A salt-tolerant and glucose-tolerant β-glucosidase B0-BG36 and its application

Abstract

The present invention provides a salt-tolerant and glucose-tolerant beta-glucosidase B0-BG36 and its application. Belong to the field of genetic engineering technology. The beta-glucosidase is a new beta-glucosidase gene b0-bg36 of a GH1 family identified in the uncultured microbial metagenome of saline-alkali soil in Karamay, Xinjiang, and beta-glucosidase B0-BG36 was obtained by cloning and heterologous expression. B0-BG36 has very high enzyme activity and can effectively maintain enzyme activity under high temperature, different pH conditions, metal ions and inhibitors, especially has high tolerance to salt and glucose. The present invention also provides the gene of the beta-glucosidase and its application, which breaks the pattern of intracellular production of beta-glucosidase in the prior art, solves the problem that the existing beta-glucosidase enzyme activity is low and is easily inhibited by end product glucose.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and more particularly to a salt-tolerant and glucose-tolerant beta-glucosidase B0-BG36 and an application thereof. Background Art

[0002] Cellulose, a major component of plant cell walls, is a polysaccharide composed of glucose that is insoluble in water and common organic solvents. It is widely distributed in nature and is the world's most abundant renewable biomass resource. However, due to its complex structure, this vast resource has not yet been effectively utilized.

[0003] Cellulase is a general term for a class of enzymes that degrade cellulose and convert it into glucose. This class includes endo-1,4-β-glucanases (endo-glucanases, EC 3.2.1.4), exo-1,4-β-glucanases (cellulose biohydrolases, EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21). β-glucosidases are essential for the complete bioconversion of cellulose. They hydrolyze cellobiose to glucose, relieving the inhibitory effect of the cellobiose product on endo- and exo-glucanases, making them the rate-limiting enzyme in cellulose degradation. β-glucosidases are widely used in industries such as food, cosmetics, biofuels, and pharmaceuticals. In recent years, with the development of lignocellulosic ethanol, β-glucosidases have garnered increasing attention. β-glucosidase is commonly found in most microorganisms, but many microorganisms produce β-glucosidase as an intracellular enzyme with very low yields and are easily inhibited by the end product glucose. Moreover, due to the limitations of laboratory pure culture technology, more than 99% of microorganisms cannot be cultured, which greatly restricts the development of β-glucosidase.

[0004] Uncultured microorganisms are Earth's largest untapped biological resource. Metagenomics technology, independent of microbial culture, can directly obtain the nucleotide sequences of most genes from environmental DNA. Therefore, metagenomics technology has great potential for extracting β-glucosidases from uncultured extreme environmental microorganisms.

[0005] Therefore, how to use metagenomic technology to provide a salt-tolerant and glucose-tolerant β-glucosidase is a technical problem that those skilled in the art urgently need to solve. Summary of the Invention

[0006] In view of this, the present invention provides a salt-tolerant and glucose-tolerant β-glucosidase B0-BG36 and its application. The β-glucosidase is a new GH1 family β-glucosidase gene b0-bg36 identified from the Xinjiang Karamay saline-alkali soil metagenome using metagenomic technology, and introduced into Escherichia coli for cloning and heterologous expression. It has high enzyme activity and can effectively maintain enzyme activity under high temperature, different pH conditions, metal ions and inhibitors, especially with high tolerance to salt and glucose. It breaks the mode of intracellular production of β-glucosidase in the prior art and solves the problem that the existing β-glucosidase has low enzyme activity and is easily inhibited by the end product glucose.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A salt-tolerant and glucose-tolerant beta-glucosidase B0-BG36, the amino acid sequence of which is shown in SEQ ID NO.4.

[0009] Another object of the present invention is to provide a gene encoding the salt-tolerant and glucose-tolerant β-glucosidase B0-BG36, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0010] Another object of the present invention is to provide use of the gene of β-glucosidase B0-BG36 in encoding β-glucosidase B0-BG36.

[0011] Another object of the present invention is to provide an expression vector containing a gene encoding β-glucosidase B0-BG36.

[0012] Another object of the present invention is to provide a microorganism containing the above expression vector.

