Low-temperature glucose-tolerant beta-glucosidase and application thereof

Abstract

The invention discloses low-temperature glucose-tolerant beta-glucosidase and application thereof, and relates to the field of enzyme engineering. The beta-glucosidase is separated from Streptomyces microflavus CLSD-1 and belongs to a GH1 family, and the amino acid sequence of the beta-glucosidase is shown as SEQ ID No: 1 or SEQ ID No: 2. The application of the beta-glucosidase is that the beta-glucosidase is applied to simultaneous saccharification and fermentation production of ethanol from cellulose. The beta-glucosidase (Bgl2350 and Bgl5676) provided by the invention has the characteristics of low temperature and high activity, the optimal reaction temperatures are respectively 40 DEG C and 30 DEG C, and both the beta-glucosidase and the Bgl5676 have excellent glucose and xylose tolerance and can tolerate 600mM glucose and 2M xylose environments. Wherein the enzyme activity of the Bgl2350 is relatively high under a low-temperature condition; the Bgl5676 shows the stability and the salt tolerance which are obviously superior to those of the traditional GH1 family beta-glucosidase. Compared with the commercially available Novozymes Cellic CTec3 commercial enzyme, the Bgl2350 and the Bgl5676 show more prominent application potential in the field of low-temperature cellulose saccharification, and can partially replace the beta-glucosidase component in the commercial enzyme in practical application.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering, and more particularly to a low-temperature glucose-tolerant β-glucosidase and its applications. Background Technology

[0002] Cellulose is a polysaccharide compound composed of d-glucose units linked by β-1,4-glycosidic bonds, with the chemical formula (C6H2O). 10 O5)n is the most abundant renewable biopolymer in nature, mainly stored in straw, with a content as high as 35%-45%. According to statistics and estimates from the Food and Agriculture Organization of the United Nations (FAO), the world's annual straw production is around 6 billion tons. As a major agricultural country, China produces as much as 650-1 billion tons of straw annually, of which about 30% is burned or discarded, resulting in a significant waste of cellulose resources. Currently, the widely used straw treatment methods are mainly dumping, burning, and steam explosion. These methods are slow, energy-intensive, and complex to operate, and cannot recover the sugar resources in straw.

[0003] Microbial remediation technology, with its advantages of energy conservation, environmental protection, and resource regeneration, has attracted widespread attention in the field of cellulose recycling. Currently, cellulose is mainly used for bioethanol production, which involves two steps: cellulose saccharification and anaerobic fermentation of glucose by yeast to produce ethanol. Cellulose saccharification depends on β-1,4-endoglucanase, β-1,4-exoglucanase, and β-glucosidase, with β-glucosidase being the rate-limiting step, determining the efficiency of the saccharification process. The optimal temperature for industrially applied β-glucosidase is 50℃, while the anaerobic fermentation temperature of yeast is 30℃. This temperature difference results in high cooling costs and time consumption, reducing production efficiency. Low-temperature β-glucosidase can simultaneously perform saccharification and fermentation processes, thereby saving cooling costs and improving production efficiency. Currently, there are few reports on low-temperature β-glucosidase, only about ten types, and none have commercial applications.

[0004] Therefore, those skilled in the art are dedicated to developing a low-temperature glucose-tolerant β-glucosidase. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to improve the ability of β-glucosidase to saccharify cellulose at low temperatures.

[0006] To achieve the above objectives, the present invention provides a low-temperature glucose-tolerant β-glucosidase.

[0007] Furthermore, the β-glucosidase is isolated from Streptomyces microflavus CLSD-1 belongs to the GH1 family, and its amino acid sequence is shown in SEQ ID No: 1 or SEQ ID No: 2.

[0008] Furthermore, the optimal reaction temperature for the β-glucosidase is 30°C or 40°C.

[0009] Furthermore, a gene encoding the aforementioned low-temperature glucose-tolerant β-glucosidase, wherein the nucleotide sequence of the gene encoding the β-glucosidase with the amino acid sequence as shown in SEQ ID No: 1 is shown in SEQ ID No: 3, and the nucleotide sequence of the gene encoding the β-glucosidase with the amino acid sequence as shown in SEQ ID No: 2 is shown in SEQ ID No: 4.

