specific enzyme activity (U / mg is the number of enzyme activity units per mg of enzyme protein, where U is the amount of enzyme required to catalyze the production of 1 μmol of product per minute under specified reaction conditions), G is the product content (μmol / L), M is the dilution factor of MbBgl3 enzyme solution, V is the volume of MbBgl3 enzyme solution (mL), T is the reaction time (min), and N is the concentration of MbBgl3 enzyme (mg / mL).

[0085]

[0086] Table 1. Specific enzyme activities of β-glucosidase MbBgl3 for different substrates.

[0087] The results showed that β-glucosidase MbBgl3 had a certain enzymatic ability to hydrolyze the natural substrate cellobiose. β-glucosidase MbBgl3 had the highest catalytic activity for β-D-glucopyranoside substrate, with a specific activity of 9.27±0.20 U / mg. It could also hydrolyze β-D-xylopyranoside and β-D-cellobiose. It had no catalytic activity for α-D-glucopyranoside and β-D-galactopyranoside, showing obvious substrate preference.

[0088] The glucose tolerance results showed that the enzyme activity gradually decreased as the glucose concentration in the reaction system increased; more than 60% of the relative enzyme activity was retained under 0.5 mol / L glucose conditions, and catalytic activity could still be detected under high glucose concentration of 2.0 mol / L, indicating that it has a certain glucose tolerance ability.

[0089] Example 7: Metal ion and chemical reagent tolerance of β-glucosidase MbBgl3

[0090] Prepare Na solutions with final concentrations of 1 mM and 10 mM. + K + Mg 2+ Fe 3+ Ca 2+ Zn 2+ Co 2+ Cu 2+ Mn 2+ Ni 2+ Li + Al 3+Twelve metal ion solutions (diluted with citrate-disodium hydrogen phosphate buffer at pH 7.0); solutions containing 0.1% and 1% EDTA or SDS, and 1% and 10% methanol, ethanol, isopropanol, and β-mercaptoethanol were prepared using the same buffer. 50 μL of the above buffer containing the test reagents was added to 40 μL of 2 mg / mL p-nitrophenyl-β-D-glucopyranoside and 10 μL of enzyme solution to form the reaction system. The relative enzyme activity under each condition was calculated with the enzyme activity without added reagents as 100%, and the results are shown below. Figure 8 As shown in Table 2.

[0091] Table 2. Tolerance of enzyme solutions to chemical reagents and alcohol reagents

[0092]

[0093] according to Figure 8 As shown in Table 2, Na + K + Mg 2+ Ca 2+ Co 2+ Mn 2+ Ni 2+ Metal ions had little effect on the activity of β-glucosidase MbBgl3 within the tested concentration range, while high concentrations of Fe... 3+ Cu 2+ Al 3+ It exhibits a significant inhibitory effect on enzyme activity. Among chemical reagents, low concentrations of EDTA have no significant effect on enzyme activity, while high concentrations of SDS have an inhibitory effect. 1% and 10% ethanol, isopropanol, and β-mercaptoethanol have little effect on enzyme activity, while 10% methanol shows an inhibitory effect. This indicates that β-glucosidase MbBgl3 has good tolerance to most metal ions and common organic solvents, and possesses potential for industrial application.

[0094] As can be seen from the above embodiments, the alkali-resistant β-glucosidase MbBgl3 provided by the present invention has good tolerance to pH, temperature, metal ions and chemical reagents, and has good application prospects in the fields of biomass conversion, bioenergy production, papermaking, textiles and detergents.

[0095] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A cellulase, characterized in that, The amino acid sequence of the cellulase is shown in SEQ ID NO.1; or An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as shown in SEQ ID NO.1, and having the same enzyme activity as the amino acid sequence shown in SEQ ID NO.

1.

2. The cellulase according to claim 1, characterized in that, Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids as shown in SEQ ID NO.1 include: The amino acid sequence of the cellulase is shown in SEQ ID NO.3; or The amino acid sequence of the cellulase is shown in SEQ ID NO.4; or The amino acid sequence of the cellulase is shown in SEQ ID NO.

5.

3. The cellulosic enzyme according to claim 1, characterized in that, The pH range for the cellulose-degrading enzyme to perform its enzymatic hydrolysis function is 3.0 to 11.

0.

4. The cellulase according to claim 1, characterized in that, The temperature range in which the cellulose-degrading enzyme performs its enzymatic hydrolysis function is 20~100℃.

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

2.

6. A recombinant vector, characterized in that, It includes the coding gene as described in claim 5.

7. A recombinant bacterial strain, characterized in that, It includes the recombinant vector as described in claim 6.

8. The use of the cellulase according to any one of claims 1-4; or the encoding gene according to claim 5; or the recombinant vector according to claim 6; or the recombinant strain according to claim 7 in the decomposition of cellulosic oligosaccharides and glycosides.

9. The cellulase according to any one of claims 1-4; or the encoding gene according to claim 5; or the recombinant vector according to claim 6; or the recombinant strain according to claim 7 in catalyzing β-glucosidic and β-xyloside bonds.

10. The application of the cellulase according to any one of claims 1-4; or the encoding gene according to claim 5; or the recombinant vector according to claim 6; or the recombinant strain according to claim 7 in the fields of biomass conversion, bioenergy, food, feed, textiles and medicine.

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