[0013] Another object of the present invention is to provide the use of the salt-tolerant and glucose-tolerant β-glucosidase B0-BG36 in cellulose degradation.

[0014] Another object of the present invention is to provide the use of the salt-tolerant and glucose-tolerant β-glucosidase B0-BG36 in food, feed, textile or cellulosic ethanol production.

[0015] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) The present invention uses metagenomic technology to identify a new β-glucosidase gene b0-bg36 of the GH1 family in the uncultured microbial metagenome of saline-alkali soil in Karamay, Xinjiang, and obtains β-glucosidase B0-BG36 by cloning and heterologous expression. It is a new salt-tolerant and glucose-tolerant β-glucosidase with strong pH tolerance and metal ion tolerance, and is relatively thermophilic and acidophilic. It has potential application value in the fields of food, feed, textile and cellulosic ethanol production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0018] Figure 1 : Phylogenetic tree of B0-BG36 and neighboring β-glucosidase protein sequences;

[0019] Figure 2 :SDS-PAGE gel analysis of recombinant β-glucosidase B0-BG36, Note: 1. Protein marker; 2. E. coli DH5α / pSHY211-B0-BG36 total protein; 3. Purified B0-BG36 protein;

[0020] Figure 3 :Effects of temperature and pH on the activity and stability of recombinant β-glucosidase B0-BG36;

[0021] Figure 4 :Effects of glucose on the activities of recombinant β-glucosidase B0-BG36 and industrial cellulase;

[0022] Figure 5 :Effect of NaCl on the activity of recombinant β-glucosidase B0-BG36;

[0023] Figure 6 : Lineweaver-Burk double reciprocal plot of B0-BG36 using cellobiose as substrate;

[0024] Figure 7 : Lineweaver-Burk double reciprocal plot of B0-BG36 using α-lactose monohydrate as substrate. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] The strains and plasmids used in the examples of the present invention are as follows:

[0027] E. coli DH5α: purchased from Shenzhen Kangti Life Science Technology Co., Ltd., used for cloning and expression of β-glucosidase gene;

[0028] Vector plasmid: The constitutive expression vector pSHY211, independently constructed in our laboratory, was used for the construction of recombinant plasmids. The construction process of pSHY211 was published in Scientific Reports (Yin et al., 2023) in September 2023. The article citation information is Yin, YR., Li, XW., Long, CH. et al. Characterization of a GH10 extremely thermophilic xylanase from the metagenome of hot spring for prebiotic production. Sci Rep 13, 16053 (2023).

[0029] Example 1

[0030] Sample collection, metagenomic DNA extraction, and sequencing

[0031] Samples were collected from saline-alkali soil in Karamay, Xinjiang, frozen in ice, and then enriched and cultured. Metagenomic DNA was extracted using a PowerSoil Kit (MOBIO DNA PowerSoil Kit, New York, NY, USA) according to the manufacturer's instructions. A 1344-bp β-glucosidase functional gene of the GH1 family was predicted from the metagenomic database provided by the sequencing company. The gene was named b0-bg36 and its nucleotide sequence is as follows:

[0032] >b0-bg36

[0033]

[0034] Example 2

[0035] Gene amplification, cloning and expression, recombinant screening, and expression, purification and identification of B0-BG36

[0036] (1) Gene amplification: Targeting the β-glucosidase gene b0-bg36, primers b0-bg36-F and b0-bg36-R were designed and PCR amplified. After amplification, the product was identified by 1.0% agarose gel electrophoresis and recovered using a DNA gel recovery kit.

[0037] PCR amplification conditions: 95°C pre-denaturation for 3 min, 98°C denaturation for 15 s, 60°C annealing for 20 s, 72°C extension for 30 s, 32 cycles, and a final extension at 72°C for 5 min.

[0038] Primer sequences:

[0039] b0-bg36-F:5'- CATCATCATCATCATCATGAA ATGGAATCAAAGATATTTCCA-3',SEQ IDNO.2

[0040] b0-bg36-R:5'- GTGCTCGAGTGCGGCCGCAAG TAATCCGTTCTTTTCTATTACC-3',SEQ IDNO.3

[0041] The underlined sequences represent homologous recombinant fragments with the pSHY211 vector previously digested with EcoRI and HindIII.