[0010] Furthermore, the method for preparing the low-temperature glucose-tolerant β-glucosidase includes the following steps: Step 1: Construct the recombinant expression vector of the β-glucosidase; Step 2: Transform the constructed recombinant expression vector into... E. coli Recombinant engineered bacteria were obtained from BL21(DE3) competent cells; Step 3: Cultivate the recombinant engineered bacteria and add IPTG to induce the expression of β-glucosidase; Step 4: Purify the supernatant using Ni-NTA affinity chromatography to obtain the target β-glucosidase.

[0011] Furthermore, in step three, the recombinant engineered bacteria are cultured in LB liquid medium at 37°C. 200 Cultivate at rpm until bacterial culture OD 600 When the value reached 0.8, IPTG with a final concentration of 0.2 mM was added, the induction temperature was 16℃, and the induction time was 16 h.

[0012] Furthermore, the method for preparing the low-temperature glucose-tolerant β-glucosidase also includes a method for detecting the optimal catalytic temperature of the β-glucosidase. This method specifically involves: preparing an in vitro catalytic system with a pH of 6 and a total volume of 400 μL; the system containing 200 μL of 5 mM pNPG dissolved in 25 mM citrate buffer and 200 μL of 25 mM Tris-HCl buffer containing 50-400 μg of the β-glucosidase at pH 7.4; preheating all other reagents except the β-glucosidase to their respective reaction temperatures; subjecting the system to catalytic reactions at different temperatures for 1 min each; then terminating the reaction by adding 400 μL of 1 M Na₂CO₃ solution; detecting the absorbance at a wavelength of 410 nm; calculating the relative activity with the highest absorbance group as 100%; and determining the optimal catalytic temperature.

[0013] Furthermore, the method for preparing the low-temperature glucose-tolerant β-glucosidase also includes a method for determining the tolerance of the β-glucosidase to sodium chloride, specifically as follows: the β-glucosidase is placed in a 0-2M NaCl system and incubated at 4°C for 30 minutes, and then its catalytic activity is detected at the optimal catalytic temperature of the β-glucosidase; then it is incubated in the NaCl system at 4°C for 2 hours and 24 hours respectively, and the remaining activity is measured again, thereby analyzing the effect of sodium chloride concentration on enzyme stability.

[0014] Furthermore, the method for preparing the low-temperature glucose-tolerant β-glucosidase also includes a method for determining the glucose tolerance of the β-glucosidase, specifically as follows: the β-glucosidase is placed in a 0-2M glucose or xylose system and incubated at 4°C for 3 hours. After incubation, the remaining enzyme activity is measured at 40°C, 30°C, and 50°C, respectively, at pH 6, to analyze the enzyme's glucose tolerance.

[0015] Furthermore, the method for preparing the low-temperature glucose-tolerant β-glucosidase also includes a method for determining the stability of the β-glucosidase. This method specifically involves: placing the β-glucosidase in a 25 mM pH 7.4 Tris-HCl buffer solution and incubating it at different temperatures; taking samples every 30 minutes during incubation; using 5 mM pNPG as a substrate after sampling, completing the catalytic reaction at the optimal catalytic temperature and determining the remaining activity; calculating the relative activity at each sampling time point with the enzyme activity at the initial incubation time as 100% to evaluate the stability of the enzyme.

[0016] Furthermore, the application of the aforementioned low-temperature glucose-tolerant β-glucosidase is in the simultaneous saccharification and fermentation of cellulose to produce ethanol.

[0017] In a preferred embodiment 1 of the present invention, the purification process of β-glucosidases Bgl2350 and Bgl5676 is described in detail.

[0018] In another preferred embodiment 2 of the present invention, the process for detecting the optimal temperature for in vitro catalysis of β-glucosidases Bgl2350 and Bgl5676 is described in detail.

[0019] In another preferred embodiment 3 of the present invention, the process for determining the stability of β-glucosidases Bgl2350 and Bgl5676 is described in detail.

[0020] In another preferred embodiment 4 of the present invention, the process for determining the tolerance of β-glucosidases Bgl2350 and Bgl5676 to sodium chloride and sugar is described in detail.

[0021] Technical effects: 1. The two β-glucosidases (Bgl2350 and Bgl5676) provided in this invention were both isolated from low-temperature strains. Streptomyces microflavus CLSD-1. Both exhibit high activity at low temperatures, with optimal reaction temperatures of 40℃ and 30℃ respectively. Both also demonstrate excellent glucose and xylose tolerance, tolerating environments with 600mM glucose and 2M xylose. Specifically, Bgl2350 shows higher enzyme activity at low temperatures, while Bgl5676 exhibits significantly better stability and salt tolerance than traditional GH1 family β-glucosidases.

[0022] 2. Compared with the commercially available Novozymes Cellic CTec3 enzyme, Bgl2350 and Bgl5676 demonstrate greater application potential in the field of low-temperature cellulose saccharification, and can partially replace the β-glucosidase component in commercial enzymes in practical applications. Furthermore, the two β-glucosidases of this invention have advantages such as simple purification steps, low catalytic temperature, and tolerance to high-concentration products, which are expected to significantly reduce cooling energy consumption in bioethanol production, improve glucose production efficiency, and are suitable for simultaneous saccharification and yeast anaerobic fermentation processes (30°C process conditions), showing broad application prospects.

[0023] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0024] Figure 1 This is a diagram showing the construction of heterologous vectors of Bgl2350 and Bgl5676 screened from the transcriptome in a preferred embodiment 1 of the present invention: where A represents the expression of Bgl2350 and Bgl5676 in the differential transcriptome; B represents the agarose gel electrophoresis of Bgl2350 and Bgl5676 constructed on pET-28a(+) to verify the PCR results; and C represents the sequence alignment results to verify the sequencing results.

[0025] Figure 2 The following are relevant detection diagrams of purified Bgl2350 and Bgl5676 in a preferred embodiment 1 of the present invention: where A is the electrophoretic purification result of Bgl2350 and Bgl5676; B is the standard curve of substrate pNP; C and D are the optimal catalytic temperature curves of Bgl2350 and Bgl5676, respectively. Figure 3 The results of stability determination of purified Bgl2350 and Bgl5676 in a preferred embodiment 3 of the present invention are shown in Figures A and B, respectively, which are the catalytic stability curves of Bgl2350 and Bgl5676 at different temperatures.

[0026] Figure 4This is a graph showing the effect of sodium chloride on purified Bgl2350 and Bgl5676 in a preferred embodiment 4 of the present invention: where A and C are the activity curves of Bgl2350 and Bgl5676 under different concentrations of sodium chloride, respectively; and B and D are bar graphs showing the changes in the thermal stability of Bgl2350 and Bgl5676 under different concentrations of sodium chloride, respectively.

[0027] Figure 5 This is a graph showing the results of glucose tolerance testing in a preferred embodiment 4 of the present invention: where A is the tolerance curve of Bgl2350, Bgl5676 and the commercial enzyme Cellic CTec3 to different concentrations of glucose; and B is the tolerance curve of the above three enzymes to different concentrations of xylose. Detailed Implementation

[0028] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0029] Example 1: Purification of β-glucosidases Bgl2350 and Bgl5676

[0030] (1) Screening of β-glucosidases Bgl2350 and Bgl5676 and construction of heterologous expression vectors