[0042] (2) Preparation of recombinant plasmid: The PCR product recovered from gel was ligated with the pSHY211 vector using the pEASY-Uni Seamless Cloning and Assembly Kit (Beijing Quanshijin Biotechnology Co., Ltd., China, Cat. No. CU101-01) to obtain the recombinant plasmid pSHY211-B0-BG36;

[0043] (3) Gene cloning and expression: The recombinant plasmid pSHY211-B0-BG36 was introduced into the competent E. coli DH5α strain for cloning and expression;

[0044] (4) Screening of recombinants: The E. coli clones containing the recombinant plasmid were screened using the double-layer plate method. The E. coli clones were cultured in the first layer of culture medium (LB medium, containing 50 μg / mL kanamycin, 2% agar) at 37°C for 16 h. Then, a second layer of culture medium (PBS buffer, containing 1% agarose, 1‰ esculin, 1% lysozyme, containing 50 μg / mL kanamycin) was added to completely cover the clones. After incubation at 37°C for 2 h, the colony color of the E. coli clones was observed, and the recombinant E. coli clones with dark brown hydrolysis circles were screened, which were the recombinant E. coli DH5α / pSHY211-B0-BG36.

[0045] (5) Expression, purification and identification of B0-BG36:

[0046] E. coli DH5α / pSHY211-B0-BG36 was activated by inoculating a single colony onto an LB plate containing kanamycin (50 μg / mL). After activation, a single colony was selected and placed into 100 mL of LB liquid medium. The culture was incubated at 37°C with continuous shaking at 180 rpm for 8 hours. The culture was then transferred to 25°C and shaken at 180 rpm for another 12 hours. After the incubation period, the cells were harvested by centrifugation at 4°C and 12,000 rpm for 20 minutes and disrupted by ultrasound. After centrifugation again, the recombinant protein was purified by Ni-NTA chromatography, the protein concentration was measured by Bradford method, the protein size of the enzyme was identified and analyzed by SDS-PAGE electrophoresis, the DNA and protein sequences were analyzed using BLASTx and BLASTp online software (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the signal peptide was predicted using SignalP (http: / / www.cbs.dtu.dk / services / SignalP / ), and the theoretical isoelectric point and molecular weight were predicted using ExPASy (https: / / web.expasy.org / compute_pi / ). A phylogenetic tree was constructed using MEGA 7 software using the maximum likelihood method (ML) and Poisson correction model ( Figure 1 ).

[0047] After analysis, the amino acid sequence of β-glucosidase B0-BG36 is as follows:

[0048] >B0-BG36

[0049] MESKIFPKNFIWGTATASYQIEGAWKEDGKGESIWDRFTHIPGKIYNDDNGDVACDHYHRYE

[0050] EDVKLMKELGIKSYRLSLSWPRLFPDGTGEPNQKGVEFYKKLITMIKENGIVPCVTLFHWDLP

[0051] QKLQEKGGWANRDSVQWFEDYARFVFEQFGDQVPYWITHNEPYVTSFMGHWRGVFAPGIS

[0052] DISTGILTAHHLLLSHGRAVAAYREMGFTGDIGITLNLSPFYTLTDKEEDKRAARLEDGHLNR

[0053] WFLDPVLKGKYPEDMIDFYSTKEGITLPEFSEEDMKTISIPTDFLGLNYYFGSYLAKGDSWPF

[0054] GSQWVDAGYQKTEMGWNITPEAFYDLLVRLHKEYDGVKIVVTENGMAVNDRVDRNGEVKD

[0055] FDRIDYLYTHFEQAHRAINEGVNLAGYYVWSLMDNFEWASGYSKRFGLIHINYKTLKRTPKESFYWYKTVIEKNGL, SEQ ID NO.4.

[0056] Result analysis:

[0057] According to protein sequence alignment, B0-BG36 had a similarity of 62.05% with the GH1 family β-glucosidase (NLU52715.1) from Clostridiaceae bacterium, 63.33% with the β-glucosidase (MBZ4645305.1) from Clostridia bacterium, and 63.33% with the β-glucosidase (MDK2809480.1) from Petroclostridium sp.