[0031] Will Streptomyces microflavusCLSD-1 was inoculated at a 3% seed culture in 100 mL glycerol medium (composition: NaNO3 1.25g, KH2PO4 0.5g, MgSO4·7H2O 0.25g, CaCl2 0.05g, NaCl 10g, CMC-Na 10g, with deionized water to a final volume of 1L) at 25℃ and in 100 mL sodium carboxymethyl cellulose medium (composition: NaNO3 1.25g, KH2PO4 0.5g, MgSO4·7H2O 0.25g, CaCl2 0.05g, NaCl 10g, glycerol 5%, with deionized water to a final volume of 1L) at 25℃. The cultures were incubated with shaking at 200 rpm for 2 and 3 days respectively to allow the strain to reach the late logarithmic growth phase. Subsequently, 50 mL of culture was collected by centrifugation at 4000 rpm for 10 min at 4℃. The supernatant was removed, and 2 mL of PBS was added and mixed thoroughly by pipetting. After centrifugation at 4000 rpm for 10 min at 4℃, the cells were washed with 2 mL of PBS. This step was repeated twice. After the last centrifugation, the supernatant was removed, and the bacterial cells were retained. After being flash-frozen in liquid nitrogen, the cells were sent to Shanghai Paisenno Biotechnology Co., Ltd. for differential transcriptome analysis. Based on the changes in gene expression levels in the transcriptome, the β-glucosidase encoding genes Bgl2350 (nucleotide sequence as shown in SEQ ID NO:3) and Bgl5676 (nucleotide sequence as shown in SEQ ID NO:4) that were significantly upregulated in a medium with sodium carboxymethyl cellulose as the carbon source were screened. Primers were then designed based on the nucleotide sequences of genes Bgl2350 and Bgl5676. The target gene fragment and the pET vector linear fragment were obtained by PCR. The primer sequences and PCR methods are shown in Table 1. Using seamless cloning technology, 120 ng of the linear vector fragment and 60 ng of the target gene were added, along with 5 μl of ClonExpress II One Step Cloning Kit seamless cloning enzyme. The volume was brought up to 10 μL with deionized water, mixed well, and incubated at 50℃ for 10 min. The mixture was then transformed into [a specific culture medium]. E. coli DH5α. The following day, single clones were selected for PCR verification and sent to the company for first-generation sequencing. Single clones with correct sequencing results were selected and cultured overnight at 37°C with 50 μg / mL kanamycin sulfate in LB liquid medium. The next day, 1 mL of bacterial culture was used to extract the recombinant plasmid using a kit and transformed into DH5α. E. coli BL21(DE3) cells.

[0032] Result: As Figure 1 As shown in Figure A, by analyzing the differential expression levels of the transcriptome, the genes Bgl2350 and Bgl5676, which encode β-glucosidase, were identified as significantly upregulated in a culture medium with sodium carboxymethyl cellulose as the carbon source. It is speculated that these two genes may be involved in the β-1,4-glycosidic bond hydrolysis of cellobiose, a downstream product of sodium carboxymethyl cellulose hydrolysis.

[0033] like Figure 1 As shown in Figure B, agarose gel electrophoresis revealed that the gene fragments Bgl2350 and Bgl5676 are approximately 1.5 kb in size, which is consistent with the theoretical values.

[0034] Figure 1 C indicates that the heterologous expression vectors pET-28a-Bgl2350 and pET-28a-Bgl5676 containing Bgl2350 and Bgl5676 were successfully constructed.

[0035] Table 1 Primer usage table for constructing heterologous expression vectors

[0036] (2) Purification of β-glucosidases Bgl2350 and Bgl5676

[0037] The selected expression vectors for Bgl2350 and Bgl5676 (pET-28a-Bgl2350 and pET-28a-Bgl5676) were transformed into... E. coli Proteins were purified in BL21(DE3) cells using Ni-NTA affinity chromatography. The proteins were cultured in LB liquid medium containing 50 μg / mL kanamycin sulfate at 37°C. 200 E. coli carrying the recombinant vector were cultured at rpm, and when OD 600 When the concentration was 0.8, IPTG was added to a final concentration of 0.2 mM to induce protein expression, and the protein was stored at 16°C. 200 Induction was performed at rpm for 16 h. After induction, the cells were collected by centrifugation at 4000 rpm for 20 min at 4 °C. Cells were collected after adding buffer (25 mM Tris-HCl pH 7.4, 300 mM sodium chloride, and 20 mM imidazole) and then lysed at 4000 rpm for 20 min at 4 °C. The cells were then hyperbarically disrupted (800 bar, 40 mL / 1.5 min) and centrifuged at 10000 rpm for 60 min. The supernatant was passed through a Ni-NTA affinity chromatography column, and high-purity protein was obtained by elution with different concentrations of imidazole. The apparent size of the protein was verified by SDS-PAGE gel imaging, and protein purity was ensured by band uniformity. Protein concentration was determined using a Thermo Scientific NanoDrop microspectrophotometer. Finally, the purified protein was stored in a suitable buffer (25 mM Tris-HCl pH 7.4 and 5% glycerol).