[0058] The theoretical isoelectric point (pI) and molecular weight (Mw) of B0-BG36 predicted on the ExPASy website (https: / / web.expasy.org / compute_pi / ) are 5.45 and 51.98 kDa, respectively. The gene was expressed in E. coli DH5α, and the recombinant protein with a His-tagged N-terminus was purified using a Ni chelate affinity column. SDS-PAGE electrophoresis analysis showed that the molecular weight of the recombinant β-glucosidase B0-BG36 protein was consistent with the theoretical prediction, indicating that the recombinant target protein ( Figure 2 ).

[0059] Example 3

[0060] (1) Optimal temperature and temperature tolerance of purified β-glucosidase B0-BG36

[0061] Under the condition of pH 7, the relative enzymatic activity of the purified β-glucosidase was measured at different temperatures (10-75°C), with a gradient of 5°C being set to determine the optimal reaction temperature.

[0062] Thermal stability analysis The residual enzyme activity of B0-BG36 was determined by incubating the pure enzyme solution at different temperatures (50°C, 55°C and 60°C) for 60 min and sampling every 20 min.

[0063] Results: At the optimum pH, B0-BG36 showed high activity between 45-60°C, with activity maintained above 40%. The optimum temperature for cellobiose and α-lactose monohydrate was 55°C, and the enzyme activity decreased rapidly at 65°C. At 65°C, the enzyme activity with α-lactose monohydrate as substrate was almost lost. The enzyme activity with cellobiose as substrate was inactivated at 75°C ( Figure 3 A) When cellobiose is used as substrate, the half-life at 55°C is 36 min. The residual activity of the enzyme is above 70% after incubation at 50°C for 60 min, while the enzyme loses activity after incubation at 60°C for 20 min. Figure 3 Middle C).

[0064] (2) Optimal reaction pH and pH stability

[0065] Enzymatic reactions with B0-BG36 were carried out at the optimal temperature using citric acid-sodium hydrogen phosphate buffers of varying pH values ​​(4.0, 4.6, 5.0, 5.6, 6.0, 6.6, 7.0, 7.6, and 8.0) and glycine-sodium hydroxide buffers of varying pH values ​​(8.0, 8.6, 9.0, 9.6, and 10.0), and the optimal pH values ​​were recorded. To analyze pH stability, the residual enzyme activity of the recombinant enzyme was measured after incubation at 4°C for 12 and 24 hours in buffers with varying pH values ​​(3.0-10.0).

[0066] Result analysis: The optimum pH of B0-BG36 was 5.0 when using cellobiose and α-lactose monohydrate as substrates. The optimum pH range of B0-BG36 when using cellobiose as substrate was 4.6 to 8.6. The pH range was wide and stable. Figure 3B). However, when α-lactose monohydrate was used as substrate at different pH values, the enzyme stability was poor, and the enzyme activity decreased rapidly with the increase of pH value. When cellobiose was used as substrate, the enzyme activity of B0-BG36 was stable in the pH range of 5-10 after incubation for 12h and 24h in different pH buffers at 4°C, and basically maintained above 60%. In particular, it had strong alkaline resistance under alkaline conditions, and the activity was maintained above 75% at pH 10.0 ( Figure 3 Middle D).

[0067] (3) Effects of different metal ions and inhibitors on recombinase

[0068] Different metal ions (K + Mg 2+ 、Fe 3+ , Ca 2+ 、Zn 2+ 、Co 2+ 、Cu 2+ 、Ag + 、Mn 2+ , Pb 2+ and Ni 2+ ) with a final concentration of 1 mM and 10 mM, and chemical reagents [disodium ethylenediaminetetraacetic acid (EDTA), Tween-80 (Tween-80), phenylmethylsulfonyl fluoride (PMSF), sodium dodecyl sulfate (SDS), dithiothreitol (DTT), ethanol (Ethanol) with a final concentration of 1% and 0.1%], the enzymatic reaction was carried out under the optimal reaction conditions, and the effects of these additives on the enzyme activity were recorded. The experimental results are shown in Table 1.