[0038] Result: As Figure 2 As shown in Figure A, high-purity β-glucosidase proteins Bgl2350 and Bgl5676 can be obtained by Ni-NTA affinity chromatography.

[0039] Example 2: Determination of the optimal temperature for in vitro catalysis of β-glucosidases Bgl2350 and Bgl5676 (pure enzymes)

[0040] 1. Establishment of pNP standard curve: Stock solutions of p-nitrophenol (pNP) at different concentrations were prepared. PNP was dissolved in 25 mM citrate buffer (pH 5.5), i.e., 0.8 mM, 1.6 mM, 2.4 mM, 3.2 mM, 4 mM, 6 mM, and 8 mM substrate pNP. Then, 200 μL of the stock solution was added to 200 μL of 25 mM Tris-HCl (pH 7.4), followed by 400 μL of 1 M Na₂CO₃. The mixture was centrifuged at 12000 rpm for 1 min, diluted 8-fold, and the absorbance was measured at 410 nm. A standard curve was plotted with pNP concentration on the x-axis and absorbance on the y-axis for subsequent quantitative calculation of the pNP product.

[0041] 2. The total in vitro catalytic volume was 400 μL, and the system pH was 6. Specific components included 200 μL of 5 mM pNPG (dissolved in 25 mM citrate buffer) and 200 μL of 25 mM Tris-HCl (pH 7.4) containing either 50 μg Bgl2350 or 400 μg Bgl5676 (reagents, except for the enzyme, were incubated at their respective reaction temperatures beforehand). The blank control group consisted of enzymes inactivated at high temperatures; all other components were the same as the experimental group.

[0042] 3. The catalytic system was incubated at different temperatures for 1 min, and then 400 μL of 1M Na2CO3 was added to terminate the reaction. The absorbance was measured at 410 nm. Enzyme activity is defined as the amount of enzyme required by Bgl2350 and Bgl5676 to catalyze the hydrolysis of pNPG to produce 1 μmol of product pNP within 1 min under optimal conditions, i.e., pH 6 and temperatures of 40℃ (Bgl2350) and 30℃ (Bgl5676).

[0043] Result: As Figure 2 As shown in Figure B, the equation of the pNP standard curve is y = 9.8541x (R0). 2 =0.9987). For example... Figure 2 As shown in C and 2D, both β-glucosidases Bgl2350 and Bgl5676 can catalyze the cleavage of the β-1,4-glycosidic bond in pNPG, indicating that they are active β-glucosidases. Optimum temperature determination revealed that Bgl2350 exhibits maximum activity at 40℃, while Bgl5676 exhibits maximum activity at 30℃. Both have relatively low optimum temperatures, confirming that they are cold-adapted β-glucosidases.

[0044] Table 2 shows that Bgl2350 and Bgl5676 have catalytic activity at low temperatures, with Bgl2350 having a specific enzyme activity as high as 10.38±0.15 U / mg.

[0045] Table 1. Results of enzyme activity assays for purified Bgl2350 and Bgl5676

[0046] Example 3: Determination of β-glucosidase stability

[0047] (1) Determination of β-glucosidase stability (pure enzyme)

[0048] 10 μL of the purified β-glucosidase Bgl2350 (5 mg / mL) obtained in Example 1 was placed in 25 mM Tris-HCl (pH 7.4) without sodium chloride, and 40 μL of Bgl5676 (10 mg / mL) was placed in 25 mM Tris-HCl (pH 7.4) without sodium chloride. The remaining activity was measured at different temperatures, with three replicates for each group. Bgl2350 was incubated in water baths at 50°C, 40°C, and 30°C for 3 h, and the catalytic activity was measured every 30 min using 5 mM pNPG as a substrate, according to the method in Example 2. Bgl5676 was incubated in water baths at 40°C, 30°C, and 20°C, and the remaining enzyme activity was measured every 5 min in the 40°C group, and every 6 h or 12 h in the 30°C and 20°C groups, according to the method in Example 2.

[0049] Results analysis: such as Figure 3 As shown, Bgl2350 maintains its activity for a long time at low temperatures but is rapidly inactivated at high temperatures. It retains over 90% activity after incubation at 30℃ for 3 hours, but is rapidly inactivated after incubation at 50℃ for 20 minutes. Bgl5676 retains 50% activity after incubation at 30℃ for 24 hours and over 50% activity after 3 days at 20℃. Experimental results demonstrate that both Bgl2350 and Bgl5676 are cold-adapted enzymes, rapidly inactivated at high temperatures, but retaining high activity for a long time at low temperatures. Both exhibit highly efficient and stable low-temperature saccharification, and in industrial applications, the reaction can be terminated promptly by raising the temperature to a moderate level (50℃), saving production costs.

[0050] Example 4: Determination of the tolerance of β-glucosidases Bgl2350 and Bgl5676 to sodium chloride and sugar (pure enzymes)

[0051] (1) Determination of the sodium chloride tolerance of β-glucosidases Bgl2350 and Bgl5676

[0052] The purified Bgl2350 (5 mg / mL) and Bgl5676 (10 mg / mL) obtained in Example 1 were added at doses of 10 μL and 40 μL respectively to 190 μL and 160 μL of 25 mM Tris (pH 7.4) buffer containing 0 M to 2 M NaCl. The incubation volume was 200 μL, and the mixture was incubated at 4 °C for 30 min. Subsequently, the catalytic activity of Bgl2350 and Bgl5676 was measured at their optimal temperatures using the enzyme activity assay method of Example 2. Bgl2350 and Bgl5676 were then incubated at 4 °C for 2 h and 24 h respectively in 25 mM Tris (pH 7.4) buffer containing 0 M to 2 M NaCl. The residual activity was then measured, and the effect of sodium chloride concentration on enzyme stability was analyzed.

[0053] Results analysis: such as Figure 4 As shown, Bgl2350 exhibits maximum activity at 0.5 M NaCl, while Bgl5676 exhibits maximum activity at 0.3 M NaCl. Sodium chloride impairs the thermal stability of Bgl2350 but significantly enhances the stability of Bgl5676, which is most stable at 0.3 M NaCl. These results indicate that Bgl5676 is a salt- and cold-resistant low-temperature β-glucosidase with commercial application potential.

[0054] (2) Determination of glucose tolerance to β-glucosidases Bgl2350 and Bgl5676

[0055] Purified Bgl2350 (5 mg / mL), Bgl5676 (10 mg / mL), and Novozymes commercial enzyme Cellic CTec3 (10 mg / mL) were added in 10 μL, 40 μL, and 10 μL respectively to a 25 mM Tris buffer at pH 7.4 containing 0 M to 2 M glucose or xylose. The final reaction volume was 200 μL, and the mixture was incubated at 4 °C for 3 h. Subsequently, the residual enzyme activity of the three enzymes was measured at 40 °C, 30 °C, and 50 °C at pH 6, respectively, using the method described in Example 2.

[0056] Results analysis: such as Figure 5 As shown, Bgl2350 and Bgl5676 retained 50% relative activity at 600 mM glucose, while the commercial enzyme was completely inactivated at 200 mM glucose. In the presence of 2 M xylose, Bgl2350 and Bgl5676 retained 50% relative activity, while the commercial enzyme retained only 25%.

[0057] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A low-temperature glucose-tolerant β-glucosidase, characterized in that, The β-glucosidase was isolated from Streptomyces microflavus CLSD-1 belongs to the GH1 family, and its amino acid sequence is shown in SEQ ID No: 1 or SEQ ID No:

2.

2. The low-temperature glucose-tolerant β-glucosidase as described in claim 1, characterized in that, The optimal reaction temperature for the β-glucosidase is 30°C or 40°C.