[0069] Table 1 Effects of metal ions and chemical reagents on the enzyme activity of B0-BG36

[0070]

[0071] Results: B0-BG36 has a strong tolerance to metal ions. Although the enzyme activity is affected to a certain extent with the increase of ion concentration, most metal ions have no obvious inhibitory effect on its enzyme activity. After incubation at 55℃ for 30 minutes at 10mM and 1mM ion concentrations, the residual activity of the enzyme in most ions can still be maintained above 80%. Among the 11 ions involved in the experiment, Ag +The inhibitory effect was strongest, with a strong inhibitory effect on enzyme activity at a concentration of 10mM, almost inactivating the enzyme. High concentrations (1%) of SDS completely inhibited the enzyme activity of B0-BG36. In the presence of other chemical reagents, the enzyme activity decreased with increasing solvent concentration. With the exception of Tween-80 and SDS, which had a strong inhibitory effect on enzyme activity at a concentration of 1%, the remaining chemical reagents had little effect on the enzyme activity of B0-BG36. Overall, B0-BG36 is stable in the presence of most metal ions and chemical reagents, and its enzyme activity is not affected by them.

[0072] (4) Effect of glucose concentration on enzyme activity

[0073] Using p-nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate, 10 μL of B0-BG36 and 10 μL of industrial cellulase (final concentration 1 mg / mL, containing a β-glucosidase component) were added to 125 μL of glucose solution containing 10 mM pNPG (15 μL) at varying concentrations (0 mM, 500 mM, 1000 mM, 1500 mM, 2000 mM, 2500 mM, 3000 mM, 3500 mM, and 4000 mM). No enzyme was added as a control. B0-BG36 and industrial cellulase were incubated at 55°C for 10 minutes, respectively, and the reaction was terminated by adding 450 μL of 1 M Na₂CO₃. The absorbance was measured at 405 nm using a microplate reader.

[0074] Results: The glucose tolerance of B0-BG36 and industrial cellulases was determined using 1 mM pNPG as a substrate. The activities of B0-BG36 and industrial cellulases were measured in the absence of exogenous glucose, with this value set as 100%. Glucose activated B0-BG36 activity when the added glucose concentration was 0 mM < ≤ 2500 mM. In particular, at a glucose concentration of 500 mM, the enzyme activity was activated to 222.3%, with an IC50 of 3302.5 mM. B0-BG36 maintained over 30% activity in the presence of 4000 mM glucose. However, glucose significantly inhibited the activity of industrial cellulases, with the relative activity dropping below 30% at 500 mM and becoming nearly inactive at 1000 mM. The new recombinant β-glucosidase B0-BG36 in this study has a high tolerance to glucose compared with industrial cellulase and is an ideal glucose-tolerant β-glucosidase for further research and industrial application ( Figure 4 ).

[0075] (5) Effect of salt concentration on recombinase

[0076] To determine the effect of salt concentration on B0-BG36 enzyme activity, various buffer solutions with varying salt concentrations were used at 55°C and pH 5.0 to examine the effects of salt concentration on the enzyme solution. NaCl solutions of varying concentrations (0 mM, 500 mM, 1000 mM, 1500 mM, 2000 mM, 2500 mM, and 3000 mM) were prepared and dissolved in 1 mL of buffer. A reaction mixture without NaCl was used as a control (100%).

[0077] Result analysis: Figure 5 As shown, B0-BG36 exhibited good enzyme activity in high concentration NaCl (0-3000 mM) solution, retaining more than 80% of its activity in the presence of 500 mM NaCl and more than 30% of its activity in the presence of 1000 mM NaCl. The enzyme activity gradually decreased with increasing salt concentration.

[0078] (6) Determination of substrate specificity and kinetic parameters

[0079] Different substrates (cellobiose, 4-nitrophenyl-D-pyranoside, α-lactose monohydrate, gentiobiose, maltose, trehalose, melibiose, raffinose, sucrose, sodium carboxymethyl cellulose, microcrystals, beechwood xylan, corncob xylan) were added to the enzyme reaction system and placed under the optimal reaction conditions to determine the amount of hydrolysis product produced.

[0080] Kinetic parameters were determined by using different concentrations of cellobiose and α-lactose monohydrate (0.2%-2%) at the optimal temperature and pH for 5 min and 10 min, respectively. The kinetic parameters (Vmax and Km) of B0-BG36 were calculated using the Michaelis-Menten equation, and the Lineweaver-Burk double reciprocal plot was drawn.