3. A gene encoding the low-temperature glucose tolerance β-glucosidase of claim 1, characterized in that, The nucleotide sequence of the gene encoding β-glucosidase with the amino acid sequence as shown in SEQ ID No: 1 is shown in SEQ ID No: 3, and the nucleotide sequence of the gene encoding β-glucosidase with the amino acid sequence as shown in SEQ ID No: 2 is shown in SEQ ID No:

4.

4. The method for preparing low-temperature glucose-tolerant β-glucosidase according to claim 1, characterized in that, Includes the following steps: Step 1: Construct the recombinant expression vector of the β-glucosidase; Step 2: Transform the constructed recombinant expression vector into... E. coli Recombinant engineered bacteria were obtained from BL21(DE3) competent cells; Step 3: Cultivate the recombinant engineered bacteria and add IPTG to induce the expression of β-glucosidase; Step 4: Purify the supernatant using Ni-NTA affinity chromatography to obtain the target β-glucosidase.

5. The method for preparing low-temperature glucose-tolerant β-glucosidase according to claim 4, characterized in that, In step three, the recombinant engineered bacteria are cultured in LB liquid medium at 37°C. 200 Cultivate at rpm until bacterial culture OD 600 When the value reached 0.8, IPTG with a final concentration of 0.2 mM was added, the induction temperature was 16℃, and the induction time was 16 h.

6. The method for preparing low-temperature glucose-tolerant β-glucosidase according to claim 4, characterized in that, The method also includes a method for detecting the optimal catalytic temperature of the β-glucosidase. Specifically, the method involves preparing an in vitro catalytic system with a pH of 6 and a total volume of 400 μL. This system contains 200 μL of 5 mM pNPG dissolved in 25 mM citrate buffer and 200 μL of 25 mM Tris-HCl buffer at pH 7.4 containing 50-400 μg of the β-glucosidase. All reagents except the β-glucosidase are preheated to their respective reaction temperatures. The system is subjected to catalytic reactions at different temperatures for 1 min each. The reaction is then terminated by adding 400 μL of 1 M Na₂CO₃ solution. The absorbance is measured at a wavelength of 410 nm. The relative activity is calculated with the highest absorbance group as 100%, and the optimal catalytic temperature is determined.

7. The method for preparing low-temperature glucose-tolerant β-glucosidase according to claim 6, characterized in that, The method also includes a method for determining the tolerance of the β-glucosidase to sodium chloride, as follows: The β-glucosidase is placed in a 0-2M NaCl system and incubated at 4°C for 30 minutes. Then, its catalytic activity is detected at the optimal catalytic temperature of the β-glucosidase. The enzyme is then incubated in the NaCl system at 4°C for 2 hours and 24 hours, respectively, and the remaining activity is measured again to analyze the effect of sodium chloride concentration on enzyme stability.

8. The method for preparing low-temperature glucose-tolerant β-glucosidase according to claim 6, characterized in that, The method also includes a method for determining the glucose tolerance of the β-glucosidase, as follows: the β-glucosidase is placed in a 0-2M glucose or xylose system and incubated at 4°C for 3 hours. After incubation, the remaining enzyme activity is measured at 40°C, 30°C and 50°C, and pH 6, respectively, to analyze the enzyme's glucose tolerance.

9. The method for preparing low-temperature glucose-tolerant β-glucosidase according to claim 6, characterized in that, The method also includes a stability assay for the β-glucosidase, which specifically involves placing the β-glucosidase in a 25 mM pH 7.4 Tris-HCl buffer solution and incubating it at different temperatures. During incubation, samples are taken every 30 minutes. After sampling, the catalytic reaction is completed at the optimal catalytic temperature using 5 mM pNPG as a substrate, and the remaining activity is measured. The relative activity at each sampling time point is calculated with the enzyme activity at the initial incubation time as 100%, thereby assessing the stability of the enzyme.

10. The application of the low-temperature glucose-tolerant β-glucosidase according to claim 1 in the simultaneous saccharification and fermentation of cellulose to produce ethanol.

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