[0081] Table 2: Hydrolysis ability of β-glucosidase B0-BG36 on different substrates

[0082]

[0083] Results: B0-BG36 showed the highest hydrolytic activity against 4-nitrophenyl-D-glucopyranoside (pNPG) (7.7±4.7 U / mg), followed by cellobiose (2.5±4.2 U / mg), α-lactose monohydrate (1.8±3.5 U / mg), and gentiobiose (0.9±1.8 U / mg). It showed no hydrolytic activity against the remaining substrates (maltose, trehalose, melibiose, raffinose, sucrose, sodium carboxymethylcellulose, microcrystalline, beechwood xylan, and corncob xylan) (Table 2). This result indicates that B0-BG36 preferentially hydrolyzes the synthetic substrate pNPG, followed by cellobiose, α-lactose monohydrate, and gentiobiose. The recombinant enzyme can hydrolyze not only the natural substrate cellobiose but also synthetic substrates, demonstrating its multifunctionality and ability to hydrolyze a variety of substrates.

[0084] Cellobiose is a natural substrate found in the natural environment, so it became the main research object. Using cellobiose as the substrate, a Lineweaver-Burk double reciprocal plot of B0-BG36 was obtained after 10 minutes of reaction with the substrate. Based on this, the Km value of the recombinant enzyme for cellobiose was calculated to be 31.39mM and the Vmax was 10.45μmol / min / mg ( Figure 6 ), when α-lactose monohydrate was used as substrate, the Km value was 109.12 mM and the Vmax was 23.87 μmol / min / mg ( Figure 7 ), which means that it has a high affinity for cellobiose, which may be due to the specificity of the enzyme. That is to say, during the long evolutionary process, microbial-derived β-glucosidase has more contact with natural substrates such as cellobiose rather than with some artificial synthetic substrates.

[0085] (7) Determination of β-glucosidase activity

[0086] β-glucosidase activity was determined using cellobiose as a substrate. 10 μL of enzyme solution was added to 90 μL of a buffer solution containing 1% (w / v) cellobiose, reacted under optimal conditions for 10 minutes, and then frozen at -80°C for 5 minutes to terminate the reaction. Subsequently, 10 μL of the reaction mixture was placed on a 96-well plate, and 200 μL of glucose oxidase-peroxidase detection kit buffer was added, incubated at 37°C for 10 minutes, and the absorbance was measured at 492 nm using a microplate reader. In the same way, a standard curve was drawn using a glucose standard for calculating the glucose concentration. One unit (U) of β-glucosidase activity is defined as the amount of enzyme required to hydrolyze cellobiose and release 2 μmol of glucose per minute. Each set of experiments was repeated three times.

[0087] Result Analysis

[0088] The activity of β-glucosidase was determined using cellobiose as a substrate, and the enzyme activity of β-glucosidase was calculated to be 2.5±4.2 U / mg (Table 2).

[0089] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other. The above description of the disclosed embodiments enables professionals and technicians in this field to implement or use the present invention. Various modifications to these embodiments will be obvious to professionals and technicians in this field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A salt-tolerant and glucose-tolerant β-glucosidase B0-BG36, characterized in that The amino acid sequence of the β-glucosidase B0-BG36 is shown in SEQ ID NO.

4.

2. A gene encoding the salt-tolerant and glucose-tolerant β-glucosidase B0-BG36 according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

3. Use of the gene according to claim 2 in encoding the salt-tolerant and glucose-tolerant β-glucosidase B0-BG36 according to claim 1.

4. An expression vector, characterized in that Contains the gene encoding the salt-tolerant and glucose-tolerant β-glucosidase B0-BG36 according to claim 2.

5. A microorganism containing the expression vector according to claim 4.

6. Use of the salt-tolerant and glucose-tolerant β-glucosidase B0-BG36 according to claim 1 in cellulose degradation.

7. Use of the salt-tolerant and glucose-tolerant β-glucosidase B0-BG36 according to claim 1 in food, feed, textile or cellulosic ethanol production.

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